WO2017055522A1

WO2017055522A1 - Stabilized env proteins of hiv

Info

Publication number: WO2017055522A1
Application number: PCT/EP2016/073377
Authority: WO
Inventors: Alba Torrents DE LA PEÑA; Rogier Sanders; Andrew B. WARD; Ian Andrew Wilson; Jean-Philippe Julien; John P. Moore
Original assignee: Academisch Medisch Centrum; Cornell University; The Scripps Research Institute
Priority date: 2015-09-29
Filing date: 2016-09-29
Publication date: 2017-04-06

Abstract

The invention relates to an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gp120 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gp125 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said gp120 or gp125 polypeptide portion and said gp41 or gp36 ectodomain polypeptide portion comprise a first cysteine residue at an amino acid position equivalent to amino acid position 49, 50, 51, 71, 72 or 73 and a second cysteine residue at an amino acid position equivalent to amino acid position of a residue selected from the group consisting of: 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569 and 570, wherein numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said first and said second cysteine residues form an intermolecular disulfide bond. The invention further relates to an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gp120 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gp 125 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one amino acid residue selected from the group consisting of: 64K and 66R; and 302F, 302W, 304F, 304W, 306L, 307F, 307W, 308L, 315Y, 315K, 3151, 315W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, and 316V; and 555K or 556K; and 137A, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1.

Description

TITLE: STABILIZED ENV PROTEINS OF HIV

TECHNICAL FIELD

The invention relates generally to novel HIV envelope proteins and to methods, compositions and products related thereto. More particularly, the invention relates to methods and compositions for the preparation, production, and administration of isolated novel HIV envelope nucleic acid and protein sequences suitable, for example, as vaccines against HIV.

BACKGROUND

An HIV vaccine is desperately needed, but no experimental vaccine has proved efficient at inducing protective immunity against HIV infection. The mature, proteolytically cleaved envelope glycoprotein trimer (Env) mediates HIV entry into target c The color code is as follows ells by undergoing a complex series of conformational changes initiated by binding to the CD4 receptor, and then the CCR5 or CXCR4 co-receptor. Therefore, the Env trimer is a potential target for an effective immune response.

The generation of an HIV envelope glycoprotein (Env) based vaccine that induces broadly neutralizing antibodies (bNAbs) is required. A major obstacle is the instability of the Env trimer, which for a long time has hampered the generation of recombinant soluble Env proteins that adequately mimic the native Env spike on virions.

There have numerous attempts to stabilize the Env proteins, inter alia by introduction of a disulfide bond between residues 501 -605 ("SOS"), which was identified over 15 years ago (Binley et al. J. Virol. 74, 627-643), supplemented by a trimer- stabilizing substitution in gp41 (I559P, combined termed SOSIP gpl40. Despite many efforts, the "SOS" disulfide bond is until now, as far as the applicants are aware of, the only successful disulfide bond to be engineered between the two subunits gpl20 and gp41. One explanation for this is that the introduction of new disulfide bonds interferes with oxidative folding and disulfide isomerization in the endoplasmic reticulum, resulting of misfolding of many candidate disulfide bond mutants. There remains a need in the art to identify an alternative and/or complementary disulfide bond capable of (further) stabilizing Env proteins. It is therefore an object of the invention to provide an alternative to the SOS mutation.

The inventors have recently described an Env spike mimic, BG505 SOSIP.664 gpl40, which is based on the above described SOS, and has in addition to the disulfide bond between gpl20 and gp41 a trimer stabilizing Ile-to-Pro substitution at position 559 in gp41. Negative stain electron microscopy (EM) studies show that the protein resembles native spikes on virions. It binds virtually all known bNAbs while occluding the epitopes for most non-NAbs, and immunogenicity studies in various animal species showed that the protein is capable of inducing potent neutralization of the autologous Tier-2 BG505 virus, something that has not been previously seen with other gpl40 immunogens. Broadly comparable, native- like SOSIP.664 trimers have also been designed for B41, ZM197M and DU422 isolates from clades B and C, respectively. All of these trimers were identified through extensive screening where many candidates were eliminated due to low expression, poor trimer formation, and high content of non-native trimers etc. As immunogens, the BG505 SOSIP.664 trimers induce NAbs to the neutralization-resistant (Tier 2) autologous virus in rabbits and macaques (Sanders et al., 2015). Broadly comparable, native-like SOSIP.664 trimers have also been produced from the B41, ZM197M and DU422 clade B or C env genes (Julien et al., 2015; Pugach et al., 2015).

The problem of the of BG505 SOSIP.664 gpl40 is that it binds minimally but reproducibly to some non- NAbs, which might explain the induction of non-NAbs by BG505 SOSIP.664 trimers in vivo.

Furthermore, HD-X studies showed that parts of the protein were exchanging with solvent relatively rapidly, indicative of local conformational flexibility. It is therefore a further object of the invention to reduce the propensity of the trimer to spontaneously sample conformations in which the CD4i epitopes associated with the co-receptor binding site are accessible. Although the V3 region is tucked away under the V1V2 domain in the cryo-EM and x-ray structures of the BG505 SOSIP.664, it can become exposed on a subset of trimers in vitro and it is immunogenic in vivo.

The native Env trimer and its SOSIP.664 mimics are conformationally flexible; they can "breathe" such that they alternate between closed and more open forms in a dynamic equilibrium (Guttman et al., 2014; Munro et al., 2014; Pugach et al., 2015). While unliganded BG505 SOSIP.664 trimers have a high propensity to remain in the closed, ground state conformation, the equilibrium for their B41 and

ZM197M counterparts is shifted. Thus, a greater proportion of more open, but still native-like, trimers can be visualized by negative- stain electron microscopy (NS-EM) (Julien et al., 2015; Pugach et al., 2015). Another manifestation of this conformational isomerism is the binding of some non-NAbs to a small fraction (presumably the partially open forms) of the SOSIP.664 trimer population; these non-NAbs recognize V3, CD4-induced (CD4i) and a subset of CD4-binding site (CD4bs) epitopes, which are normally only accessible on neutralization-sensitive (Tier-1) viruses (Sanders et al., 2013). How conformational flexibility influences the immunogenicity of Env trimers is not known. It is also an object of the invention to reduce the rate or extent of "breathing" and impede conformational changes involved in the transition to more open conformations.

There remains therefore a need for improved HIV Env based immunogens that do not have these disadvantages.

BRIEF DESCRIPTIONS OF THE FIGURES

Figure 1.1 (A-H) shows the design of amino-acid substitutions to stabilize SOSIP.664 trimers. (A) Crystal structure of BG505 SOSIP.664 trimer (Pancera et al., 2014), showing the locations of amino-acid substitutions relevant to this study. One protomer is colored according to sub-regions: gp41 in red; V1V2 in cyan; V3 in purple; gpl20 inner domain layer 1 in blue; layer 2 in yellow; layer 3 in orange; outer domain and N- and C- termini of g l20 in green. (B) Detailed view of V3 and surrounding regions showing the A316W substitution, as well as residues V120, L122 and P206 with which W316 can now make hydrophobic interactions. (C) Detailed view of layers 1 and 2 of the gpl20 inner domain, showing how E64 and H66 face the unstructured region of gp41 (residues 548-568) between a6 and al. (D) Detailed view of a CD4-liganded gpl20 monomer structure (Pancera et al., 2010) highlighting the same region as in (C) and showing how the E64 and E66 side chains face gpl20 layer 2. (E) Depiction of how the, α6-α9 helices of gp41 encircle the N- and C-termini of gpl20, with M535 and N543 located in the middle and at the N-terminus of a6, respectively. (F) Infection of TZM-bl cells by BG505.T332N Env- pseudoviruses with an A316W, E64K or H66R substitution (G) Biolayer interferometry analysis of CD4 binding to AviB-tagged BG505 SOSIP trimers (H) SPR analysis of CD4 binding (left panel) and sequential CD4 and 17b binding (right panel) to BG505 SOSIP.664-D7324 trimers and both versions of SOSIP .V4-D7324 trimers.

Figure 1.2 (A-C) shows HD-X analysis of BG505 SOSIP.664 and SOSIP.v4 trimers. Butterfly plots of PGT145-purified BG505 (A) SOSIP.664, (B) SOSIP.v4.1, (C) SOSIP.v4.2 trimers, comparing deuterium exchange levels in the presence and absence of sCD4. Regions that are less (red) or more (blue) protected upon CD4-binding are mapped on the BG505 SOSIP.664 crystal structure (Pancera et al., 2014), to depict CD4-induced conformational changes. The differences in CD4-induction between the SOSIP.664 and SOSIP .v4.1 or SOSIP .v4.2 trimers were also plotted on the crystal structure (rightmost panels of B and C). The exchange kinetics of individual peptides are shown in Fig. 1.S3. Figure 1.3 shows an EM reconstruction of AMC008 SOSIP.v4.2 trimers in complex with PGV04 and 35022 Fab. The reconstruction shows there is a very high degree of structural similarity with BG505 SOSIP.664 trimers in complex with 35022 and PGT122.

Figure 1.4 (A-N) shows the immunogenicity of AMC008 and BG505 SOSIP.v4 trimers in rabbits. (A) Immunization schedule. Rabbits were immunized at week 0, 4 and 20 (green) and the Ab responses were analyzed at week 22 (magenta). Blue symbols represent BG505 trimer-immunized animals, red symbols AMC008. The SOSIP.664 recipients are shown by closed circles, SOSIP.v4 by squares. Midpoint binding Ab titers were measured by D7324-capture ELISA against (B) SOSIP.664-D7324 or (C) SOSIP .v4- D7324 trimers. (D) The ratio of the SOSIP.664/SOSIP.v4 midpoint titers is plotted. The V3 Ab response was determined by pre-incubating the sera with a cyclized V3 peptide (BG505 or AMC008 sequence, as appropriate, either unmodified or with the A316W change present in SOSIP .v4 trimers), before determining the remaining trimer binding titers by ELISA. The relative V3 responses (as a % of the total anti-trimer responses) were calculated by comparing the midpoint titers in the absence and presence of the V3 peptide. Similar results were obtained using a WT or an A316W-substituted V3 peptide (r=0.91, PO.0001, data not shown). The ratio of the SOSIP.664/SOSIP.v4 trimer binding titers correlates with the relative V3 peptide responses (r=0.59, p=0.0006, data not shown). (E) Heterologous Tier 1A (SF162) and (F) autologous Tier 2 (BG505) or Tier IB (AMC008) neutralization titers were determined using the TZM-bl assay (LSI Table 4&5). The serum dilution at which HIV-1 infectivity is inhibited by 50% (ID₅₀) is plotted. (G) The ratio of autologous/SF162 neutralization is shown. Correlation plots between autologous BG505 NAb and (H) BG505 SOSIP.664 binding Ab titers, or (I) BG505 SOSIP.v4 binding Ab titers. Correlation plots between autologous AMC008 NAb titers and (J) AMC008 SOSIP.664 binding Ab titers, SOSIP.664 (K & M), SOSIP.v4 (L & N) binding Ab titers. The r and p values are given for non-parametric Spearman correlations.

Figure LSI (A-O), related to Figure 1.1, shows the screening of stabilizing mutations using unpurified AMC008, BG505 B41 and ZM197M SOSIP.664 proteins. BN-PAGE analysis of unpurified (A) AMC008, (B) BG505, (C) B41 SOSIP.664 variants, followed by western blotting with 2G12. In the AMC008 SOSIP.664 context, the A316W, I535M and L543N substitutions make a particularly substantial contribution to trimer formation, both individually and collectively. We note that introducing the reverse N543L substitution into BG505 SOSIP.664 reduced trimer formation (data not shown). (D) Use of a novel thermostability assay to screen unpurified BG505 SOSIP.664-D7324 variants. Culture supernatants from 293T cells were incubated for 30 min at temperatures ranging from 39-77°C, then analyzed by D7324-capture ELISA with 2G12 detection. The binding of 2G12 is plotted as a function of temperature, with the OD450 value at 39°C set at 100%). The A316W substitution increases

thermostability. (E) The unfolding pattern of the variant proteins was visualized by plotting the first derivative of the graph in (D) using Graphpad prism 5. (F) The T_m values were derived from the peaks in the first derivative curves shown in (D). The T_m values obtained in this assay are highly reproducible (standard deviation of ± 0.3°C for BG505 SOSIP.664-D7324). (G) Correlation plot between T_m values obtained using unpurified BG505 SOSIP.664-D7324 proteins in the above thermostability assay and T_m values obtained using PGT145-purified BG505 SOSIP.664-D7324 trimers in a DSC assay (Table 1.1 ; Fig. 1.S2). (H-L) ELISA binding curves for bNAbs and non-NAbs to unpurified SOSIP.664 wild type and mutant proteins based on the following genotypes: (H) AMC008; (I) BG505; (J) B41 ; (K) ZM197M. (L) Competition ELISA using wild type and A316 W-substituted BG505-V3 peptides. Increasing concentrations of wild type (light green) or A316W-substituted (dark green) peptides were incubated with the V3 non-NAb 39F (1.0 μg/ml) prior to addition to immobilized, PGT145-purified BG505 SOSIP.664- D7324 trimers. As the two peptides were equally effective at binding 39F and impeding its binding to the trimers, the A316W substitution does not directly affect the 39F epitope. Similar results were obtained with two other V3 non-NAbs, 19b and 14e (data not shown). (M) High- throughput mutagenesis screen at position 316 of BG505 SOSIP.664-D7324, with a capture ELISA endpoint. Wild type or position 316- mutated variant Env proteins in culture supernatants from transiently transfected 293T cells were captured via D7324. The bound proteins were detected using either PGT145 (0.11 μg/ml; to detect native trimers) or 14e (0.5 μg/ml; to detect V3 exposure), and the ratios of the OD450 signals were plotted. The wild type residue (alanine) is shown in grey, the designed mutant (tryptophan) in red and other substituted amino acids in black. Only bulky hydrophobic amino acids reduce V3 non-NAb binding. (N, O) Spontaneous sampling of the CD4i conformation was assessed using a D7324-capture ELISA. Exposure of CD4i epitopes was assessed by measuring the binding of high concentrations (10 μg/ml) of non-NAbs (N) 17b and (O) 412d. The OD₄₅o values obtained using the wild type BG505 SOSIP.664-D7324 protein were set at 100% and used to normalize the values for the E64K and H66R mutants. Mock indicates blank transfection supernatant. Figure 1.S2 (A-N), which is related to figure 1.1, shows the effect of stabilizing mutations on the biochemical and biophysical properties of AMC008, BG505, B41 and ZM197M SOSIP.664 proteins. (A) The PGT145-purified SOSIP.664 proteins were analyzed by BN-PAGE analysis and found to be exclusively trimeric. (B) Reducing (+DTT) and non-reducing (-DTT) SDS-PAGE analysis of the same proteins. The conversion of gpl40 bands into gpl20 when DTT is present indicates gpl20-gp41 cleavage is efficient; there are also no indications that the purified trimers form higher m.wt. disulfide- linked oligomers. (C) Negative- stain EM analyses of 2G12/SEC-purified (top left panel) or PGT145-purified (all other panels) AMC008, BG505, B41 and ZM197M SOSIP.664 trimer variants. The 2D class averages are shown. Based on loop-movement, compactness and angles between individual protomers, the trimers are classified as closed native-like, partially open native-like or non-native (Pugach et al., 2015). Native-like trimers are regularly shaped and have the highest concentration of electron density at the particle center (usually shaped like a triangle because Env is trimeric). The absence or presence of additional density around this center of mass determines whether trimers are classified as closed native-like or partially open native-like, respectively. Non-native forms are often elongated and no triangular center of density is visible. The classifications are quantified below each panel. The percentages of closed and partially open native-like trimers are in green, and of non-native forms in red. The total number of particles classified is defined as 100%. (D) DSC analysis of PGT145-purified AMC008, BG505, B41 and ZM197M

SOSIP.664 trimer variants. The unfolding patterns were fitted using a non-two state model, revealing three individual unfolding peaks. The T_m values of the individual peaks are listed in Table LSI . For simplification, the data were also fitted to a two-state model and the T_m values of those analyses are provided in Table 1.1. (E) SAXS analysis of PGT145-purified BG505 SOSIP.664 and SOSIP.v4 trimers. The guinier plot show the radius of gyration for the BG505 trimers (upper panel). The Kratky plots (middle panel) show Gaussian-like peaks for all three trimers at the low angle (low q) region dipping towards 0, indicating that these proteins are well folded globules . Lower panel: summary of SAXS experiments as well as DLS experiments. The radii of gyration (R_g) were derived from SAXS

experiments using both Guinier analysis with a q*Rg range of 1 - 1.3, and from the particle distance distribution plots obtained from indirect transformation using GNOM (Svergun, 1992). The standard deviations of the R and R_g values were small, and similar between the different constructs (+/- 0.2-0.5 A). (F) Glycan profile of PGT145-purified SOSIP.664 variants. The percentages of mannose-5 to mannose-9 glycans (M5-M9) derived from the glycan profiles are listed in Table 1.1 (G-J) ELISA binding of bNAbs and non-NAbs to D7324-captured, PGT145-purified (G) AMC008; (H) BG505; (I) B41 ; (J) ZM197M SOSIP.664-D7324 trimer variants. (K-M) V3 Fab 19b binding to (K) AMC008, (L) BG505 and (M) B41 trimers was also assessed by ITC. The enthalpy changes (ΔΗ), dissociation constants (Κ_Ό) and stoichiometrics of binding (molar ration; N) are listed in panel (Ν).

Figure 1.S3 (A-C), which is related to Figure 1.2, shows the effect of stabilizing mutations on HD-X profiles of BG505 SOSIP.664 trimers. (A, B) Left panels: Butterfly plots of HD-X exchange levels in the absence of CD4. (A) PGT145-purified BG505 SOSIP.664 vs. SOSIP.v4.1 trimers; (B) PGT145-purified BG505 SOSIP.664 vs. SOSIP.v4.2 trimers. Right panels: The differences in exchange patterns are mapped onto the BG505 SOSIP.664 structure (Pancera et al., 2014). Regions that are less or more protected from deuterium exchange are colored red and blue, respectively. The positions of the stabilizing mutations are indicated in green. (C) BN-PAGE gel-shift analysis of BG505 SOSIP.664 wild type and double mutant trimers in the presence of sCD4 (0.55 mg/ml; i.e., in 3-molar excess per protomer). For all three trimers, there is a complete band-shift in the presence of sCD4, which shows that saturating binding of CD4 occurs in each case. However, note that the wild type trimers dimerize when CD4 binds, but the two double mutants do not. The large conformational changes upon CD4 binding in the WT protein might result in dimerization via the flexible variable loops, which is not the case in the mutants, as

conformational changes are largely prevented upon CD4 binding.

Figure 2.3 (A-C) shows the HIV- 1 SOSIP Env trimer structure at 3.0 A resolution. (A) Ribbon representation of gp41 in the context of the BG505 SOSIP.664 trimer. The gpl20 subunits are shown in surface representation while gp41 is shown in ribbon representation. (B) Ribbon representation of one protomer with the new region of 20 residues, located within the HR1 helix, and connecting the 1548 and 1568 residues, highlighted with a yellow surface. The position of the I559P mutation in the trimer is also indicated. On the right hand side is an enlarged image of the region between residues 1548 and 1568 (in red) and a 2Fo-Fc electron density map contoured at 0.5σ (yellow mesh) (C). (D) shows the detailed representation of mutations introduced into the gp41 structure to stabilize the SOSIP.664 trimer that improved trimer yield or thermostability (increased melting temperature). Figure 3.1 (A) shows the antigenicity and structure of BG505 SOSIP .v5 trimers containing a novel disulfide bond between gpl 20 and gp41. (a) SPR analysis of quaternary bNAbs PG16, PGT145, PGT151 and 35022 (upper panel) and CD4, 17b and sequential CD4 and 17b binding (lower panel) to BG505 SOSIP.664, BG505 SOSIP.664 H72C-H564C, BG505 SOSIP.664 A73C-A561 and BG505 SOSIP.v5.

Figure 3.2 (A-D) shows the dynamics of BG505 SOSIP.v5 trimers. Butterfly plots comparing the HDX- MS profiles of wild-type (a) with A73C-A561 C (b), E64K A316W A73C-A561 C (c), and E64K A316W H72C-H564C (d) BG505 SOSIP.664 trimers upon sCD4 binding. The percent exchange for each observable peptide is plotted at the position of the center of the peptide on the primary sequence for each time point (3 s to 20 h). Differences are mapped onto the trimer crystal structure by coloring regions with faster (red) or slower (blue) deuterium exchange upon sCD4 binding. Segments that were unresolved in the crystal structure (V2, V4, and a portion of gp41) are shown as dashed lines. Point mutations are indicated and shown as green spheres. Figure 3.3 (A-D) shows the immunogenicity of BG505 SOSIP.v5 trimers in rabbits, (a) Midpoint Ab binding titers were measured by D7324-capture ELISA, using SOSIP.664 for the SOSIP.664 immunized rabbits and SOSIP.664 containing A316W mutation for the SOSIP.v5 immunized animals. The V3 antibody response was determined by incubating a V3 peptide with the sera before determining the anti- trimer binding titer. The V3 peptide was used as in the BG505 SOSIP.664 and as in the BG505 SOSIP.v5 depending on the trimer used as an immunogen. (b) The relative V3 responses were calculated by comparing the midpoint titers with and without using the V3 peptide, (c) Neutralization of SF162 TierlA and (d) BG505 viruses was assessed by using the TZM-bl assay. The plots show the serum dilution at which 50% of the infectivity is inhibited. Figure 3.4 shows the Immunogenicity of BG505 and ZM197M SOSIP.v5 trimers in rabbits, (a) Schematic representation of vaccination schedule, (b) Color coding for the various immunogens tested, (c and i) Midpoint Ab binding titers were measured by D7324-capture ELISA using sera dilutions for the trimer variants. Neutralization of HIV-1 viruses by animals immunized with BG505 (d-g, left panels) and ZM197M (j-m, right panels) (see bottom of figure for the specific immunogens). Neutralization of (d and j) the autologous virus, (e and k) SF162 (Tier 1A), and (f, g, 1 and m) heterologous Tier 2 viruses, 92RW and SHIV162P3, as assessed by the TZM-bl assay. The plots show the ID50, the serum dilution at which 50% infectivity is inhibited by antibodies present in the sera, (h and n) Number of heterologous hits per rabbit with an ICso >40 are shown.

Figure 3.S1(A-D) : Introducing a novel disulfide bond between gpl20 and gp41. a, Five regions in gpl20 and gp41 that are in reasonably close contact are shown in the crystal structure of BG505 SOSIP.664.

These regions constitute residues in CI, C2 and C5 in gpl20 and HR1 (a6, a7, and the unresolved region connecting these helices) in gp41. The gpl20 subunit of a BG505 heterodimer is shown in grey and its gp41 in dark red. The original disulfide bond, located between residues 501 and 605 is colored blue. A close view of the interface is represented in the inset. Residues in gpl20 that might contact residues in gp41 are shown in blue spheres, b, A panel of unpurified His-tagged BG505 gpl40 proteins was screened in which the original disulfide bond was replaced by two new cysteine residues in the regions highlighted in panel A (i.e. the A501C-T605C "SOS" disulfide bond was absent during screening). Screening was based on recently published BG505 crystal structures. Trimerization efficiency was analyzed by BN- PAGE followed by western blotting. The majority of the mutants showed less efficient trimerization. The antigenic profile of the newly generated BG505 gpl40 trimers was determined by His-tag ELISA. Since BG505 gpl40 lacking a disulfide bond between gpl20 and gp41 falls apart in its constituents resulting in loss of reactivity with anti-gpl20 MAbs, binding of anti-gpl20 MAb (2G12) was evidence for the formation of an intermolecular disulfide bond. A number of mutant proteins, particularly those with a new cysteine at position 72 or 73 in the loop between αϊ and βθ combined with one in residues 554-568 in the loop between a6 and a7, showed strong reactivity with 2G12 suggesting that they formed a covalent bond

(i.e. new disulfide bond) between gpl20 and gp41. Two bNAbs that recognize distinct quaternary epitopes (PGT145: VlV2-apex; and PGT151 : gpl20-gp41 interface) were used to assess the antigenic structure of the mutants. Four mutants showed binding of PGT145 and PGT151 similar to the original BG505 SOSIP.664 trimer: H72C-H564C, A73C-A558C, A73C-A561C and A73C-L568C, suggesting that these cysteine pairs preserved the antigenic structure of the BG505 gpl40 trimers. The antigenic profile of these four proteins was assessed using a larger panel of bNAbs. Quaternary structure dependent bNAbs 35022, 3BC315 and PG16 bound efficiently to these BG505 proteins. Spontaneous opening up of the trimer was investigated with the binding of CD4i non-NAbs 17b. Binding of 17bs was abrogated and the presence of sCD4 partially restored ability to bind. Non-NAb F240 binding to the disulfide loop region of gp41 showed a slight increased binding compared to original BG505 SOSIP.664, suggesting that the absence of the A501C-T605C disulfide bond opens up the epitope of F240. F240 is directed to an epitope overlapping with residue 605. Color code: red: no binding, orange: moderate binding and green: strong binding. Binding ability is also scaled from no binding (-) to strong binding (+++). The summaries are based on at least 2-3 experiments, c, The effect on stability of the Env mutants was evaluated by a thermal melting assay that can be used on unpurified Env trimers. The proteins were incubated for 1 h at varying temperatures in a graded PCR machine and the residual binding of 2G12 was assessed by ELISA (right panel). The first derivative reveals the unfolding pattern (right panel), d, The midpoints of thermal denaturation (r_m) for each mutant were determined based on the melting profiles in panel c. Mutants containing the disulfide bond between H72C-H564C and A73C-A561C showed an increase in T_m by 1.3°C and 2.0°C compared to BG505 SOSIP trimer, respectively, suggesting that locating the disulfide bond at the core of the trimer, as opposed to the membrane proximal end, increases its stability. The T_m values for double disulfide proteins are included here for reference (see Fig. 3.S2).

Figure 3.S2 shows the combining two disulfide bonds between gpl20 and gp41. A)-Cleavage and trimerization efficiency of supernatant produced gpl40 proteins that incorporated the original disulfide bond at position 501-605, or a new disulfide bond at positions H72C-H564C or A73C-A561C, or both disulfide bonds, were determined by reducing SDS-PAGE (left panel) and BN-PAGE (right panel). All the mutants were properly cleaved, but showed slightly less efficient trimerization compared to original BG505 SOSIP.664 protein. B) The antigenic phenotype of unpurified BG505 gpl40 variants was assessed by ELISA and summarized. bNAbs 2G12, VRCOl, PGT145 and PGT151 bound very strongly to all variants. The BG505 gpl40s containing only the H72C-H564C or A73C-A561C disulfide bond but not the original A501C-T605C bond showed decreased reactivity with non-NAb 17b to a CD4i-epitope, but increased reactivity with non-NAb F240, which is directed to an epitope overlapping with residue 605, suggesting that the replacement of the original bond with the new bonds closed up non-NAb epitopes in the membrane distal parts of the trimer, but opened-up non-NAb epitopes at in gp41. The double disulfide mutants adopted a combined phenotype. Thus, binding of CD4i non-NAb 17b were abrogated, a property shared with the H72C-H564C and A73C-A561C mutants, and the binding of the gp41 non-NAb F240 was also very low, a property conferred by the original A501 C-T605C disulfide bond. Additional Cys substitutions at residues 558-569 were screened for their ability to pair with 72C and 73C, and improve native like BG505 SOSIP.664 trimers that also contained the A501C-T605C disulfide bond. Each unpurified protein was tested for reactivity with 2G12, PGT145 and PGT151 and its thermostability was tested as described in Figure 3. SI . A number of different disulfide bonds were tolerated without disturbing the overall conformation of the trimer, pointing at a high degree of plasticity in this region. In general, the mutants that were binding quaternary- dependent bNAbs to a level similar or higher than the original BG505 SOSIP.664 gpl40 also showed high trimer stability. The presence of an extra disulfide bond in the core of the trimer slightly enhanced trimer stability. Color code: red: no binding, orange: moderate binding and green: strong binding. Binding ability is also scaled from no binding (-) to strong binding (+++). The summaries are based on 3 experiments. Figure 3. S3 shows the biochemical, biophysical and antigenic characterization of PGT145-purified stabilized BG505 SOSIP.664 trimers. a, Cleavage efficiency and disulfide bond formation was determined by SDS-PAGE under reducing and non-reducing conditions, respectively. Under reducing conditions all proteins showed a gpl20 band, indicating that they were completely cleaved. Under non- reducing conditions the proteins all showed a gpl40 band, but they migrated differently through the gels, indicative of different levels of compactness (compact proteins take up less SDS and therefore migrate slower through SDS-PAGE gels). The double disulfide variants all showed a slower migration pattern due to the presence of the extra disulfide bond, b, BG505 SOSIP.664, SOSIP.v4.1 and SOSIP.v5 gpl40 proteins purified by PGT145 affinity chromatography were exclusively trimeric as determined by BN- PAGE. c, The formation of native-like trimers was assessed by negative stain electron microscopy. The 2D reference free class averages of the two double disulfide bond proteins (BG505 SOSIP.v5.1 and BG505 SOSIP.V5.2) compared to BG505 SOSIP.664 and SOSIP.v4.1 are shown. The percentage of closed native-like and open native-like trimers is shown in green and the percentage of non-native trimers in red. d, The thermal stability of BG505 SOSIP.664, BG505 SOSIP.v4.1 and BG505 SOSIP.v5 was measured by DSC. The independent non-two state best fit curves are depicted in dashed red line and the T_m values of each peak are given in the graphs. See materials and methods section for more details on curve modeling, e, SAXS scattering curve (upper panel) and Kratky plot (lower panel) show the consistency of the scattering pattern among the wild-type BG505 and disulfide mutants, indicating that they have the same solution structure, f, The antigenicity of BG505 SOSIP.664, BG505 SOSIP.664 H72C-H564C or A73C-A561C, BG505 SOSIP.664 E64K A316W (BG505 SOSIP.v4.1) and BG505 SOSIP.664 E64K A316W H72C-H564C (BG505 SOSIP.v5.1) or A73C-A561C (BG505 SOSIP.v5.2) was determined by Ni-NTA ELISA. Binding curves of a panel of broadly neutralizing and non- neutralizing antibodies are shown.

Figure 3.S4 shows the HDX-MS profiles of PGT145 purified wild-type and stabilized BG505 SOSIP.664. Butterfly plots comparing the HDX-MS profiles of wild-type with A73C-A561C (A), BG505

SOSIP.V5.2. (B) BG505 SOSIP.v5.1 and (C) BG505 SOSIP.664 trimers. The percent exchange for each observable peptide is plotted at the position of the center of the peptide on the primary sequence for each time point (3 s to 20 h). The difference plots below each primary plot reveal regions undergoing slower exchange (more protected, above the zero) and faster exchange (less protected, below the zero). The corresponding difference plots are shown below, with any major changes highlighted and labeled.

Differences are mapped onto one lobe of trimer crystal structure (PDB: 4VTP). Segments unresolved in the crystal structure (V2, V4, and a portion of gp41) are shown as dashed lines. Point mutations are indicated and shown as green spheres. Binding of sCD4 to BG505 SOSIP.664 trimers was assessed by Native-PAGE analysis. Upon sCD4 binding the wild-type BG505 SOSIP forms dimers (of trimers), which is not seen with either mutant. The laddering effect in the 50-200 kDa range is from sCD4.

Figure 3.S5D shows the antigenicity of AMC008, B41 and ZM197m SOSIPs was determined by D7324- ELISA. Representative curves of a panel of bNAbs and non-NAbs are shown. The plots are representative of two or three experiments.

Fig. 4.1 shows the design and biochemical characterization of BG505 SOSIP.664-ferritin nanoparticles. A) Top: model of eight BG505 SOSIP.664 trimers (PDB: 4TVP) with gpl20 subunits in blue and gp41 subunits in green, displayed on the H. Pylori ferritin nanoparticle (in violet, PDB: 3BVE), viewed down one of the threefold axes of the ferritin particle. The figure was drawn using Pymol. Bottom: the BG505 SOSIP.664-gpl40-ferritin construct. The hexa-arginine furin cleavage site (R6), the SOS disulfide bond between gpl20 and gp41 (C501-C605), and the I559P substitution that facilitates trimerization are indicated on the SOSIP.664 component, to which the ferritin moiety is linked via a Gly-Ser-Gly (GSG) spacer. B) Coomassie-stained reducing and non-reducing SDS-PAGE (left) and BN-PAGE (right) gels comparing soluble SOSIP.664 trimers and SOSIP.664-ferritin nanoparticles. The nanoparticles were too large to enter BN-PAGE gels efficiently, but were visible at the top of the lanes (Fig. lb, right panel, right lane). C) Representative ELISA binding curves of a panel of antibodies to SOSIP.664 trimer (2.0 μg/ml) and SOSIP.664-ferritin (0.45 μg/ml) with 2G12 as loading control. D) Unprocessed electron micrograph showing individual SOSIP.664-ferritin particles (indicated by arrows). Protein samples were prepared on carbon-coated copper grids. Imaging was carried out using an FEI Tecnai T12 microscope operating at 120 keV. Images were collected using a Tietz TemCam-F416 CMOS camera at 1 μηι defocus with an average dose of 25 electrons/A2 and a magnification of x52,000. e 84 NS-EM 2D class averages of SOSIP.664-ferritin particles. The SOSIP.664 spikes (blue arrows) and the ferritin cage (magenta arrow) are highlighted in the top right 2D class average image (E).

Fig. 4.2 shows the induction of increased antibody responses by BG505 SOSIP.664-ferritin in mice and rabbits, a Eight BALB/C mice were immunized three times (at weeks 0, 4 and 12) with either 2.8 μg of BG505 SOSIP.664 trimer or BG505 SOSIP.664-ferritin protein formulated with 25 μg MP LA adjuvant. The midpoint binding (EC50) titers to BG505 SOSIP.664 trimer were determined at week 14 by NiNTA ELISA [2]; the median titers are denoted by horizontal lines. Statistical analysis was performed using a two-tailed Mann- Whitney U test, b Two groups of five New Zealand White rabbits received

intramuscular immunizations at weeks 0, 4 and 12 with 200 μg of a non-adjuvanted DNA plasmid via electroporation of the quadriceps, followed by a protein boost at week 24 with 17 μg of protein in ISCOMATRIX™ adjuvant (75 units per rabbit) [24]. The DNA plasmids encoded either the soluble BG505 SOSIP.664 gpl40 or the BG505 SOSIP.664 gpl40-ferritin nanoparticles; none of the plasmids encoded furin. The protein boost was, correspondingly, either soluble SOSIP.664 trimers or SOSIP.664- ferritin particles, in both cases purified by a PGT145 bNAb column. The four historic control rabbits (indicated by circles in panel b) received identical DNA priming, but were then boosted with

ISCOMATRIX™ adjuvanted (75 units per rabbit) soluble BG505 SOSIP.664 trimers (40 μg) that had been purified using 2G12-affinity chromatography followed by size exclusion chromatography (SEC) [2], which are antigenically identical to PGT145-purified BG505 SOSIP.664 trimers [25]. Anti-trimer serum binding titers over the course of the experiment were tested in D7324-capture ELISA using 2G12/SEC purified D7324-tagged BG505 SOSIP.664 trimers (0.5 μg/ml), essentially as described before [2, 9]. The medians of the midpoint binding titers (±error) are plotted. Asterisks indicate significant differences at specific time points (two-tailed Mann- Whitney U test; *P < 0.05). c Midpoint neutralization (IC50) titers against the autologous neutralization-resistant (tier 2) virus, BG505, and against the negative control, MLV, at week 26. d IC50 titers against a panel of heterologous neutralization-sensitive (tier 1A and tier IB) viruses at week 26. The IC50 titers in c and d were determined using the TZM-bl neutralization assay. The pre-bleed samples lacked neutralization activity (not shown). Neutralization assays were performed either at the Academic Medical Center (SF162, 6535.3, ZM197M, HXB2, DJ268.3, BaL, ZM109F, 94UG103, 92RW020, Q23envl7 and MLV) or the Duke University Medical Center (DUMC) (BG505.T332 N, MN.3, MW965.26, Q259.d2.17, Cel 176_A3, Q769.d22, Q842.dl2, YU2, Q23envl7 and MLV). The fold difference in median IC50 titer (horizontal lines) is depicted below the graphs. The dotted horizontal lines in the BG505 SOSIP.664 group represent the median titers for the five animals from the current experiment, i.e. excluding the four control sera. The titers were very similar when the four control sera were included or excluded. Statistical differences between the nine trimer-immunized rabbits and the five nanoparticle-immunized rabbits were determined using a two-tailed Mann- Whitney U test

SUMMARY OF THE INVENTION

The invention is based on the surprising identification of a stabilizing disulfide bond resulting from a point mutation of a residue located within the alpha-0 and a residue located within the HR1 region of the Env protein of both HIV-1 and HIV-2. The modified HIV envelope (Env) glycoprotein complexes of the invention are more structurally stable than native Env complexes, which are characteristically more labile or unstable in order to be capable of efficiently undergoing conformational changes during the process of virus-cell fusion. In addition, the HIV envelope (Env) glycoprotein complexes of the invention display less reactivity to non-Nab 17b. The introduction of the disulfide bond as described herein greatly enhances trimer stability, decreases conformational flexibility, and improves the presentation of bNAb epitopes, while diminishing the exposure of non-NAb epitopes. X-ray crystallographic analyses showed that the stabilized trimers assume the unliganded conformation and Hydrogen-Deuterium Exchange studies indicated that conformational transitions towards the liganded state are blocked. The Env proteins of the invention comprising the new disulfide bonds also stabilized and improved Env trimers from diverse virus isolates. These stabilized Env trimers allow for the design of multivalent vaccines aimed at inducing bNAbs. The inventors have shown that higher global and local stability and further occlusion of non-NAb epitopes might improve the performance of Env trimers as immunogens. It was found that trimers of the stabilized Env proteins as taught herein induced weaker immune responses directed against non-Nab V3 epitopes in rabbits.

The invention therefore provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus, the Env polypeptide comprising:

- a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1, or

- a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2;

wherein said gpl20 or gpl25 polypeptide portion comprises a first cysteine residue at an amino acid position equivalent to amino acid position 49, 50, 51, 71, 72 or 73, and wherein said gp41 or gp36 ectodomain polypeptide portion comprises a second cysteine residue at an amino acid position equivalent to an amino acid position selected from the group consisting of: 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569 and 570, wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said first and said second cysteine residues form a disulfide bond between said gpl20 and said gp41 polypeptide portions, or between said gpl25 and said gp36 polypeptide portions. These disulfide bonds have several advantages. An advantage is that these disulfide bond result in trimer variants that were stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes to more open forms. These disulfide bonds also further increase stability and result in a higher melting temperature. In addition, these disulfide bonds reduce or abolish non-neutralizing V3-directed antibody binding to stabilized trimers, while quaternary- dependent bNAb epitopes is maintained or improved. Consequently, the trimers stabilized with these disulfide bonds elicit reduced V3 -directed Tier 1 A NAb responses, while autologous Tier 2 or Tier IB NAb responses are maintained. Preferably, said first and second cysteinse are at the amino acid positions equivalent to a pair of amino acid positions selected from the group consisting of: a. position 49 and a position selected from the group consisting of positions 555, 556, and 560, and

b position 50 and a position selected from the group consisting of positions 555 and 556, and

c position 51 and position 554, and d. position 72 and a position selected from the group consisting of positions 554, 555, 556, 558, 559, 560, 561, 562, 563, 564, 565, 566, and 567, and

e. position 73 and a position selected from the group consisting of positions 555, 556, 558, 559 ,560, 561, 562, 563, 565, 566, 567, 568, and 569 wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1.

In a preferred embodiment, said first cysteine is at a position selected from the group consisting of positions 49, 50 and 51 of SEQ ID NO:l, and wherein said second cysteine is at a position selected from the group consisting of positions 554, 555, 556 and 560 of SEQ ID NO: 1. The inventors observed that after introduction of a disulfide bond between 555C and 49C, binding of the 17b non-bnAb to its CD4-induced epitope was decreased 5-fold (Figure 2.S10). In a preferred embodiment, said first cysteine is at position equivalent to position 49 of SEQ ID NO:l and said second cysteine is present at position equivalent to position 555 of SEQ ID NO:l . Another advantage is that such isolated, recombinant or synthetic polypeptides have a high melting temperature. The inventors found that a SOSIP.664-L555C/Q49C double mutant has a melting temperature of 75.2°C, which is the highest that is observed to date for a SOSIP.664 trimer variant (Figure 2.5C). Therefore, in a preferred embodiment, said isolated, recombinant or synthetic polypeptide comprises cystein residues at a postion equivalent to postion 555, 49, 501, 605, and a P at a position equivalent to postion 559 of SEQ ID NO:l .

In a preferred embodiment, said second cysteine residue is at an amino acid position equivalent to amino acid position of a position selected from the group consisting of: 558 (A558), 561 (A561), 564 (H564), or 568 (L568). The amino acids between brackets refer to the amino acids in SEQ ID NO:l .

In another preferred embodiment, said first cysteine is at a position selected from the group consisting of positions 72 and 73 of SEQ ID NO:l, and wherein said second cysteine is at a position selected from the group consisting of positions 556, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567 and 568 of SEQ ID NO: 1. The advantage is that these disulfide bonds provide a high yield (see Table 3.3).

In another preferred embodiment, said first and second cysteine are at the amino acid positions equivalent to amino acid positions of a pair of amino acid residues selected from the group consisting of: residues 72 and 564, residues 73 and 558, residues 73 and 561, residues 73 and 568 of SEQ ID NO: 1. These residues create a disulfide bond which has a number of advantages, including: increasing the number of trimers to in a closed state, generating a native-like conformation in trimers and improving thermostability of trimers, keeping the trimer trapped in the closed, unliganded state, reducing binding to non-NAbs against the V3 and CD4i epitopes and improving the binding of the quaternary structure dependent bNAbs PG16, PGT145 and PGT151. The inventors observed that after introduction of a disulfide bond between 72C and 564C or between 73C and 561C in an HIV Env polypeptide, the fraction of trimers of such Env polypeptides fraction having the closed native-like conformation was increased. In a preferred embodiment therfore, said first cysteine is at position equivalent ot position 72 or 73 of SEQ ID NO:l and said second cysteine is present at a position equivalent to position 561 or 564 of SEQ ID NO:l . The fraction of closed native-like trimers was further increased when the 64K and 316W amino acid residues were also present see Table 3.1 and Fig. 3S3c). Therefore, in a preferred embodiment, said isolated, recombinant or synthetic polypeptide comprises the cystein residues at postions equivalent to postions72, 564, 501, 605, and a P at position equivalent to postion 559 of SEQ ID NO: 1. In another preferred embodiment, said isolated, recombinant or synthetic polypeptide comprises the cystein residues at positions 73, 561, 501, 605, and a P at position 559, wherein said amino acid residues are at amino acid positions equivalent to the amino acid positions of SEQ ID NO: 1.

The invention is further based on the identification of a number of mutants which improve the performance of Env trimers and in particular BG505 SOSIP trimers as immunogens. These mutations have stabilizing effect on Env trimers higher global, improve affinity of neutralizing antibodies, improve and local stability and/or reduce the binding to non-NAb epitopes.

It was found that the presence of a K at a postion equivalent to position 64 and an R equivalent to postion to SEQ ID NO:l in an isolated, recombinant or synthetic polypeptide of the invention stabilizes the trimers in the closed, pre- fusion ground state, impeding or even preventing their spontaneous sampling of the CD4-induced conformation, and reducing the opening of their variable loops. They do not affect Env folding or structure. These amino acid residues substantially reduced, or even eliminated, binding of the CD4i non-NAbs to the Env proteins of the invention. In addition, they improve the thermostability of the trimer, increases the number of losed trimers, and enhance binding to bNAbs PG16 and PGT145. The invention therefore provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one amino acid residue selected from the group consisting of a K at a postion equivalent to postion 64 of SEQ ID NO:l or an R at a position equivalent to position 66 in SEQ ID NO:l . The invention further provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one amino acid residue selected from the group consisting of 302F, 302W, 304F, 304W, 306L, 307F, 307W, 308L, 315Y, 315K, 3151, 315 W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, and 316V, wherein said amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1, and wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1. These residues reduce the binding to non-NABs in the V3 domain, in particular to 14e (see Table 1.2).

It was further found that presence of the 316W amino acid residue in isolated, recombinant or synthetic polypeptide of the invention stabilizes the trimers in the closed, pre-fusion ground state, impeding or even preventing their spontaneous sampling of the CD4-induced conformation, and reducing the opening of their variable loops. Therefore, said isolated, recombinant or synthetic polypeptide comprises a W at a position equivalent to position 316 of SEQ ID NO:l A further advantage is that it increases

thermostability. Therefore, in a preferred embodiment, said at least one amino acid residue comprises 316W of SEQ ID NO: 1.

The invention further provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one amino acid residue selected from the group consisting of 555K or 556K, wherein said amino acid residue is at amino acid position equivalent to the amino acid position of SEQ ID NO: 1, and wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1. These residues create a hydrogen bond between HR1 in gp41 and a-helix 0 in gpl20 within a protomer. Trimers having these residues have an increase by ~3 and ~2-fold compared to wild type (see Figure 2.3C).

The invention is further based on the finding that the affinity and stoichiometry of antibody-trimer binding was increased when the Nl 37 glycan of the HIV Env polypeptide was absent (Figure 2.1 and Figure 2.S2). Hence, the N137 glycan impedes Env binding of PGT121 bNAb antibody family members, implying that accommodating this glycan is the major driving force behind affinity maturation. Indeed, the Kd of 35 nM for binding of early precursor 3H+3L Ab to the SOSIP.664-N137A trimer represents an ~15-fold decrease vs. SOSIP.664 (i.e., wild-type trimer) and is comparable to the Kd of 32 nM for the mature PGT124 bnAb and wild-type trimer (Figure 1 and Figure S2). The 32H+3L antibody, the more mature PGT124 precursor, also exhibits a decrease in Kd (~16-fold) upon N137 removal, which represents a slightly lower Kd (27nM) than the PGT124 mature antibody. Thus, the Kd of PGT124 for the SOSIP.664-N137A trimer was also decreased to 6nM (Figure 2.1 and Figure 2.S2). The same trend was also observed for the PGT122 branch, where the intermediate 9H+3L and the mature PGT122 bnAb had -11- and ~5-fold Kd decreases, respectively, for SOSIP.664-N137A vs. wild-type trimer (Figure 2.1 and Figure 2.S2). The invention therefore further provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gp 125 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide wherein the N137 glycan of the HIV Env polypeptide was absent, preferably comprising 137A or wherein N137 is deleted, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1. This residue improves the affinities of all the intermediate and mature Abs for the trimer carrying this residue (see Figure 2.1 and Figure 2.S2). Preferably, said gp41 ectodomain polypeptide portion comprises the amino acid sequence of the amino acids of positions 512-664 of SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto.

In a preferred embodiment, said 120 polypeptide portion comprises the amino acid sequence of the amino acids of positions 31-511 of SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto.

In a preferred embodiment, said isolated, recombinant or synthetic polypeptide which comprises at least one amino acid residue selected from the group consisting of:

64K, 66R, 302F, 302W, 304F, 304W, 306L, 307F, 307W, 308L, 315Y, 315K, 3151, 315W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, 316V, 555K, 556K, and 137A, as described above, further comprises an intermolecular disulfide bond as defined above.

Preferably, said isolated, recombinant or synthetic polypeptide comprises the amino acid sequence according to any of the sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5.

In a preferred embodiment, said isolated, recombinant or synthetic polypeptide is based on the envelope sequences from elite neutralizers. These envelope sequences have proven to be able to induce bNAb responses in patients. These sequences present multiple bNAb epitopes while occluding most non-NAb epitopes. In the Amsterdam Cohort Studies on HIV-1 and AIDS (ACS), the inventors identified three HIV-1 elite neutralizers. From those three patients, they obtained multiple full-length envelope sequences from viral DNA isolated from PBMCs, obtained at multiple timepoints after infection. These sequences are listed herein as SEQ ID NO: 10-99. These sequences can form the basis of envelope-based vaccines. In a preferred embodiment, said isolated, recombinant or synthetic polypeptide has therefore at least the amino acid sequence corresponding to the gpl40 portion of any of the isolated, recombinant or synthetic polypeptides having the amino acid sequence of any of SEQ ID NO: 10-99.

In a highly preferred embodiment, said isolated, recombinant or synthetic polypeptide has at least the amino acid sequence corresponding to the gpl40 portion of any of the isolated, recombinant or synthetic polypeptides having the amino acid sequence of any of SEQ ID NO: 10-29. Sera from these elite neutralizers very potently neutralized the parental BG505, 94UG103 and MGRM-C026 viruses (ID50 values of 484, 453 and 967, respectively, at 40 months post-SC). In aspects of the isolated, recombinant or synthetic polypeptides discussed above, said isolated, recombinant or synthetic polypeptide of the invention has a further disulfide bond present between cysteine residues at positions equivalent to the residues of 501 and 605 of SEQ ID NO 1, preferably by the point mutations A501C and T605C. An advantage thereof is that these cysteine residues that form a further disulfide bond between gpl20 and gp41 and these isolated, recombinant or synthetic Env proteins showed higher percentages of closed trimers, which indicates further stabilization of the trimer. Another advantage of this further disulfide bond is that these isolated, recombinant or synthetic Env protein display an increased binding of the bNAb 3BC315 to gp41 and quaternary bNAbs PGT145 and PG16 against the trimer apex, as well as quaternary bNAbs PGT151 and 35022 against the gpl20-gp41 interface, suggesting that these trimers have an improved quaternary native-like conformation.

In a preferred embodiment said isolated, recombinant or synthetic polypeptide has the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

In a preferred embodiment, the isolated, recombinant or synthetic polypeptide of the invention, comprises further at least one amino acid residue selected from the group consisting: 25 W, 51C, 54C, 60E, 64K, 66N, 66R, 66A, 71C, 112A, 112S, 112R, 114C, 120C, 121 W, 121F, 121C, 122C, 125W, 127F, 127W, 128C, 129F, 129C, 134C, 136C, 136N, 139W,140I, 151C, 152C, 153C, E153F, 153W, 154C, 154F, 154W, 155C, 156C, 161C, 162F, 162W, 163F, 163W, 164F, 164W, 165F, 165W, 165Y, 166Y, 166F, 166W, 166C, 167C, 167F, 167E, 167N, 172F, 172W, 175C, 175F, 175W, 176W, 177C, 177W, 178C, 178C, 179C, 179F, 179W, 180F, 180W, 181F, 181W, 182F, 182W, 191F, 191W, 193C, 193F, 193W, 194F, 194W, 194W, 198F, 198W, 199C, 200C, I201C, 202C, 202F, 202W, 203C, 204F, 204W, 210A, 210S, 210R, 21 IK, 220C, 221C, 244C, 293F, 293S, 302F, 302W, 304F, 304W, 306L ,307F, 307W, 308L, 313C, 315Y, 315K, R315I, R315W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, 316V, 317C, 318C, 319W, 320C, 420C, 421C, 421F, 421W, 422F, 422W, 423F, 423W, 430F, 430W, 432F, 432W, 432P, 433P, I433C, M436F, 436W, 437C, 520W, 523C, 535M, 543N, 554K, 555K, 556K, 559C, 560C, 561C, 562C, 570C, 571C, 574C, 578C, 582C, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1. These amino acid residues further improve the antigenic properties of the isolated, recombinant or synthetic Env protein or result in a better stability. In a preferred embodiment, the isolated, recombinant or synthetic Env proteins comprise the amino acid residues 201C and 433 C, which also reduce binding of non-Nabs, increase thermostability and constrain conformational flexibility of the Env proteins.

Preferably, said at least further at least one amino acid residue or mutation (the substituted residue is indicated by brackets) is selected from the group consisting of (N)302F, (N)302W, (R)304F, (R)304W, (R)306L, (I)307F, (I)307W, (R)308L, (R)315Y, (R)315K, (R)315I, (R)315W, (R)315F, (R)315V, (A)316Y, (A)316K, (A)316I, (A)316F, (A)316W, (A)316V, most preferably (A)316F, (A)316W, (R)315W, (R)315F, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1. An advantage thereof is that these mutations fill the hydrophobic cavity underneath the V3 and prevents the V3 from popping out of its hidden location underneath VI V2 and enhances thermostability of the isolated, recombinant or synthetic Env protein of the invention.

In another preferred embodiment, said at least one amino acid residue is selected from the group consisting of: 64K, 66R, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1. These amino acid residues substantially reduced, or even eliminated, binding of the CD4i non-NAbs to the Env proteins of the invention and therefore reduce spontaneously sampling of the CD4-bound conformation.

Preferably, said isolated, recombinant or synthetic polypeptide has at least one amino acid residue selected from the group consisting of 302F, 302W, 304F, 304W, 306L, 307F, 307W, 308L, 315Y, 315K, 3151, 315W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, 316V and at least one amino acid residue selected from the group consisting of: 64K, 66N, 66R, 66A, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1 , and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1. An advantage thereof is that the combination of these mutations result in further improvement of the thermostability and an increase in compactness of the isolated, recombinant or synthetic polypeptide of the invention in comparison to unmodified counterparts (see Fig. 1.S1B-G). In addition, it reduces the binding to non-NAbs 19b and 14e. In each case, binding of non-NAbs to CD4i- epitopes (targets of for example the E64K or H66R substitutions) and V3 epitopes (targets of the A316W change and equivalents thereof) to the double mutants was diminished or abolished, while bNAb epitopes were unchanged (Fig. 1.S1H-K). In a highly preferred embodiment, the isolated, recombinant or synthetic Env protein comprises E64 or H66, which appear to be critical for facilitating CD4-induced

conformational changes within the trimer that are essential for function, and that are accompanied by formation of the CD4i epitopes. In a highly preferred embodiment, said isolated, recombinant or synthetic polypeptide comprises the combination of amino acid residues selected from the group consisting of: 64K and 316W; 125W and 316W; 535M and 543Q; 535M and 543N; 64K, 316W and 543N; 535M, 543N, 316W and 64K;535M, 543N, 316W and 66R; 535M, 543N, 66R and 316W; 64K and 535M; 64K, 316W and 535M; 66R, 316W and 535M; 535M and 543Q; 535M and 543N; 316W, 535M and 543N; 64K, 316W, 535M and 543N, 66R, 316W, 535M and 543N, wherein numbering of said combination of amino acid residues is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1.

In a preferred embodiment, said isolated, recombinant or synthetic polypeptide has the amino acid sequence of SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:l l l, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:119, SEQ ID

NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:180, and SEQ ID NO:181, or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Preferably, said isolated, recombinant or synthetic polypeptide is of HIV- 1 or HIV-2.

In another aspect, the invention further provides a trimer comprising an isolated, recombinant or synthetic polypeptide according to the invention.

In another aspect, the invention further provides a virus like particle (VPLs) or pseudoparticle comprising the isolated, recombinant or synthetic polypeptide according to the invention or the trimer according to the invention.

The invention is further based on the finding that ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-b earing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer-b earing nanoparticles induced significantly higher neutralizing antibody responses against most tier 1A viruses, and higher responses, to several tier IB viruses and the autologous tier 2 virus than when the same trimers were delivered as soluble proteins. Nanoparticles that display hemagglutinin trimers from influenza virus on their surface are described in WO2013044203A2. However, it was unknown whether such nanoparticles also improve immunogenicity of Env proteins of HIV. Therefore, the invention further provides a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to a HIV Env protein, such that the nanoparticle comprises HIV Env trimers on its surface.

Preferably, the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin, an insect ferritin, a fungal ferritin and a mammalian ferritin. Preferably, the monomeric ferritin subunit protein is a monomeric subunit of a Helicobacter pylori ferritin protein. Preferably, the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence of the isolated, recombinant or synthetic polypeptide as described above.

Preferably, the monomeric ferritin subunit protein comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, and 181. Preferably, the fusion protein comprises a linker sequence, preferably having the amino acid sequence GSG.

Further provided is a pharmaceutical composition comprising the isolated, recombinant or synthetic polypeptide according to the invention, the trimer according to the invention, the VLP or the nanoparticle as described above and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant. In an embodiment, said pharmaceutical composition further comprises an anti- retroviral agent.

In another aspect, the invention provides the isolated, recombinant or synthetic polypeptide according to the invention, the trimer according to the invention, the VLP, the nanoparticle or the pharmaceutical composition according to the invention or for use as a medicament.

The invention further provides the isolated, recombinant or synthetic polypeptide according to the invention, the VLP, the nanoparticle or the pharmaceutical composition according to the invention or the trimer according to the invention for use in the treatment of an HIV infection or in the prevention of an HIV infection. In a preferred embodiment, said HIV infection is a HIV- 1 infection. The invention further provides a nucleic acid encoding the isolated, recombinant or synthetic polypeptide according to the invention.

The invention further provides a vector comprising the nucleic acid according to the invention. In an embodiment, said nucleic acid is a cDNA.

The invention further provides an eukaryotic or prokaryotic host cell comprising the vector of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. The following standard abbreviations are used throughout the specification to indicate specific amino acids: A=ala=alanine; R=arg=arginine; N=asn=asparagine; D=asp=aspartic acid; C=cys=cysteine; Q=gln=glutamine; E=glu=glutamic acid; G=gly=glycine;

H=his=histidine; I=ile=isoleucine; L=leu=leucine; K=lys=lysine; M=met=methionine; F=phe=phenylalanine; P=pro=proline; S=ser=serine; T=thr=threonine; W=trp=tryptophan; Y=tyr=tyrosine; V=val=valine.

"HIV" refers to the human immunodeficiency virus. HIV includes, without limitation, HIV-1 and HIV-2. The HIV-1 virus may represent any of the known major subtypes or clades (e.g., Classes A, B, C, D, E, F, G, J, and H) or outlying subtype (Group 0). Also encompassed are other HIV-1 subtypes or clades that may be isolated. There are two distinct types of HIV, HIV-1 and HIV-2, which are distinguished by their genomic organization and their evolution from other lentiviruses. Based on phylogenetic criteria (i.e., diversity due to evolution), HIV-1 can be grouped into three groups (M, N, and O). Group M is subdivided into 11 clades (A through K). HIV-2 can be divided into six distinct phylogenetic lineages (clades A through F) (Human Retroviruses and AIDS 1998: A compilation and analysis of nucleic acid and amino acid sequences (Los Alamos National Laboratory, Los Alamos, NM, 1998, http://hiv- web.lanl.gov).

The term "env gene", as used herein, refers to the polynucleotide of the viral genome that encodes the envelope protein of HIV. The related terms "Env polypeptide", "Env protein'Or "envelope polypeptide" refers to a molecule derived from an HIV envelope protein. The HIV envelope glycoprotein complex (Env) is displayed on the surface of the virus and is the target of neutralizing antibodies. Two different proteins comprise the Env complex: The HIV-1 comprises gpl20, the surface component, and gp41, the transmembrane component. The HIV-2 Env polypeptide comprises the Env gpl25 subunit, which is highly homologous to gpl20 of HIV-1 and the HIV-2 transmembrane subunit gp36, which is highly homologous to HIV-1 gp41. Each functional Env complex consists of three copies of each of these two proteins in a trimer of heterodimers. The glycoproteins are initially produced during virus infection as a polyprotein precursor, designated gpl60. Cellular proteases cleave gpl60 into the two subunits, gpl20 and gp41, which remain non-covalently associated with each other in the Env complex. The gpl40 is a modified gpl60, that lacks the segments of gp41 that normally are imbedded in the viral membrane (transmembrane or TM segment) or in the interior of the virus or cell (cytoplasmic tail, CT).

The term "Env polypeptide" encompasses without limitation a gpl40 envelope polypeptide, gpl45 envelope and gpl60 envelope polypeptide.

As used herein, the term "mature Env monomer" refers to both a HIV-1 gpl60 Env glycoprotein, comprising the HIV-1 Env gpl20 subunit and the HIV-1 Env transmembrane subunit gp41, and to a HIV- 2 gpl40 glycoprotein, comprising the HIV-2 Env gpl25 subunit and the HIV-2 transmembrane subunit gp36. The term "soluble Env monomer" refers to both the soluble HIV-1 Env glycoprotein (termed gpl40), comprising the HIV-1 Env gpl20 subunit and the extracellular region of the HIV-1 Env gp41 subunit, and the soluble HIV-2 Env glycoprotein, comprising the HIV-2 Env gpl25 subunit and the extracellular region of the HIV-2 Env gp36 subunit. The heterotrimeric nature of these HIV Env proteins are likely to result in the presentation of neutralization epitopes that differ from those on homotrimers, which may lead to the elicitation of broader neutralizing antibody responses upon immunization.

The term "gpl40 envelope" or "gpl40 envelope polypeptide" refers to a protein having two polypeptide chains, the first chain comprising the amino acid sequence of the HIV gpl20 glycoprotein and the second chain comprising the amino acid sequence of the water-soluble portion of HIV gp41 glycoprotein ("gp41 portion"). HIV gpl40 protein includes, without limitation, proteins wherein the gp41 portion comprises a point mutation such as I559P. A gpl40 envelope comprising such mutation is encompassed by the terms "HIV SOS gpl40", as well as "HIV gpl40 monomer" or "SOSIP gpl40".

The term "gpl60 envelope" or "gpl60 envelope polypeptide" refers to a protein having two polypeptide chains, the first chain comprising the amino acid sequence of the HIV gpl20 glycoprotein and the second chain comprising the amino acid sequence of the complete (thus including the transmembrane portion of gp41) HIV gp41 glycoprotein ("gp41 portion").

The term "gpl45 envelope" or "gpl45 envelope polypeptide" refers to a protein having two polypeptide chains, the first chain comprising the amino acid sequence of the HIV gpl20 glycoprotein and the second chain comprising the amino acid sequence of a truncated HIV gp41 glycoprotein ("gp41 portion"), wherein the cytoplasmic tail is deleted.

The term "gp41 " includes, without limitation, (a) the entire gp41 polypeptide including the

transmembrane and cytoplasmic domains (also referred herein as "complete polypeptide portion"); (b) gp41 ectodomain (gp4lECTo); (c) gp41 comprising the ectodomain and the transmembrane domains, but without the cytoplasmic tail; (d) gp41 modified by deletion or insertion of one or more glycosylation sites; (e) gp41 modified so as to eliminate or mask the well-known immunodominant epitope; (f) a gp41 fusion protein; and (g) gp41 labeled with an affinity ligand or other detectable marker.

As used herein, "ectodomain" means the extracellular region of a transmembrane protein exclusive of the transmembrane spanning and cytoplasmic regions. gp41 polypeptides or polypeptide portions also include "gp41 -derived molecules", which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three-dimensionally similar chemicals) of the native gpl20 structure, as well as protein sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gp41, or combinations thereof.

As used herein, "gp41 portion" encompasses any gp41 polypeptides as defined above, including consensus peptides, which are derived from the HR2 domain of gp41 from any HIV isolate. These peptides can include gp41 homo logs that have at least one amino acid substitution, deletion or insertion. The term "g l20" as used herein refers to an envelope protein from HIV-1. The envelope protein is initially synthesized as a longer precursor protein of 845-870 amino acids in size, designated as gpl60. gpl60 forms a homotrimer and undergoes glycosylation in the endoplasmic reticulum and within the Golgi apparatus. It is then cleaved by a cellular protease into gpl20 and gp41. Gp41 contains a transmembrane domain and remains in a trimeric configuration; it interacts with gp 120 in a non-covalent manner. gpl20 contains most of the external, surface-exposed, domains of the envelope glycoprotein complex, and it is gpl20 which binds both to the cellular CD4 receptor and to the cellular chemokine receptors (such as CCR5).

The mature gpl20 wildtype polypeptides have about 500 amino acids in the primary sequence. gpl20 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kD. Exemplary sequence of wt gpl60 polypeptides are shown on GENBANK®, for example accession numbers AAB05604 and AAD12142 incorporated herein by reference in their entirety as available on Feb. 25, 2009.

The gpl20 core has a unique molecular structure, which comprises two domains: an "inner" domain (which faces gp41) and an "outer" domain (which is mostly exposed on the surface of the oligomeric envelope glycoprotein complex). The two gpl20 domains are separated by a "bridging sheet" that is not part of either domain. The gpl20 core typically comprises 25 beta strands, 5 alpha helices, and 10 defined loop segments. The 10 defined loop segments include five conserved regions (C1-C5) and five regions of high variability (VI -V5).

As used herein, "gpl20 polypeptide" or "gpl20 polypeptide portion" encompasses peptides, including consensus peptides, which are derived from the HR2 domain of gpl20 from any HIV isolate. These peptides can include gpl20 homo logs that have at least one amino acid substitution, deletions or insertions. In a preferred embodiment, the gpl20 polypeptide has the amino acid sequence corresponding to the amino acids of positions 31-511 of HxB2 SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, or 99%, wherein the positions are numbered according to the HxB2 reference sequence. In another preferred embodiment, said gpl20 polypeptide or gpl20 polypeptide portion refers to residues 31-511 of BG505 SOSIP SEQ ID NO:2 or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, wherein numbering is according to SEQ ID NO:l . gpl20 polypeptides and portions thereof also include "gpl20-derived molecules" which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three-dimensionally similar chemicals) of the native gpl20 structure, as well as protein sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gpl20, or combinations thereof.

As used herein, a variant gpl20 polypeptide is a gpl20 polypeptide in which one or more amino acids have been altered (e.g., inserted, deleted or substituted). In one example, a variant gpl20 polypeptide is a gpl20 polypeptide in which at least 8 consecutive residues, such as 9, 10, 11 or 12 consecutive residues, of the fourth conserved loop (C4) between residues 419 and 434 of gpl20 of SEQ ID NO:l have been deleted. In a particular example, a variant gpl20 polypeptide includes a gpl20 polypeptide in which residues 424-432 are deleted. Additional variant gpl20 polypeptides include deletions of INMWQKVGK (residues 434-442 of SEQ ID NO:l), INMWQKVGKA (residues 434-443 of SEQ ID NO:l),

INMWQKVGKAM (residues 434-444 of SEQ ID NO: 1), RIKQIINMWQKVGK (residues 429-442 of SEQ ID NO:l), IKQIINMWQKVGK (residues 430-442 of SEQ ID NO:l), KQIINMWQKVGK

(residues 431 -442 of SEQ ID NO : 1 ), QIINMWQKVGK (residues 422-442 of SEQ ID NO : 1 ),

IINMWQKVGK (residues 433-442 of SEQ ID NO:l). In other embodiments, variant gpl20 polypeptides include combinations of the amino and carboxyl ends between residues 429 and 444. Any of the disclosed variant gpl20 polypeptide including deletions in C4 can also include a deletion in the V1V2 loop region (with an amino acid sequence set forth in SEQ ID NO:l); see S R Pollard and DC Wiley, EMBO J. 11 :585-91, 1992 which is hereby incorporated by reference in its entirety.

Unless the amino acid numbering of gpl40/145/160/120/41/125/36 polypeptides disclosed herein is directly referred to a specific sequence, the numbering is relative to the HxB2 numbering scheme as shown for SEQ ID NO:l in this application.

The term "fragment", as used herein, refers to a unique portion of the polynucleotide encoding the HIV-1 envelope polypeptide of the present invention shorter in length than the parent sequence. Similarly, the term "fragment" refers to an HIV-1 envelope polypeptide of the present invention comprising up to the entire length of the defined peptide sequence minus one amino acid residue and the coding nucleotide sequence thereof. For example, a fragment may comprise from 5 to 2500 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250, 500 or at least 700 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25 percent or 50 percent) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.

The term "truncated Env polypeptide" as used herein refers to a fragment of the Env polypeptide, which comprises at least a gpl20 portion and an ectodomain portion of gp41. As used herein, the terms "heptad repeat 1 " and "HRl " are used indistinctly to refer to an heptad repeat region that is located at the amino terminus of wild- type gp41. A heptad repeat is a motif in which a hydrophobic amino acid is repeated every seven residues; such motifs are designated a through g. See Lupas A, Trends Biochem. Sci. 1996; 21 :375-382. Heptad repeats which contain hydrophobic or neutral residues at the a and d positions can form alpha helices and are able to interact with other heptad repeats by forming coiled coils. See Chambers P, et al., J. Gen. Virol. 1990; 71 :3075-3080; Lupas A, supra. The gp41 HRl and HR2 sequences are well known in the art. See Miller M, et al., Proc. Natl. Acad. Sci. USA 2005; 102: 14759- 14764. In the particular case of the HxB2 env protein, the HRl region corresponds to amino acids 542 to 591 of the polypeptide depicted in SEQ ID NO: l .

The term "I559P" refers to a point mutation wherein the isoleucine residue at position 559 of a polypeptide chain is replaced by a proline residue. Thus, the invention encompasses an HIV- 1 isolate in which a proline residue replaces, or is substituted for, a non- proline (e.g., isoleucine) amino acid at an amino acid position equivalent to position 559 in the SEQ ID NO: l , for example. Illustratively, e.g., equivalent amino acid position(s) in other HIV- 1 strains or clades may be determined by reference to SEQ ID NO: 1. In a preferred embodiment, the gpl40 of HIV-1 BG505 containing said I559P point mutation has the amino acid sequence of SEQ ID NO:2.

The term " H564C" refers to a point mutation wherein the histidine residue at position 564 of a polypeptide chain is replaced by a cysteine residue. Thus, the invention encompasses an HIV- 1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., histidine) amino acid at an amino acid position equivalent to position 564 in the SEQ ID NO: l , for example SEQ ID NO: 102, SEQ ID NO: l l l , SEQ ID NO: 1 13, SEQ ID NO: 160, SEQ ID NO: 165 and SEQ ID NO: 180. Other examples of isolated, recombinant or synthetic polypeptides according to the invention have the amino acid sequence of Illustratively, e.g., equivalent amino acid position(s) in other HIV- 1 strains or clades may be determined by reference to SEQ ID NO: l .

The term "A73C " refers to a point mutation wherein the alanine residue at position 73 of a polypeptide chain is replaced by a cysteine residue. Thus, the invention encompasses an HIV- 1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., alanine) amino acid at an amino acid position equivalent to position 73 in the SEQ ID NO: l , for example SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 1 12, SEQ ID NO: 1 14, 137- 150, SEQ ID NO: 161 , SEQ ID NO: 166 and SEQ ID NO: 181. Illustratively, e.g., equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO: l

The term "H72C " refers to a point mutation wherein the histidine residue at position 72 of a polypeptide chain is replaced by a cysteine residue. Thus, the invention encompasses an HIV- 1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., histidine) amino acid at an amino acid position equivalent to position 72 in SEQ ID NO: l , example SEQ ID NO: 102, SEQ ID NO: l 1 1 , SEQ ID NO: 1 13, SEQ ID NO: 123-136, SEQ ID NO: 160, SEQ ID NO: 165 and SEQ ID NO: 180. Illustratively, e.g., equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO:l.

The term "A316 W" refers to a point mutation wherein the alanine residue at position 316 of a polypeptide chain is replaced by a tryptophan residue. Thus, the invention encompasses an HIV-1 isolate in which any hydrophobic amino acid residue (e.g. tyrosine, phenylalanine, valine or isoleucine, but preferably a tryptophan), preferably bulky residue replaces, or is substituted for, a smaller amino acid residue (e.g., alanine) amino acid at an amino acid position equivalent to position 316 in SEQ ID NO:l, for example SEQ ID NO:5, SEQ ID NO:108-110, SEQ ID NO:113-114, SEQ ID NO:154, SEQ ID NO:156-161, SEQ ID NO: 170, SEQ ID NO: 174, and SEQ ID NO:177-181. Illustratively, e.g., equivalent amino acid position(s) in other HIV- 1 strains or clades may be determined by reference to SEQ ID NO: 1.

The term "A561C" refers to a point mutation wherein the alanine residue at position 561 of a polypeptide chain is replaced by a cysteine residue. Thus, the invention encompasses an HIV-1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., alanine) amino acid at an amino acid position equivalent to position 561 in SEQ ID NO:l, for example SEQ ID NO:104, SEQ ID NO:l 12, SEQ ID NO:l 14, SEQ ID NO:131, SEQ ID NO:143, SEQ ID NO:161, SEQ ID NO:166, and SEQ ID NO:181. Illustratively, e.g., equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO:l .

The term "E64K " refers to a point mutation wherein the glutamic acid residue at position 64 of a polypeptide chain is replaced by a lysine residue. Thus, the invention encompasses an HIV-1 isolate in which a lysine residue replaces, or is substituted for, a non- lysine (e.g., glutamic acid) amino acid at an amino acid position equivalent to position 64 in the SEQ ID NO:l, for example SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:152, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160-163, SEQ ID NO:168, SEQ ID NO:174, and SEQ ID NO:178. Illustratively, e.g., equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO: 1. The term "H66R" refers to a point mutation wherein the histidine residue at position 66 of a polypeptide chain is replaced by an arginine residue. Thus, the invention encompasses an HIV-1 isolate in which an arginine residue replaces, or is substituted for, a non- arginine (e.g., histidine) amino acid at an amino acid position equivalent to position 66 in the SEQ ID NO:l, for example. Illustratively, e.g., equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO: l . The term "pharmaceutically acceptable carriers" are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer, phosphate-buffered saline (PBS), or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may include, but are not limited to, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers, diluents and excipients include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Solid compositions may comprise nontoxic solid carriers such as, for example, glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate. For administration in an aerosol, such as for pulmonary and/or intranasal delivery, an agent or composition is preferably formulated with a nontoxic surfactant, for example, esters or partial esters of C6 to C22 fatty acids or natural glycerides, and a propellant. Additional carriers such as lecithin may be included to facilitate intranasal delivery.

Preservatives and other additives, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like may also be included with all the above carriers.

Adjuvants are formulations and/or additives that are routinely combined with antigens to boost immune responses. Suitable adjuvants for nucleic acid based vaccines include, but are not limited to, saponins, Quil A, imiquimod, resiquimod, interleukin-12 delivered in purified protein or nucleic acid form, short bacterial immunostimulatory nucleotide sequences such as CpG- containing motifs, interleukin-2/Ig fusion proteins delivered in purified protein or nucleic acid form, oil in water micro- emulsions such as MF59, polymeric microparticles, cationic liposomes, monophosphoryl lipid A, immunomodulators such as Ubenimex, and genetically detoxified toxins such as E. coli heat labile toxin and cholera toxin from Vibrio. Such adjuvants and methods of combining adjuvants with antigens are well known to those skilled in the art.

Adjuvants suitable for use with protein immunization include, but are not limited to, alum; Freund' s incomplete adjuvant (FIA); saponin; Quil A; QS-21 ; Ribi Detox; monophosphoryl lipid A (MPL) adjuvants such as Enhanzyn(TM); nonionic block copolymers such as L- 121 (Pluronic; Syntex SAF); TiterMax Classic adjuvant (block copolymer, CRL89-41, squalene and microparticulate stabilizer;

Sigma- Aldrich); TiterMax Gold Adjuvant (new block copolymer, CRL- 8300, squalene and a sorbitan monooleate; Sigma- Aldrich); Ribi adjuvant system using one or more of the following: monophosphoryl lipid A, synthetic trehalose, dicorynomycolate, mycobacterial cell wall skeleton incorporated into squalene and polysorbate-80; Corixa); RC-552 (a small molecule synthetic adjuvant; Corixa) ; Montanide adjuvants (including Montanide IMS1I1X, Montanide IMS131x, Montanide IMS221x, Montanide IMS301x, Montanide ISA 26A, Montanide ISA206, Montanide ISA 207, Montanide ISA25, Montanide ISA27, Montanide ISA28, Montanide ISA35, Montanide ISA50V, Montanide ISA563, . Montanide ISA70, Montanide ISA 708, Montanide ISA740, Montanide ISA763A, and Montanide ISA773; Seppic Inc., Fairfield, NJ) ; and N-Acetylmuramyl-L-alanyl-D-isoglutamine hydrate (Sigma- Aldrich) . Methods of combining adjuvants with antigens are well known to those skilled in the art. Because current vaccines depend on generating antibody responses to injected antigens, commercially available adjuvants have been developed largely to enhance these antibody responses. To date, the only FDA-approved adjuvant for use with human vaccines is alum. However, although alum helps boost antibody responses to vaccine antigens, it does not enhance T cell immune responses. Thus, adjuvants that are able to boost T cell immune responses after a vaccine is administered are also contemplated for use. It is also known to those skilled in the art that cytotoxic T lymphocyte and other cellular immune responses are elicited when protein-based immunogens are formulated and administered with appropriate adjuvants, such as ISCOMs and micron-sized polymeric or metal oxide particles. Certain microbial products also act as adjuvants by activating macrophages, lymphocytes and other cells within the immune system, and thereby stimulating a cascade of cytokines that regulate immune responses. One such adjuvant is monophosphoryl lipid A (MPL), which is a derivative of the gram- negative bacterial lipid A molecule, one of the most potent immunostimulants known. The Enhanzyn(TM) adjuvant (Corixa Corporation, Hamilton, MT) consists of MPL, mycobacterial cell wall skeleton and squalene.

Adjuvants may be in particulate form. The antigen may be incorporated into biodegradable particles composed of poly- lactide-co-glycolide (PLG) or similar polymeric material. Such biodegradable particles are known to provide sustained release of the immunogen and thereby stimulate long-lasting immune responses to the immunogen. Other particulate adjuvants include, but are not limited to, micellular particles comprising Quillaia saponins, cholesterol and phospholipids known as

immunostimulating complexes (ISCOMs; CSL Limited, Victoria AU), and superparamagnetic particles. Superparamagnetic microbeads include, but are not limited to, [mu]MACS(TM) Protein G and

[mu]MACS(TM) Protein A microbeads (Miltenyi Biotec) , Dynabeads(R) Protein G and Dynabeads(R) Protein A (Dynal Biotech) . In addition to their adjuvant effect, superparamagnetic particles such as [mu]MACS(TM) Protein G and Dynabeads(R) Protein G have the important advantage of enabling immunopurification of proteins.

A "prophylactically effective amount" is any amount of an agent which, when administered to a subject prone to suffer from a disease or disorder, inhibits or prevents the onset of the disorder. The

prophylactically effective amount will vary with the subject being treated, the condition to be treated, the agent delivered and the route of delivery. A person of ordinary skill in the art can perform routine titration experiments to determine such an amount. Depending upon the agent delivered, the prophylactically effective amount of agent can be delivered continuously, such as by continuous pump, or at periodic intervals (for example, on one or more separate occasions) . Desired time intervals of multiple amounts of a particular agent can be determined without undue experimentation by one skilled in the art.

"Inhibiting" the onset of a disorder means either lessening the likelihood of the disorder's onset, preventing the onset of the disorder entirely, or in some cases, reducing the severity of the disease or disorder after onset. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely.

"Reducing the likelihood of a subject's becoming infected with HIV" means reducing the likelihood of the subject's becoming infected with HIV by at least two-fold. For example, if a subject has a 1% chance of becoming infected with HIV, a twofold reduction in the likelihood of the subject becoming infected with HIV would result in the subject having a 0.5% chance of becoming infected with HIV. In the preferred embodiment of this invention, reducing the likelihood of the subject's becoming infected with HIV means reducing the likelihood of the subject's becoming infected with the virus by at least ten-fold. "Subject" means any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, goats, pigs, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds and fowl, such as chickens and turkeys. Artificially modified animals include, but are not limited to, transgenic animals or SCID mice with human immune systems. In the preferred embodiment, the subject is a human. "Exposed" to HIV means contact or association with HIV such that infection could result. A

"therapeutically effective amount" is any amount of an agent which, when administered to a subject afflicted with a disorder against which the agent is effective, causes the subject to be treated. "Treating" a subject afflicted with a disorder shall mean causing the subject to experience a reduction, diminution, remission, suppression, or regression of the disorder and/or its symptoms. In one embodiment, recurrence of the disorder and/or its symptoms is prevented. Most preferably, the subject is cured of the disorder and/or its symptoms.

"HIV infected" means the introduction of viral components, virus particles, or viral genetic information into a cell, such as by fusion of cell membrane with HIV. The cell may be a cell of a subject. In the preferred embodiment, the cell is a cell in a human subject. "Host cells" include, but are not limited to, prokaryotic cells, e.g., bacterial cells (including gram-positive cells), yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to HeLa cells, COS cells, CV1 cells and various primary mammalian cells. Numerous mammalian cells can be used as hosts, including, but not limited to, mouse embryonic fibroblast NIH-3T3 cells, CHO cells, HeLa cells, L(tk-) cells, PER.C6 and COS cells. Mammalian cells can be transfected by methods well known in the art, such as calcium phosphate precipitation, electrop oration and microinjection.

Electroporation can also be performed in vivo as described previously (see, e.g., U.S. Patent Nos.

6, 1 10,161 ; 6,262,281 ; and 6,610,044).

"Immunizing" means generating an immune response to an antigen in a subject. This can be

accomplished, for example, by administering a primary dose of an antigen, e.g., a vaccine, to a subject, followed after a suitable period of time by one or more subsequent administrations of the antigen or vaccine, so as to generate in the subject an immune response against the antigen or vaccine. A suitable period of time between administrations of the antigen or vaccine may readily be determined by one skilled in the art, and is usually on the order of several weeks to months. Adjuvant may or may not be coadministered. "Nucleic acid" refers to any nucleic acid or polynucleotide, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, T, G and U, as well as derivatives thereof. Derivatives of these bases are well known in the art and are exemplified in PCR Systems, Reagents and Consumables (Perkin-Elmer Catalogue 1996- 1997, Roche Molecular Systems, Inc., Branchburg, NJ, USA) .

A "vector" refers to any nucleic acid vector known in the art. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors and bacteriophage vectors. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as animal papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTC or MoMLV) , Semliki Forest virus or SV40 virus. The eukaryotic expression plasmid PPI4 and its derivatives are widely used in constructs described herein. However, the invention is not limited to derivatives of the PPI4 plasmid and may include other plasmids known to those skilled in the art. In accordance with the invention, numerous vector systems for expression of recombinant proteins may be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), Semliki Forest virus or SV40 virus. Additionally, cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide (e.g., antibiotic) resistance, or resistance to heavy metals such as copper or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals. The cDNA expression vectors incorporating such elements include those described by (Okayama and Berg, 1983). The term "Protein conformation" refers to the characteristic 3 -dimensional shape of a protein, including the secondary (helices, sheet), supersecondary (motifs), tertiary (domains) and quaternary (multimeric proteins) structure of the peptide chain.

The term "native conformation" as used herein refers to the characteristic state, formation, shape or structure of a protein in the biologically active form in a living system in which it is folded to a global minimum of Gibbs free energy as defined by C.B. Anfinsen (Nobel Lecture, Dec. 1 1 , 1972).

The skilled person is capable of determining whether a protein comprising disulfide bonds is folded properly and in the native conformation. Such determinations may for instance comprise the measurement of the properly folded, oxidized and digested Lys-Arg-Insulin intermediate, by HPLC analysis. In embodiments, such determinations comprise X-ray crystallographic analyses as described herein. A method of the present invention is used preferably for the isolation of recombinantly produced proteins that comprise disulfide-bonds in the native conformation. Recombinantly produced proteins can be either directly expressed or expressed as a fusion protein. Detection of the expressed protein is achieved by methods known in the art such as, for instance, radioimmunoassays, Western blotting techniques or immunoprecipitation.

Based on loop-movement, compactness and angles between individual protomers, the Env trimers may be classified as closed native-like, partially open native-like or non-native (Pugach et al., 2015). Native-like trimers are regularly shaped and have the highest concentration of electron density at the particle center (usually shaped like a triangle because Env is trimeric). The absence or presence of additional density around this center of mass determines whether trimers are classified as closed native-like or partially open native-like, respectively. Non-native forms are often elongated and no triangular center of density is visible.

The term "closed native conformation" or "closed conformation" when referring to an Env trimer, refers to the visual shape of the Env trimer as previously described for BG505 SOSIP.664 (Sander et al. 2013, A next-generation cleaved, soluble HIV- 1 Env trimer, BG505 SOSIP. 664 gpl40, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog 9:el 003618.

http://dx.doi.org/10.1371/iournal.ppat.100361838). Based on visual inspection of 2D images, an Env trimer has a closed native conformation if it has a compact triangular propeller shape with no additional density surrounding the trimer. Trimers containing triangular propeller density of an intensity and size similar to the closed native conformation group, but displaying one, two, or three smaller spheres of density at the distal ends of the triangular density, are defined as having an open native conformation.

The term "nonnative conformation" refers to the visual shape of an Env trimer particle which does not clearly show a central, triangular mass. Typically, a trimer having a nonnative conformation resembles previously described images of uncleaved, non-SOSIP gpl40 proteins (Ringe RPet al. 2013. Cleavage strongly influences whether soluble HIV- 1 envelope glycoprotein trimers adopt a native-like

conformation. Proc Natl Acad Sci U S A 1 10: 18256-18261. http://dx.doi.org/l 0.1073/pnas

.1314351 110.).

The conformation as described above of the isolated, recombinant or synthetic Env protein may suitably be characterized by negative stain electron microscopy (EM) and Reducing SDS-PAGE as described herein. Embodiments of the Invention

The isolated, recombinant or synthetic Env proteins of the invention may be made of any Env polypeptide and may be of any type or clade of HIV. The HIV-1 Env polypeptide comprises a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion. The Env polypeptides are modified to comprise a first cysteine residue at an amino acid position equivalent to amino acid position 49, 50, 51, 71, 72 or 73 and a second cysteine residue at an amino acid position equivalent to amino acid position of a residue selected from the group consisting of: 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570 of the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, resulting in an intermolecular disulfide bond between said first and said second cysteine residue.

The present invention encompasses HIV envelope (Env) glycoprotein complexes, which comprise covalently associated surface gpl20 and transmembrane gp41 glycoprotein subunits, and soluble forms thereof. The HIV envelope (Env) glycoprotein complexes of the invention are more structurally stable than native Env complexes, which are characteristically more labile or unstable in order to be capable of efficiently undergoing conformational changes during the process of virus-cell fusion. In addition, the HIV envelope (Env) glycoprotein complexes of the invention display less reactivity to non-Nab 17b.

In accordance with the present invention, the structural instability of the native HIV Env complex, or soluble forms thereof, is overcome by the introduction of amino acid sequence changes designed to stabilize inter-subunit interactions between gpl20 and gp41 or between their HIV-2 equivalents gpl25 and gp36. Such changes according to this invention include the introduction of a disulfide bond between gpl20 and gp41 or their HIV-2 equivalents, between a residue located within the alphaO and a residue located within the HR1 region of said Env polypeptide. The introduction of the disulfide bond as described herein greatly enhances trimer stability, decreases conformational flexibility, and improves the presentation of bNAb epitopes, while diminishing the exposure of non-NAb epitopes. X-ray

crystallographic analyses showed that the stabilized trimers assume the unliganded conformation and Hydrogen-Deuterium Exchange studies indicated that conformational transitions towards the liganded state are blocked. The Env proteins of the invention comprising the new disulfide bonds also stabilized and improved Env trimers from diverse virus isolates. These stabilized Env trimers allow for the design of multivalent vaccines aimed at inducing bNAbs. The inventors believe that higher global and local stability and further occlusion of non-NAb epitopes might improve the performance of Env trimers as immunogens.

The gp41 portion encompasses any gp41 polypeptides or fragments or truncated forms thereof, including consensus peptides, which are derived from the HR2 domain of gp41 from any HIV isolate. These peptides can include gp41 homo logs that have at least one amino acid substitution, deletions or insertions. Gp41 polypeptides or polypeptide portions also include "gp41 -derived molecules" which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three- dimensionally similar chemicals) of the native gpl20 structure, as well as proteins sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gp41, or combinations thereof.

In a preferred embodiment, the gp41 ectodomain polypeptide portion has the amino acid sequence of the amino acids of positions 512-664 of HxB2 SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In another preferred embodiment, said gp41 ectodomain polypeptide portion refers to residues 516-672 of BG505 SOSIP SEQ ID NO:2 or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

The "gpl20 polypeptide" or "gpl20 polypeptide portion" encompasses peptides, including consensus peptides, which are derived from gpl20 from any HIV isolate. These peptides can include gpl20 homologs that have at least one amino acid substitution, deletions or insertions. In a preferred embodiment, the gpl20 polypeptide has the amino acid sequence corresponding to the amino acids of positions 31-511 of HxB2 SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, wherein the positions are numbered according to the HxB2 reference sequence.

In another preferred embodiment, said gpl20 polypeptide or gpl20 polypeptide portion refers to residues 37-515 of BG505 SOSIP SEQ ID NO:2 or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Because of sequence numbering variability among different HIV strains and isolates, it will be appreciated that the amino acid positions of the residues between which a disulfide bond of the invention may be formed may not be the same in all HIV isolates. For example, residue 73 and residue 561 in SEQ ID NO:l are located at different positions in SEQ ID NO:2. Therefore, as used herein the term "position equivalent to a certain residue of a certain sequence", refers to the position of the same residue a can be identified after alignment of the different sequences. For example position equivalent to the amino acid 73 (which is an A) of SEQ ID NO 1, refers to the A in position 78 of SEQ ID NO:2. It will be appreciated that once a certain amino acid is identified in, for example, a given gpl60 sequence, the corresponding position in the gpl60 sequence of other HIV-1 or HIV-2 isolates can be easily identified by aligning the sequence of both gpl60 variants or by performing multiple alignment of the gpl60 wherein the position of the disulfide bond is to be identified within a plurality of other gpl60 sequences.

For sequence comparison, typically one sequence acts as a reference, to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of HIV envelope glycoproteins, fusion proteins comprising envelope glycoproteins and nucleic acid sequences encoding the same, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used. The term "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for instance, by the Smith- Waterman local homology algorithm, by the

Needleman-Wunsch homology alignment algorithm, by the Pearson-Lipman similarity search method, by computerized implementations of these algorithms or by manual alignment and visual inspection. See Smith T, Waterman M, Adv. Appl. Math. 1981 ; 2:482-489; Needleman S, Wunsch C, J. Mol. Biol. 1970; 48:443-453; Pearson W, Lipman D, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448; the GAP,

BESTFIT, FASTA and TFASTA programs, Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, USA; Ausubel F, et al., Eds" "Short Protocols in Molecular Biology", 4th Ed. (John Wiley and Sons, Inc., New York, NY, USA).

The BLAST and BLAST 2.0 algorithms are suitable for determining percent sequence identity and sequence similarity. See Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402; Altschul S, et al., J. Mol. Biol. 1990; 215:403-410. The BLAST and BLAST 2.0 programs are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.

Software for performing BLAST analyses is publicly available through the National Center for

Biotechnology Information (http://blast.ncbi.nlm.nih.gov/blast.cgi, August 2010). This algorithm involves first identifying high scoring sequence pairs (HSPs) through the recognition of short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. See Altschul S, et al., 1997, 1990, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix alignments (B) of 50, expectation (E) of 10, M=5, N4, and a comparison of both strands. See Henikoff S, Henikoff J, Proc. Natl. Acad. Sci. USA 1989; 89:10915-10919. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See Karlin S, Altschul S, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The Env polypeptides according to the invention also encompass HIV-2 Env polypeptides. HIV-2 Env proteins comprise a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion. By alignment, the positions equivalent to residues mentioned herein, such as at residues 72 and 73 can be identified. For example, in SEQ ID NO:9, which is an exemplary HIV-2 gpl60 protein, the equivalent residues are residues V and D of amino acid sequence RDTWGTVQCLPDNGDYTEIRLN of SEQ ID NO:9. These residues correspond to the H and A of the amino acid sequence

HNVWATHACVPTDPNPQEWLV of SEQ ID NO:l . The equivalent residues of residues 561 and 564 can also be identified by alignment as residues R and E of amino acid sequence

QQPVDWKRQQELLRLTVWGTKNLQ of SEQ ID NO:9. These residues correspond to residues A and H of amino acid NNLLRAIEAQQHLLQLTVWGIKQLQ of SEQ ID NO:l .

In a preferred embodiment, the isolated, recombinant or synthetic polypeptide of the invention has an affinity for an bNAb including but not limited to 2G12, PGT145, PGT151, VRCOl, PGT121, PG16. In another preferred embodiment, the isolated, recombinant or synthetic polypeptide is capable of forming homotrimers, preferably also of heterodimers. In another preferred embodiment, the isolated, recombinant or synthetic polypeptide has a negligible affinity for antibodies directed against the V3 such as the antibodies 19b, 14E, 39F.

In another preferred embodiment, the isolated, recombinant or synthetic polypeptide has a negligible affinity forbinding to antibodies directed to the CD4-induced epitopes, such as the antibodiesantibody 17b, 48d and 412d.

In another preferred embodiment, the isolated, recombinant or synthetic polypeptide has a melting temperature higher than 60.0 °C, preferably higher than 63.0 °C, 63.7 °C; 65,0 °C, 68,0 °C, 68,3 °C, 68,5 °C, 69,0 °C, 70 °C , 75 °C, 75.2 °C, 80,0 °C, 85,0 °C, or 90,0 °C, preferably as determined by a DSC assay. The melting temperature may be determined using a conventional DSC assay as previously described. In another preferred embodiment, the isolated, recombinant or synthetic polypeptide is capable of folding a trimer in a closed native conformation. Preferably, as may be determined using 2D class averages imagery as described herein, at least 33%, 35%>, more preferably at least 40%>, 45%>, 50%>, 55%>, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 95% is in a native conformation.

In another preferred embodiment, the isolated, recombinant or synthetic polypeptide having at least 33%> of homotrimers in closed native conformation.

The isolated, recombinant or synthetic Env proteins of the invention also encompass Env proteins which have the disulfide bond according to the invention and which have further modifications to the Env proteins. However, it will be understood that such modifications should preferably not have detrimental effects for the above mentioned functions of the isolated, recombinant or synthetic Env protein. Preferably, the isolated, recombinant or synthetic Env protein has also a negligible affinity to CD4i-Abs such as 48d, 412d. In the context of the present invention, the capacity for adhesion to CD4 can be determined by radio-immune precipitation, by ELISA, Octet, ITC or by surface plasmon resonance, the detail of these methods being set out in the remainder of the description. These methods can be modified within the limit of current knowledge, the objective being to simply make sure that the glycoprotein according to the invention indeed forms a complex with CD4.

The CD4 molecules can be prepared in all kinds of different ways, including purification from a natural source or using genetic recombination techniques. In this context, it is possible to use the CD4 molecules described in WO 89/03222, WO 89/02922, Smith et al. (Science, 238, 1704-1707, 1987) and Littman et al. (Nature, 325, 453-455, 1987), for example. The company ERC BioServices Corporation, 649A Lofstrand Lane, Rockeville, Md. 20850, USA, also sells a CD4 produced by CHO ST4.2 cells (In: Aids Research and Reference Reagent Program Catalog, the Nat. Inst. Health U.S.D.H.H.S.), for example.

The affinity (Kd) for an antibody can suitably be measured by surface plasmon resonance and is preferably be of the order of 10<-4 >to 10<-12 >M, preferably 10<-9 >to 10<-11 >M, which is in accordance with the affinities already measured for gpl20 molecules (Smith et al., Science, 238: 1704, 1987; Lasky et al., Cell, 50: 975, 1987), for example.

The thermostability of the isolated, recombinant or synthetic Env proteins may suitably be determined using differential scanning calorimetry (DSC) as described herein. In a preferred embodiment, the isolated, recombinant or synthetic Env protein of the invention has at least a homology of 70% to SEQ ID NO:l, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein of the invention has at least a homology of 70% to SEQ ID NO:2, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein of the invention has at least a homology of 70% to SEQ ID NO:9, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein of the invention has a sequence homology of at least 70%> to a polypeptide having the amino acid sequence of any of SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In another preferred embodiment, the isolated, recombinant or synthetic Env protein of the invention comprises a polypeptide having the amino acid sequence having a sequence homology of at least 70%> to the amino acid sequence corresponding to the gpl40 portion of any of the Env polypeptides having the amino acid sequences of SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The Env polypeptides contemplated for modification may also comprise modifications that do not substantially alter the function of the Env protein. As an example of modifications contemplated to be within the scope of the present invention, certain amino acids may be substituted for other amino acids in a polypeptide structure without appreciable loss of interactive binding capacity of the structure such as, for example, the epitope of an antigen that is recognized and bound by an antibody. Since it is the interactive capacity and nature of a polypeptide that defines its biological (e.g. immunological) functional activity, certain amino acid sequence substitutions can be made in an amino acid sequence (or its underlying DNA coding sequence) and nevertheless obtain a polypeptide with comparable properties. Various changes may be made to the amino acid sequences of the antigens of the present invention without appreciable loss of immunogenic activity.

It is understood in the art that in order to make functionally equivalent amino acid substitutions, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art. See Kyte J, Doolittle R, J. Mol. Biol. 1982; 15(1):105-132. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within plus or minus 2 is preferred, those which are within plus or minus 1 are particularly preferred, and those within plus or minus 0.5 are even more particularly preferred. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics; these are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartate (-3.5), asparagine (-3.5), lysine (-3.9) and arginine (-4.5).

It also is understood in the art that the substitution of similar amino acids can be made effectively on the basis of hydrophilicity; particularly where the immunologically functional equivalent polypeptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present invention. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity. See Hopp T, US 4,554,101. In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within plus or minus 2 is preferred, those which are within plus or minus 1 are particularly preferred, and those within plus or minus 0.5 are even more particularly preferred. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate (+3.0 plus or minus 1), glutamate (+3.0 plus or minus 1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5 plus or minus 1), alanine (-0.5), histidine (- 0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5) and tryptophan (-3.4). It is well known in the art that where certain residues are shown to be particularly important to the immunological or structural properties of a protein or peptide, like for example, residues in binding regions or epitopes, such residues may not generally be exchanged. In this manner, functional equivalents are defined herein as those polypeptides, which maintain a substantial amount of their native

immunological activity. In general, the shorter the length of the molecule, the fewer changes can be made to the molecule without affecting its function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification. In a preferred embodiment, the isolated, recombinant or synthetic Env protein comprises a methionine at position equivalent to position 535 of SEQ ID NO:l(M535). Residue 535 is positioned in the middle of the a6 helix, which might explain why a methionine here has a stabilizing effect; thus, Met has a higher helical propensity in the isolated, recombinant or synthetic Env (Fig. L IE, Table 1.S2).

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises a glutamine, a methionine, or an asparagine at position equivalent to position 543 of SEQ ID NO:l .

All residues selected of 535M, 543N and 543Q improve trimer formation and a similar effect was observed for both 543N and 543Q (Fig. LSI A). In addition,

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises 535M together with 543N or 543Q. NS-EM analysis showed an unchanged percentage of closed vs. open trimers (-15% closed; Table 1.1), but thermostability was increased (see Table 1.1). Isolated, recombinant or synthetic Env proteins of the invention comprising 535M together with 543N or 543Q also have an improved binding of the quaternary structure-dependent bNAbs PGT145, 35022 and PGT151.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises an 316W, 316Y, 316V, or 3161. The advatage thereof is that such residue strengthens hydrophobic interactions between V3 and the underlying gpl20 layer 2 (i.e., V120, L122 and P206), thereby decreasing the propensity for V3 to flip out of its ground-state location (Fig. 1.1 A,B). As a result, the A316W, 316Y, 316V, or 3161 improves trimer formation, and it also increases the thermostability as assessed by a novel assay that can be used with unpurified Env proteins (Fig. 1.S1B-G). Of note is that the V3 non-NAbs 447-52D, 39F, 14e and 19b each bound markedly less well to all the A316W variant trimers than to wild- type (Fig. 1.S1H-K). For example, 14e and 19b binding to PGT145 purified BG505 SOSIP.664 A316W trimers was reduced by ~80%> and ~50%>, respectively, and for the CD4i non-NAbs 17b and 412d binding was -50% lower (Table 1.2, Fig. 1.S2F). As the 316W residue did not affect binding of the same non- NAbs to V3 peptides, we conclude that it works indirectly on the trimer, by impeding the exposure of V3 epitopes (Fig. 1.S1L). In contrast, the A316W residue has no adverse effect on the binding of multiple bNAbs to the AMC008, BG505 and B41 SOSIP.664 trimers (Fig. 1.S1H-K). A high-throughput mutagenesis screen of position 316 showed that other bulky hydrophobic amino acids (Tyr/Phe/Val/Ile) also decreased V3 exposure (>3-fold), whereas other substitutions had little, if any, effect (Fig. 1.S1M).

Further, trimers based on the modified protein of the invention comprising 316W, 316Y, 316V, or 3161 have a high proportion in the closed conformation. Their thermostability was also improved see Table 1.1). Introduced into Env-pseudovirus, the 316W, 316Y, 316V, or 3161 residue reduced infectivity by -98%, implying either that conformational flexibility is important for co-receptor interactions during entry or that the 316 residue plays a direct role in co-receptor binding (Fig. 1.S1 L).

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the residue 64K or 66R. Both 64K and 66R residues substantially reduce, or even eliminate, the binding of the CD4i non-NAbs in the isolated, recombinant or synthetic Env proteins (Fig. l .SlH-O). After trimers of the isolated, recombinant or synthetic Env protein having the 64K residue were PGT145-purified and visualized by NS-EM, the percentage of closed trimers was substantially increased for the E64K mutant (-90%) closed vs. 35% for wild-type). Each residue had also a minor (improved) effect on thermostability. In an ELISA, bNAb binding was enhanced (e.g., for the trimer apex bNAbs PG16 and PGT145).

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the residues 316W, 316Y, 316V, or 3161 combined with any of the 64K or 66R residues. These isolated, recombinant or synthetic Env proteins have acquired the beneficial properties associated with each individual residue. Each double mutant was expressed efficiently and formed fully cleaved trimers, and thermostability was slightly increased over wild-type (Fig. 1.S1B-G). For the double mutants of all genotypes tested, the binding of non-NAbs to CD4i-epitopes (targets of the 64K or 66R residues) and V3 epitopes (targets of the 316W residue) was diminished or abolished, while bNAb epitopes were unaffected (Fig. 1.S1H-K).

The term "SOSIP.v4 trimer" as used herein, refers to a trimer of the the isolated, recombinant or synthetic Env protein having the 559P amino acid residue comprising further the optimal amino acids at position 535M and 543N or 543Q, and in addition the 316W residue and either theE64K (designated SOSIP.v4.1) or the 66R (SOSIP.v4.2) residues (Table 1.S3). SOSIP.v4 trimers induced weaker anti-V3 responses in rabbits and, as a result, lower titers of V3-dependent NAbs against Tier- IA virus SF162. This effect was observed without compromising the autologous NAb response. The inventors have surprisingly found that many bNAbs bound equally well to BG505 SOSIP.664-D7324 and SOSIP.v4-D7324 trimers in ELISA, indicating their epitopes are unaffected by the stabilizing substitutions. This outcome was confirmed by SPR for the quaternary structure-dependent bNAbs PG16, PGT145, 35022 and PGT151 (Fig. 1.1H). Furthermore, in the ELISA there was a marked reduction in the binding of V3 non-NAbs 14e and 19b (by ~90%> and 50%>, respectively, compared to SOSIP.664) and CD4bs non-NAb b6 (by ~75%>), while the CD4i non-NAbs 17b and 412d bound to a negligible or undetectable extent (Table 1.2, Fig. 1.S2H). In previous ITC studies, the 19b Fab bound minimally to the BG505 SOSIP.664 trimers; the stoichiometry was 0.2 (i.e., on average one Fab per 5 trimers), (Pugach et al., 2015; Sanders et al., 2013). In contrast, 19b binding to the BG505 SOSIP.v4.1 mutant was abolished (Fig. 1.S2L), confirming that the stabilizing substitutions reduced V3 exposure. Similarly, non-NAb binding to V3 and CD4i epitopes on the B41 SOSIP.v4 trimers was strongly reduced (Table 1.2, Fig. 1.S2I). Thus, in the ITC assay, 19b binding was reduced ~10-fold (stoichiometry 0.02 vs. 0.2 for SOSIP.664; Fig. 1.S2M). Hence, as for the other SOSIP.v4 trimers, the B41 V3 region is now effectively sequestered. Comparable results were also obtained for ZM197M SOSIP.v4 trimers. The bNAb epitopes were generally unaffected, and the quaternary structure dependent bNAbs PGT145 and PGT151 actually more strongly in ELISA than to the wild type ZM197M SOSIP.664-D7324 trimers (Table 1.2, Fig. 1.S2J). Once more, the binding of non-NAbs to V3 and CD4i epitopes was greatly diminished (Table 1.2, Fig. 1.S2J).

Furthermore, the inventors have also found that SOSIP.v4 trimers have a native-like conformation. To confirm their overall native-like structure and bNAb epitope presentation, they prepared complexes of the AMC008 SOSIP.V4 trimers with bNAbs PGV04 and 35022 (added as Fabs), and visualized them by NS- EM. The stabilized trimers were all compact entities that were virtually indistinguishable from previously published low-resolution reconstructions of BG505 and B41 SOSIP.664 trimers (see Fig. 1.3). Therefore, in another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543N, and 316W. In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q, and 316W. An advantage of these 2 embodiments is that their trimers have retained the ability to bind the quaternary-dependent and various other bNAbs (2G12, PGT135, PGT121, PGT126, PG9, PG16, PGT145, PGT151, 35022, VRCOl and CHI 03). In addition, their reactivity with non-NAbs was generally reduced. Furthermore, they do not undergo CD4-induced conformational changes efficiently, as judged by the lack of induction of the 17b and 412d CD4i epitopes (Table 1.2, Fig. 1.S2G). In Isothermal Titration Calorimetry (ITC) experiments using untagged said trimers, reactivity of Fab fragments of the V3 non-NAb 19b was decreased compared to wild type.

In ELISA, CD4-IgG2 binding to AMC008, B41 and ZM197 SOSIP.v4-D7324 trimers was slightly reduced compared to SOSIP.664-D7324, but more markedly so for the BG505 trimers. Thus, if CD4- IgG2 binding to BG505 SOSIP.664-D7324 trimers is defined as 100% (see Methods), binding to the

SOSIP.v4.1 and SOSIP.v4.2 variants was reduced to 27% and 32%, respectively (Table 1.2, Fig. 1.S3). In contrast, there was no change in the binding of bNAbs CH103 and VRCOl to CD4bs epitopes. An SPR analysis showed that CD4-IgG2 association rates were comparable for the SOSIP.v4 and wild type trimers, but the ligand dissociated markedly faster from the stabilized trimers (Fig. 1.1H). Biolayer interferometry data confirmed that the affinity of sCD4 for various AviB-tagged BG505 stabilized trimers was 2- to 4-fold lower in each case, with the greatest reduction (4-fold) seen with the A316 W single mutant and the SOSIP.v4.2 double mutant (Fig. 1.1G).

The ELISA studies also showed that the sCD4-induction of the 17b and 412d CD4i epitopes was negligible for the AMC008, B41, ZM197M and BG505 SOSIP.v4 trimers (5-11% compared to 100% for their SOSIP.664 counterparts; Table 1.2, Fig. 1.S3). When 17b was added to CD4-IgG2-trimer complexes in an SPR study, it did not bind detectably to the his-tagged BG505 SOSIP.v4 trimers, in marked contrast to its strong binding to the wild-type SOSIP.664 trimer under the same conditions (Fig. 1.1H). Thus, the stabilizing substitutions that create SOSIP.v4 trimers impede CD4-induced conformational changes.

The inventors determined that the E64K A316W and H66R A316W substitutions did not alter the overall structure and conformational dynamics of the trimers in a manner that is detectable by HDX-MS.

The stabilizing substitutions blocked the ability of CD4 to induce ordering of residues 370-382 (CD4bs), 245-256 and 476-483 (layer 3), 206-226 (layer 2), 53-92 (layer 1) and 566-592 (HR2; al) (Fig. 1.2B&C, Fig. 1.S3). Furthermore, the substitutions reduced the CD4-induced disorder of residues 165-181 (V2), 286-320 (V3), 520-537 (a6) and 593-628 (gp41 disulfide loop). Thus, the E64K+A316W and

H66R+A316 W substitutions prevent the CD4-induced opening of the trimer apex, as well as the rigidification cascade that spreads from the CD4bs via layer 3, layer 2 and layer 1 to gp41 HR1 (Fig. 1.2B&C).

The inventors used a V3 peptide competition ELISA to assess the relative contribution of V3 Abs to the overall Ab response induced by the various trimers (Sanders et al., 2015). They estimated that the median anti-V3 response in the BG505 SOSIP.664 immunized rabbits was ~30%> of the total anti-Env response but only ~10%> in the SOSIP.v4 recipients (Fig. 1.4E). For the rabbits given AMC008-based trimers, the corresponding reduction in the contribution of the anti-V3 response was only minimal, however, i.e., from -50% to -40% (Fig. 1.4E).

The inventors observed strong autologous neutralization (ID50>500) in 9 of 10 BG505 SOSIP.v4- immunized rabbits, (Fig. 4G,H; Table 1.S5). The majority of the AMC008 trimer recipients induced an autologous NAb response. The inventors tested the rabbit sera against a large panel of heterologous viruses and found that cross-neutralization of Tier 2 viruses was weak and sporadic, which is consistent with our findings using BG505 SOSIP.664 trimers. This heterologous response was, however, more frequently observed in SOSIP.v4-immunized animals than in SOSIP.664-immunized animals (Table 1.S4).

Autologous BG505.T332N NAb titers and BG505 SOSIP.664-D7324 binding Ab titers were positively correlated (r=0.61, P=0.016), consistent with our previous report (Sanders et al., 2015). The correlation was slightly stronger when the NAb titer comparison was with SOSIP.v4-D7324 binding Ab titers (r=0.66 p=0.0078), reinforcing the argument that the stabilized SOSIP.v4 trimers present neutralization- relevant epitopes more efficiently compared to the SOSIP.664 immunogens (Fig. 1.4LJ). For the AMC008 trimer immunogenicity experiment, there was no correlation between autologous NAb titers and binding Ab titers against the AMC008 SOSIP.664-D7324 trimers (r=0.17 p=0.54). In contrast, there was a strong correlation when the comparison involved binding Ab titers against the SOSIP.v4 trimers (r=0.68 p=0.0052; Fig. 1.4K,L). This outcome is again consistent with the argument that SOSIP.v4 trimers present neutralization-relevant epitopes more efficiently compared to SOSIP.664 trimers.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543N, 316W and 64K. An advantage of this the isolated, recombinant or synthetic Env protein is that a very high percentage of its trimers have of closed conformation. Trimers of these Env protein have no detectable binding to 19b IgG.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q, 316W and 64K. An advantage of this the isolated, recombinant or synthetic Env protein is that a very high percentage of its trimers have of closed conformation. Trimers of these Env protein have no detectable binding to 19b IgG. In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543N, 316W and 66R. An advantage of this the isolated, recombinant or synthetic Env protein is that a very high percentage of its trimers have of closed conformation. Trimers of these Env protein have no detectable binding to 19b IgG.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q, 316W and 66R. An advantage of this the isolated, recombinant or synthetic Env protein is that a very high percentage of its trimers have of closed conformation. Trimers of these Env protein have no detectable binding to 19b IgG.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q and 567K. These residues promote trimer formation. In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 64K or 66R, 316W, 73C and 561C. These residues reduce binding to non-NAbs b6 and 14e and increase binding affinity with PGT145 and PGT151.

In another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 64K and 316W or 66R and 316W, and more preferably combined with 73C and 561 C. These residues stabilize the proteins in their closed conformation, further occluding non-NAb epitopes and increasing their thermostability. In addition, the proteins show a high percentage of underprocessed oligomannose glycans, which is typical of virus-derived Envs and soluble native-like Env trimers.

In another preferred embodiment, the the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P combined with the amino acid residues 125W and 316W; 535M and 543Q; 535M and 543N; 64K, 316W and 543N; 535M, 543N, 316W and 64K; 535M, 543N, 316W and 66R; 535M, 543N, 66R and 316W; 64K and 535M; 64K, 316W and 535M; 66R, 316W and 535M; 535M and 543Q; 535M and 543N; 316W, 535M and 543N; 64K, 316W, 535M and 543N, 66R, 316W, 535M and 543N; 559P, 316W, 64K, 73C, 561C, or 49C and 555C, wherein said residue is numbered by reference to SEQ ID NO:l .

In another preferred embodiment, the trimer of the invention is a SOSIP.v4 trimer as defined above. An advantage of these trimers is that they are fully cleaved and highly homogeneous, as assessed by reducing and non-reducing SDS-PAGE and BN-PAGE analysis (Fig. 1.S2A-B). The proportion of closed trimers in each case. These trimers were more thermostable by 2-4°C, compared to wild-type (Table 1.1, Fig. 1.S2D). Their glycosylation profiles differed little if at all from their wild- type counterparts, with oligomannose glycans again predominating (Table 1.1 , Fig. 1.S2F). In addition, these trimers are all native-like, more thermostable and adopt the fully closed conformation more frequently.

Env trimers and VLPs In an embodiment, the invention encompasses envelope trimers for the production of virus like particles (VPLs) and pseudoparticles for use as VLP -based immunogens, to generate neutralizing antibodies, for example, and VLP -based vaccines against which a subject can mount a potent immune response against HIV. In accordance with the invention, Env trimers comprising the stabilizing disulfide bonds of the invention, as well as Env trimers comprising other stabilizing mutations in gpl20 and gp41 or equivalents thereof from HIV-2 as described herein, are used to generate VPLs and pseudovirions having reduced monomer, dimer and tetramer forms and enhanced trimer forms of gpl20/gp41 Env. The N- terminal stabilizing mutations in the context of HIV- 1 virus as described herein can yield trimer forms of Env (gpl20/gp41) on VLP and pseudovirions, to the virtual exclusion of monomer, dimer and tetramer forms, thus allowing for an immunogen that more closely resembles native HIV envelope trimers. Pharmaceutical compositions

This invention provides a composition comprising the modified polypeptide of the invention and a pharmaceutically acceptable carrier, excipient, or diluent. This invention also provides a composition comprising the trimeric complex of the invention and a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the composition further comprises an adjuvant. In one embodiment, the composition further comprises an antiretroviral agent.

Compositions and immunogenic preparations, including vaccine compositions, comprising the polypeptides of the present invention capable of inducing an immunological reaction (including protective immunity) in a suitably treated animal or human, and a suitable carrier therefore, are provided. Immunogenic compositions are those which result in specific antibody production or in cellular immunity when injected into a human or an animal. Such immunogenic compositions or vaccines are useful, for example, in immunizing an animal, including a human, against infection and/or damage caused by HIV.

The vaccine preparations comprise an immunogenic amount of one or more of the isolated, recombinant or synthetic polypeptides of the invention. By "immunogenic amount" is meant an amount capable of eliciting the production of antibodies directed against the retrovirus in a mammal into which the vaccine has been administered. The route of administration and the immunogenic composition may be designed to optimize the immune response on mucosal surfaces, for example, using nasal administration (via an aerosol) of the immunogenic composition. In some embodiments, the methods and compositions of the invention also include use of another antiviral agent in addition to the one or more of the present Env polypeptides, or a combination of Env polypeptides as described herein. Thus, other antiretroviral agents or compounds, which can be administered in addition to the polypeptides and compositions of the invention include, without limitation, protease inhibitors, retroviral polymerase inhibitors, azidothymidine (AZT) , didanoside (DDI), soluble CD4, a polysaccharide sulfates, T22, bicyclam, suramin, antisense ohogonulceotides, ribozymes, rev inhibitors, protease inhibitors, glycolation inhibitors, interferon and the like. Examples include acyclovir, 3-aminopyridine-2- carboxyaldehyde thiosemicarbazone (3-AP,

Triapine(TM)) and 3-amino-4- methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) , thiamine disulfide, thiamine disulfide nitrate, thiamine disulfide phosphate, bisbentiamine, bisbutytiamine, bisibutiamine, alitiamine, fursultiamine and octotiamine.

In a preferred embodiment, the pharmaceutical composition comprises a cocktail of different Env polypeptides. Preferably, said pharmaceutical composition comprises at least 2 different Env

polypeptides. Preferably said pharmaceutical composition comprises at least 2, 3 or 4 different Env polypeptides according to the invention. An advantage thereof is that these compositions induce better heterologous responses against several Tier-2 viruses. Preferably, said pharmaceutical composition comprises at least 2, 3 or 4 different Env polypeptides of the invention. Nanoparticles

The present invention provides novel Env-ferritin nanoparticle (np) vaccines. Such nanoparticles comprise fusion proteins, each of which comprises a monomeric subunit of ferritin joined to an immunogenic portion of an Env protein. Because such nanoparticles display Env protein on their surface, they can be used to vaccinate an individual against HIV. In some embodiments of the present invention, the various protein domains (e.g., gpl40 protein, trimerization domain, etc.) may be joined directly to one another. In other embodiments, it may be necessary to employ linkers (also referred to as a spacer sequences) so that the various domains are in the proper special orientation. The linker sequence is designed to position the Env protein in such a way to that it maintains the ability to elicit an immune response to the HIV virus. Linker sequences of the present invention comprise amino acids. Preferable amino acids to use are those having small side chains and/or those which are not charged. Such amino acids are less likely to interfere with proper folding and activity of the fusion protein. Accordingly, preferred amino acids to use in linker sequences, either alone or in combination are serine, glycine and alanine. Examples of such linker sequences include, but are not limited to, SGG, GSG, GG and NGTGGSG. Amino acids can be added or subtracted as needed. Those skilled in the art are capable of determining appropriate linker sequences for proteins of the present invention.

Methods of treatment

In rabbits, the stabilized trimers induced lower levels of V3 -directed Tier lA NAbs but similar autologous Tier IB or Tier 2 NAb titers, compared to wild-type trimers. Stabilized, closed trimers might be useful components of vaccines aimed at inducing bNAbs. This invention also provides a method for preventing a subject from becoming infected with HIV-1, comprising administering to the subject an amount of the composition of the invention effective to prevent the subject from becoming infected with HIV. This invention further provides a method for reducing the likelihood of a subject becoming infected with HIV, comprising administering to the subject an amount of the composition of the invention effective to reduce the likelihood of the subject becoming infected with HIV.

This invention also provides a method for delaying the onset of, or slowing the rate of progression of, an HIV-related disease in an HIV-infected subject, which comprises administering to the subject an amount of the composition of the invention effective to delay the onset of, or slow the rate of progression of, the HIV- related disease in the subject. This invention also provides a method for eliciting an immune response against HIV or an HIV infected cell in a subject comprising administering to the subject an amount of the composition of the invention effective to elicit the immune response in the subject. This invention provides a method for eliciting an immune response against HIV or an HIV infected cell in a subject comprising administering to the subject an amount of the trimeric complex of the invention effective to elicit the immune response in the subject.

EXAMPLES

Example 1

AMC008 SOSIP.664 tnmers as a basis for studying stabilization strategies

For initial studies of how to improve the frequency of closed SOSIP.664 tnmers at the expense of partially open or non-native forms we selected the AMC008 clade B env gene from the Amsterdam

Cohort Studies. When the AMC008 SOSIP.664 Env proteins were expressed, -50% migrated as trimers on a BN-PAGE gel (Fig. 1.S 1A) but, unlike their BG505, B41 and ZM197M counterparts, only -35% of the 2G12/SEC-purified AMC008 trimers were visibly native-like in NS-EM images (Fig. 1.S2C). Thus, -25%) were closed trimers and -10% partly open trimers, but the remaining ~65%> adopted heterogeneous, splayed out shapes that resemble the typical appearance of uncleaved, non-native gpl40 proteins

(Pritchard et al., 2015; Ringe et al., 2013). The midpoint of thermal denaturation (T_m), determined by Differential Scanning Calorimetry (DSC), for AMC008 SOSIP.664 trimers was 59.6°C, much lower than their BG505 counterparts (68.1°C; Table 1.1). Finally, the antigenicity profile of the AMC008 SOSIP.664 trimers showed that they bound quaternary structure- dependent bNAbs very poorly, but multiple non- NAbs efficiently (Figs. 1.S 1H). As such defects are fairly typical of what we have seen with many Env sequences, the AMC008 SOSIP.664 trimers seemed suitable for testing new, structure-guided

stabilization strategies. To assess their general applicability, we also tested the same strategies on the BG505, B41 and ZM197M trimers.

Positive selection of native-like trimers by PGT145 purification We first investigated whether we could isolate native-like AMC008 SOSIP.664 trimers by affinity chromatography using the quaternary-dependent bNAb PGT145, which does not recognize non-native Env proteins (Pugach et al., 2015). Even without a subsequent SEC step, PGT145-purification yielded homogeneous, fully cleaved trimers (Fig. 1.S2A & Fig. 1.2B) that were—100% native-like when visualized by NS-EM (Fig. 1.S2C). In their glycan profile, oligomannose glycoforms were predominant (~67%>), with a high Man9GlcNAc2 content (~34%>), properties that are hallmarks of native and native-like trimers (Table 1.1 ; Fig. l .S2F)(Bonomelli et al., 201 1 ; Pritchard et al., 2015). However, NS-EM imaging also showed that most (~85%>) of the PGT145-purified trimers were in a partly open conformation (Table 1.1 , Fig. 1.S2C), and their thermal stability was not substantially improved (T_m = 60.2°C; Table 1.1). And, of course, eliminating the non-native proteins (~65%> of the total) via the PGT145 column reduced the overall Env yield by -3 -fold.

When BG505 SOSIP.664 Env proteins were similarly purified via PGT145 the trimers were still fully native-like, but the proportion in the closed conformation was reduced from >95%> (2G12/SEC) to ~35%>. Thus, binding to and/or elution from PGT145 causes the BG505 trimers to partially open up, which may be attributable to the known, albeit quite subtle allosteric effects of PGT145 on the trimer structure (Derking et al., 2015). The stability of PGT145-purified BG505 SOSIP.664 trimers (T_m = 66.7°C) was also slightly reduced compared to 2G12/SEC (T_m = 68.1°C; Table 1.1), although their glycan content (Table l .l)(Pritchard et al., 2015) and antigenicity profiles were unaltered, compared to when 2G12/SEC purification was used (Derking et al., 2015). When other trimers were purified via PGT145 columns, B41 SOSIP.664 trimers were similar to their 2G12/SEC-purified counterparts in that they were partly open (45-60%) in both cases (Pritchard et al., 2015; Pugach et al., 2015). However, for ZM197M SOIP.664, the closed trimer content was reduced from 80% (2G12/SEC) to -15% (Table 1.1, Fig. 1.S2A-D) (Julien et al., 2015).

Thus, PGT145-purification has paradoxical effects: it can positively select native-like trimers from a mixture of native and non-native Env forms, but it can also open up previously closed, native-like trimers (at least for BG505 and ZM197M). Below, we exploit these contrasting properties by simultaneously using PGT145-affmity chromatography as both a purification strategy and a "stress-test"; i.e., to monitor whether stabilized trimers now resist PGT145-induced opening.

Mutation of gp41 residues 535 and 543 to make SOSIP.v3 trimers

A previous comparison of JR-FL, a clade B Env that forms SOSIP trimers inefficiently, with K H1144, a clade A Env that does so more efficiently, showed that having methionine at position 535 (M535) and glutamine at 543 (Q543) in HR1 of gp41 benefits trimer formation (Fig. L IE, Table l .S2)(Dey et al., 2008) . Similar findings were made when HIV-1 was cultured under harsh conditions (Leaman and Zwick, 2013). Residue 535 is positioned in the middle of the a6 helix, which might explain why a methionine here has a stabilizing effect; thus, Met has a higher helical propensity than the He present in AMC008 Env (Fig. L IE, Table 1.S2) (Levitt and Greer, 1977). Position 543 is located at the end of the same helix; a Gin residue here has a lower helical propensity than the unfavorable Leu residue and might prevent the extension of the a6 helix and thereby the initiation of the fusion process (Levitt and Greer, 1977).

We noted that the BG505 sequence contains M535 and N543. Based on the high quality BG505 trimers we consider N543 to have the same stabilizing effect on the gp41 prefusion structure as Q543. In contrast, the AMC008 sequence contains neither M535 nor N/Q543, but instead 1535 and L543 (Fig. LIE, Table 1.S2). We therefore constructed AMC008 SOSIP.664 trimer mutants containing one or more of the I535M, L543N and L543Q substitutions (Fig. 1.S1A &1H). All three individual changes improved trimer formation and a similar effect was observed for L543N and L543Q (Fig. 1.S1A). When the AMC008 SOSIP.664 I535M+L543N double mutant was PGT145-purified, NS-EM analysis showed an unchanged percentage of closed . open trimers (~15%> closed; Table 1.1), but thermostability was slightly increased from 60.2°C (wild-type) to 61.6°C (Table 1.1). The I535M+L543N substitutions also modestly improved binding of the quaternary structure-dependent bNAbs PGT145, 35022 and PGT151, but otherwise did not affect trimer antigenicity (Table 1.2, Fig. 1.S2H). The B41 and ZM197M sequences also both lack one of the beneficial amino acids at position 535 or 543, so we introduced the L543N substitution into B41 and V535M into ZM197M. Both changes improved trimer formation (Fig. 1.S1J &1K).

From here on, we refer to SOSIP.664 proteins containing the optimal amino acids at position 535 (i.e., Met) and 543 (i.e., Asn/Glu) as SOSIP version 3 (SOSIP.v3). Specifically, SOSIP.v3.1 contains the 543Q change, SOSIP.v3.2 contains 543N. For more details on nomenclature, see Table 1.S3.

Reducing V3 exposure by enhancing hydrophobic packing

Stabilizing the closed conformation of SOSIP.664 trimers requires the V3 region to be sequestered and a reduction in the exposure of non-NAb epitopes (for Tier-2 viruses), including CD4i epitopes. Although the V3 region is tucked beneath the VI V2 domain in the BG505 SOSIP.664 cryo-EM and x-ray structures (Julien et al., 2013b; Lyumkis et al., 2013; Pancera et al., 2014), it can become exposed on a subset of trimers in vitro and is immunogenic in vivo (Sanders et al., 2013, 2015). One way to readily gauge V3 exposure is MAb reactivity with SOSIP.664-D7324 trimers under ELISA conditions in which V3 epitopes become readily accessible (Sanders et al., 2013). We designed an A316 W substitution to strengthen hydrophobic interactions between V3 and the underlying gpl20 layer 2 (i.e., V120, L122 and P206), thereby decreasing the propensity for V3 to flip out of its ground-state location (Fig. Ι .ΙΑ,Β). For AMC008 and BG505 SOSIP.664-D7324 proteins, the A316W change did improve trimer formation, and it also increased the thermostability of the BG505 trimers as assessed by a novel assay that can be used with unpurified Env proteins (Fig. 1.S1B-G). Of note is that the V3 non-NAbs 447-52D, 39F, 14e and 19b each bound markedly less well to all the

A316W variant trimers than to wild-type (Fig. 1.S1H-K). For example, 14e and 19b binding to PGT145 purified BG505 SOSIP.664 A316W trimers was reduced by -80% and ~50%>, respectively, and for the CD4i non-NAbs 17b and 412d binding was -50% lower (Table 1.2, Fig. 1.S2F). As the A316W substitution did not affect binding of the same non-NAbs to V3 peptides, we conclude that it works indirectly on the trimer, by impeding the exposure of V3 epitopes (Fig. 1.S1L). In contrast, the A316W substitution had no adverse effect on the binding of multiple bNAbs to the AMC008, BG505 and B41 SOSIP.664 trimers (Fig. 1.S1H-K). A high-throughput mutagenesis screen of position 316 showed that other bulky hydrophobic amino acids (Tyr/Phe/Val/Ile) also decreased V3 exposure (>3-fold), whereas other substitutions had little, if any, effect (Fig. 1.S1M). When BG505 SOSIP.664 A316W trimers were purified on PGT145 columns and viewed by NS-EM, the proportion in the closed conformation increased from ~35%> to ~80%>. Their thermostability was also greater (T_m = 69.0°C vs. 66.7°C; Table 1.1). Introduced to the BG505 Env-pseudovirus, the A316W substitution reduced infectivity by ~98%>, implying either that conformational flexibility is important for co-receptor interactions during entry or that A316 plays a direct role in co-receptor binding (Fig. S1L). Reducing spontaneous sampling of the CD4-bound conformation We next evaluated two substitutions (E64K and H66R) in layer 1 of the g l20 inner domain that we had identified in a study of how HIV-1 escapes from the entry inhibitor VIR165 (Fig. 1A,C,D;). HIV-1 isolates containing either sequence change not just resist VIR165 but now require it for infectivity; the substitutions appear to impede native trimers from spontaneously sampling the CD4-bound conformation, but adding VIR165 enables them to now undergo CD4-induced conformational changes. In the unliganded BG505 SOSIP.664 trimer, the side chains of residues 64 and 66 are positioned to interact with HR1 of gp41 (Finzi et al., 2010; Do Kwon et al., 2015; Pancera et al., 2010), but the high crystallographic B-values around residues 64 and 66 imply there is local flexibility (Fig. 1.1C) (Pancera et al., 2014). Hydrogen-Deuterium Exchange (HD-X) studies confirmed that this region does not adopt a stable secondary structure, but becomes ordered after CD4-binding (Guttman et al., 2014). Indeed, in the structure of a CD4-liganded gpl20 core protein, the side chains of residues 64 and 66 have reoriented to interact with gpl20 layer 2 (Fig. 1.1. ID) (Pancera et al., 2010). Thus, these residues may be part of a switch that transduces CD4-induced conformational changes from the CD4bs and layers 3 and 2 of gp 120 to gp41, via layer 1. We hypothesize that the E64K and H66R substitutions impede this allosteric process. Exploratory studies on unpurified culture supernatants indicated that the E64K and H66R changes each substantially reduced, or even eliminated, the binding of the CD4i non-NAbs to AMC008, BG505 and B41 Env proteins (Fig. l .SlH-O). When mutant BG505 SOSIP.664 trimers were PGT145-purified and visualized by NS-EM, the percentage of closed trimers was substantially increased for the E64K mutant (-90% closed . 35% for wild-type), but not H66R (~30-35%> closed). Each substitution had only a minor effect on thermostability (T_m= 67.9°C for E64K, 67.5°C for H66R vs. 66.7°C for wild-type). In an ELISA, bNAb binding was unaltered or sometimes enhanced (e.g., for the trimer apex bNAbs PG16 and PGT145), whereas the 17b and 412d non-NAbs no longer bound (Table 1.2). Overall, the SOSIP.664 trimer data are consistent with the earlier report that the E64K and H66R substitutions impede viral Env from spontaneously sampling the CD4-bound conformation (Egginkl5). Combining A316W with E64K or H66R substitutions: SOSIP.v4 trimers

The E64K or H66R substitutions were combined with A316W, and unpurified supernatants from the resulting AMC008, BG505, B41 and ZM197M SOSIP.664 double mutant proteins were analyzed on top of the SOSIP.v3 proteins. In each case, the E64K+A316W and H66R+A316W double mutants had acquired the beneficial properties associated with each individual change. Each double mutant was expressed efficiently and formed fully cleaved trimers, and thermostability was slightly increased over wild-type (Fig. 1.S1B-G). For the double mutants of all four genotypes, the binding of non-NAbs to CD4i-epitopes (targets of the E64K or H66R substitutions) and V3 epitopes (targets of the A316W change) was diminished or abolished, while bNAb epitopes were unaffected (Fig. 1.S1H-K).

Biophysical properties of PGT145-purifled, multiply stabilized SOSIP.v4 trimers The various PGT145-purified SOSIP.v4 trimers were fully cleaved and highly homogeneous, as assessed by reducing and non-reducing SDS-PAGE and BN-PAGE analysis (Fig. 1.S2A-B). The proportion of closed trimers in each case, most notably for AMC008 SOSIP.v4.2 (-90% closed vs. -15%; Table 1.1 , Fig. 1.S2C). Except for ZM197M SOSIP.v4 (no change), the SOSIP.v4 trimers were more thermostable by 2-4°C, compared to wild-type (Table 1.1 , Fig. 1.S2D). Their glycosylation profiles differed little if at all from their wild-type counterparts, with oligomannose glycans again predominating (Table 1.1 , Fig. 1.S2F).

Dynamic Light Scattering (DLS) experiments showed that BG505 wild-type and both SOSIP.v4 variant trimers were all monodisperse and had the same hydrodynamic radius (R ) of -69 A (Fig. 1.S2E).

Furthermore, in Small Angle X-ray Scattering (SAXS) studies, these trimers were all well folded, with a radius of gyration (R_g) of -53 A (Fig. 1.S2E). On Kratky plots, the Gaussian-like peak in the low angle (low q) region, that dips towards zero, is typical seen with well-folded globular proteins (Fig. 1.S2E).

Overall, the multiply stabilized, PGT145-purified SOSIP.v4 trimers derived from 4 different env genes from 3 clades are all native-like. Moreover, compared to unmodified, earlier generation SOSIP.664 trimers, the SOSIP.v4 variants are more thermostable and adopt the fully closed conformation more frequently.

Antigenicity of stabilized, PGT145-purifled SOSIP.v4 trimers

D7324-tagged versions of wild type (i.e., SOSIP.664) and stabilized trimer variants were PGT145 affinity-purified and studied by ELISA. The AMC008 SOSIP.v4-D7324 trimers retained the ability to bind the quaternary-dependent and various other bNAbs (2G12, PGT135, PGT121, PGT126, PG9, PG16, PGT145, PGT151 , 35022, VRCOl and CH103). However, in contrast to SOSIP.v3, their reactivity with non-NAbs was generally reduced compared to wild-type trimers, in some cases by >90%>. Furthermore, although the AMC008 SOSIP.v4-D7324 trimers still bound CD4-IgG2, they did not undergo CD4- induced conformational changes efficiently, as judged by the lack of induction of the 17b and 412d CD4i epitopes (Table 1.2, Fig. 1.S2G). In Isothermal Titration Calorimetry (ITC) experiments using untagged AMC008 trimers, reactivity of Fab fragments of the V3 non-NAb 19b was decreased compared to wild type; the stoichiometry of the 19b-trimer complex was 0.3 for wild type (i.e., 1 Fab per 3 trimers), but 0 (i.e., no complexes were detected) for SOSIP.v4.1 and negligible (0.03; i.e., -1 Fab per 30 trimers) for SOSIP.V4.2 (Fig. 1.S2K). Many bNAbs bound equally well to BG505 SOSIP.664-D7324 and SOSIP.v4-D7324 trimers in ELISA, indicating their epitopes are unaffected by the stabilizing substitutions. This outcome was confirmed by SPR for the quaternary structure-dependent bNAbs PG16, PGT145, 35022 and PGT151 (Fig. 1.1H). Furthermore, in the ELISA there was a marked reduction in the binding of V3 non-NAbs 14e and 19b (by -90% and 50%, respectively, compared to SOSIP.664) and CD4bs non-NAb b6 (by -75%), while the CD4i non-NAbs 17b and 412d bound to a negligible or undetectable extent (Table 1.2, Fig. 1.S2H). In previous ITC studies, the 19b Fab bound minimally to the BG505 SOSIP.664 trimers; the stoichiometry was 0.2 (i.e., on average one Fab per 5 trimers), (Pugach et al., 2015; Sanders et al., 2013). In contrast, 19b binding to the BG505 SOSIP.v4.1 mutant was abolished (Fig. 1.S2L), confirming that the stabilizing substitutions reduced V3 exposure. Similarly, non-NAb binding to V3 and CD4i epitopes on the B41 SOSIP.v4 trimers was strongly reduced (Table 1.2, Fig. 1.S2I). Thus, in the ITC assay, 19b binding was reduced ~10-fold (stoichiometry 0.02 . 0.2 for SOSIP.664; Fig. 1.S2M). Hence, as for the other SOSIP.v4 trimers, the B41 V3 region is now effectively sequestered. Comparable results were also obtained for ZM197M SOSIP.v4 trimers. The bNAb epitopes were generally unaffected, and the quaternary structure dependent bNAbs PGT145 and PGT151 actually more strongly in ELISA than to the wild type ZM197M SOSIP.664-D7324 trimers (Table 1.2, Fig. 1.S2J). Once more, the binding of non-NAbs to V3 and CD4i epitopes was greatly diminished (Table 1.2, Fig. 1.S2J).

NS-EM analyses of bNAb complexes with stabilized trimers

To confirm their overall native-like structure and bNAb epitope presentation, we prepared complexes of the AMC008 SOSIP.v4 trimers with bNAbs PGV04 and 35022 (added as Fabs), and visualized them by NS-EM. The stabilized trimers were all compact entities that were virtually indistinguishable from previously published low-resolution reconstructions of BG505 and B41 SOSIP.664 trimers (Fig. 1.3).

CD4 binding and CD4-induced conformational changes

In ELISA, CD4-IgG2 binding to AMC008, B41 and ZM197 SOSIP.v4-D7324 trimers was slightly reduced compared to SOSIP.664-D7324, but more markedly so for the BG505 trimers. Thus, if CD4- IgG2 binding to BG505 SOSIP.664-D7324 trimers is defined as 100% (see Methods), binding to the SOSIP.v4.1 and SOSIP.v4.2 variants was reduced to 27% and 32%, respectively (Table 1.2, Fig. 1.S3). In contrast, there was no change in the binding of bNAbs CH103 and VRCOl to CD4bs epitopes. An SPR analysis showed that CD4-IgG2 association rates were comparable for the SOSIP.v4 and wild type trimers, but the ligand dissociated markedly faster from the stabilized trimers (Fig. 1.1H). This finding is consistent with a report that the introduction of the H66N substitution into a gpl20 core protein leads to a faster rate of sCD4 dissociation but no change in the association kinetics (Kassa et al., 2009a). Biolayer interferometry data confirmed that the affinity of sCD4 for various AviB-tagged BG505 stabilized trimers was 2- to 4-fold lower in each case, with the greatest reduction (4-fold) seen with the A316 W single mutant and the SOSIP.v4.2 double mutant (Fig. 1.1 G).

Conformational flexibility assessed by Hydrogen-Deuterium Exchange

We used Hydrogen-Deuterium Exchange (HD-X) coupled with mass spectrometry (MS) to study PGT145-purified BG505 SOSIP.v4.1 and SOSIP.v4.2 trimers. The goal was to obtain more precise information on whether the stabilizing substitutions affected protein dynamics in the absence and presence of sCD4, and CD4-induced conformational changes (Fig. 1.2, Fig. 1.S3). The profiles for both the BG505 SOSIP.v4 trimers were very similar to ones obtained previously for 2G12/SEC-purified BG505 SOSIP.664 trimers (Fig. 1.S3) (Guttman et al., 2014). Hence, the E64K A316W and H66R A316W substitutions did not alter the overall structure and conformational dynamics of the trimers in a manner that is detectable by HDX-MS.

When sCD4 was present, the deuterium- exchange patterns now differed markedly for SOSIP.v4 trimers compared to wild type, SOSIP.664 trimers (Fig. 1.S3D). As judged by BN-PAGE gel-shift assays, BG505 SOSIP.664 trimers as well as SOSIP.v4 trimers formed complexes with sCD4, when added in molar excess, despite the slightly lower affinity (Fig. 1.S3C). CD4-binding to wild-type trimers resulted in greater protection of the CD4bs, but also of layers 1-3 in the gpl20 inner domain and HR1 of gp41 (Fig. 1.2A). Thus, CD4-binding rigidifies and/or buries these domains, as reported previously (Guttman et al., 2014). Conversely, CD4-binding led to decreased protection in the V2 and V3 loops as well as in several gp41 regions, which is consistent with a CD4-induced opening of the trimer apex that eventually increases the accessibility of the gp41 fusion machinery (Fig. 1.2A). Regions of the trimer that are less (red) or more (blue) protected upon CD4 binding are mapped onto the BG505 SOSIP.664 crystal structure (Fig. 1.2A-C).

In contrast, the above CD4-induced changes were greatly attenuated or entirely abrogated when the SOSIP.v4 trimers were studied in the same way. Thus, the stabilizing substitutions blocked the ability of CD4 to induce ordering of residues 370-382 (CD4bs), 245-256 and 476-483 (layer 3), 206-226 (layer 2), 53-92 (layer 1) and 566-592 (HR2; al) (Fig. 1.2B&C, Fig. 1.S3). Furthermore, the substitutions reduced the CD4-induced disorder of residues 165-181 (V2), 286-320 (V3), 520-537 (a6) and 593-628 (gp41 disulfide loop). Thus, the E64K+A316W and H66R+A316W substitutions prevent the CD4-induced opening of the trimer apex, as well as the rigidification cascade that spreads from the CD4bs via layer 3, layer 2 and layer 1 to gp41 HR1 (Fig. 1.2B&C). We have described this sequence of events as the allosteric "priming" network (Guttman et al., 2014).

Reduced V3 antibody and Tier 1A neutralizing antibody responses to SOSIP.v4 trimers in rabbits

Our principal goal when stabilizing the closed state of SOSIP trimers is to reduce the exposure, and by inference the immunogenicity, of non-NAb epitopes such as V3. The rationale is that reducing unwanted Ab responses may help focus the immune responses on Tier-2 NAb epitopes. Accordingly, we immunized rabbits with PGT145-purified AMC008 and BG505 SOSIP.664 trimers and their SOSIP.v4.1 and v4.2 counterparts, and quantified various Ab responses by ELISA two weeks after the third immunization (week 22; Fig. 1.4A). High titers of binding Abs against SOSIP.664-D7324 and SOSIP.v4- D7324 trimers were induced in all the rabbits. However, the binding Ab titers in the SOSIP.664 trimer- immunized rabbits were higher when determined against the SOSIP.664-D7324 trimers than against the SOSIP.v4-D7324 variants (the titer ratios were -2 for BG505 and -3 for AMC008). In contrast, for the SOSIP.v4-immunized rabbits, the binding Ab titers measured against the SOSIP.664-D7324 and

SOSIP.v4-D7324 trimers were similar for BG505 (ratio ~1) and higher to SOSIP.664-D7324 for

AMC008 (ratio of -2). Hence, some of the Abs raised in SOSIP.664 trimer-immunized rabbits do not bind the SOSIP.v4-D7324 trimers under ELISA conditions (Fig. 1.4 B-D).

We used a V3 peptide competition ELISA to assess the relative contribution of V3 Abs to the overall Ab response induced by the various trimers (Sanders et al., 2015). We estimated that the median anti-V3 response in the BG505 SOSIP.664 immunized rabbits was -30% of the total anti-Env response but only -10% in the SOSIP.v4 recipients (Fig. 1.4E). For the rabbits given AMC008-based trimers, the corresponding reduction in the contribution of the anti-V3 response was only minimal, however, i.e., from -50% to -40% (Fig. 1.4E).

Since Tier 1 A NAb responses to BG505 SOSIP.664 trimers are dominated by V3- specificities (Sanders et al., 2015), we hoped that SOSIP.v4 trimers would induce lower, or less V3-dependent, titers of such NAbs. This was indeed the case in that titers of NAbs against the Tier 1A virus SF162 were significantly lower in SOSIP.v4 immunized rabbits compared to SOSIP.664 (14- and 8-fold for BG505 and AMC008, respectively) (Fig. 1.4F; Table 1.S5). In contrast, there was no such reduction in the autologous NAb titers. Overall, strong autologous neutralization (ID50>500) was observed in 9 of 10 BG505 SOSIP.v4- immunized rabbits, with a weaker response in the 10^th (Fig. 1.4G,H; Table 1.S5), while a strong autologous response was induced in 3 of 5 BG505 SOSIP.664 recipients. The majority of the AMC008 trimer recipients induced an autologous NAb response, although these responses were less potent than the corresponding autologous responses to the BG505 trimers, despite the greater sensitivity of the AMC008 virus (i.e., Tier-IB vs. Tier- 2) (Fig. 1.4G,H; Table 1.S4). We also tested the rabbit sera against a large panel of heterologous viruses and found that cross-neutralization of Tier 2 viruses was weak and sporadic, which is consistent with our findings using BG505 SOSIP.664 trimers. This heterologous response was, however, more frequently observed in SOSIP.v4-immunized animals than in SOSIP.664-immunized animals (Table 1.S4).

Autologous BG505.T332N NAb titers and BG505 SOSIP.664-D7324 binding Ab titers were positively correlated (r=0.61, P=0.016). The correlation was slightly stronger when the NAb titer comparison was with SOSIP.v4-D7324 binding Ab titers (r=0.66 p=0.0078), reinforcing the argument that the stabilized SOSIP.v4 trimers present neutralization-relevant epitopes more efficiently compared to the SOSIP.664 immunogens (Fig. 1.4LJ). For the AMC008 trimer immunogenicity experiment, there was no correlation between autologous NAb titers and binding Ab titers against the AMC008 SOSIP.664-D7324 trimers (r=0.17 p=0.54). In contrast, there was a strong correlation when the comparison involved binding Ab titers against the SOSIP.v4 trimers (r=0.68 p=0.0052; Fig. 1.4K,L). This outcome is again consistent with the argument that

SOSIP.v4 trimers present neutralization-relevant epitopes more efficiently compared to SOSIP.664 trimers.

Methods and Experimental Procedures Trimer design, expression and purification

The AMC008 env gene is derived from a subtype B virus, isolated 8 months post-seroconversion from a participant in the Amsterdam Cohort studies on HIV/AIDS who later developed a bNAb response (patient H18818 in reference (Euler et al., 2010)). The design of AMC008 SOSIP.664 trimers is identical to the BG505, B41, and ZM197M SOSIP.664 constructs described elsewhere (Julien et al., 2015; Pugach et al., 2015; Sanders et al., 2013). C-terminally tagged SOSIP.664-D7324 and SOSIP.664-aviB trimers were made as described (Sanders et al., 2013; Sok et al., 2014). All experiments used D7324-tagged trimers, except for DLS, SAXS, HD-X and ITC studies (untagged trimers), SPR (His-tagged trimers) and Octet assays (aviB-tagged trimers).

Env proteins were expressed by PEI transient transfection in adherent HEK293T cells or suspension cultures of HEK293F or HEK293S (Gn ^/_), as described previously (Julien et al., 2013c; Sanders et al., 2013). Env proteins were purified from transfection supernatants by affinity chromatography using a PGT145- or 2G12-column (Julien et al., 2013c; Pugach et al., 2015; Sanders et al., 2013). 2G12-purified Env proteins were further fractionated by SEC, whereas the PGT145 column yielded pure trimers without the need for SEC. SDS-PAGE and BN-PAGE assays were used to assess trimer cleavage and purity (Sanders et al., 2002; Schiilke et al., 2002).

Antigenicity assays

Antigenicity was determined using SOSIP.664-D7324 trimers for SPR or D7324-capture ELISA, as described elsewhere (Derking et al., 2015; Sanders et al., 2013; Yasmeen et al., 2014). In both assays, the D7324 antibody was attached to the solid phase and used to capture the epitope-tagged trimers, which were then recognized by various solution-phase MAbs. ITC was used to generate data on MAb-trimer interactions in solution.

Thermostability assays

As a screening assay, we used a PCR machine-based system to expose unpurified SOSIP.664-D7324 trimers to a range of temperatures, before using D7324-capture ELISA to assess when the 2G12 epitope had been lost. A conventional DSC assay was used to quantify the melting of purified trimers (ref). The two methods yielded data on multiple trimers that correlated well.

Biophysical techniques Multiple biophysical assays were used to analyze the properties of various SOSIP.664 trimers, including DLS, SAXS and HDX-MS as described elsewhere ((Guttman et al., 2014)). Images of purified

SOSIP.664 trimers, either alone or as Fab complexes, were generated by NS-EM as previously described. Rabbit immunization and analysis

Rabbits (5 per group) were immunized with 22 μg of PGT145-purified trimers at week 0, 4 and 20, and NAb responses were assessed at week 22. The rabbit sera were assayed for autologous and cross-reactive NAbs using Env-pseudoviruses in the TZM-bl cell assay, and for trimer-binding antibodies by D7324- capture ELISA (Sanders et al., 2013).

References

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Table 1.1 Biophysical properties of stabilized AMC008, BG505, B41 and ZM197M SOSIP.664 trimers.

The biophysical properties of PGT145-purified SOSIP.664-D7324 trimers were assessed using NS-EM to determine native-like trimer formation, DSC to quantify thermostability (T_m), and glycan profiling to measure glycan content. For comparison, values for 2G12-SEC purified trimers are included from previous reports. We note that these reported T_m values were obtained with untagged proteins for BG505 and D7324 tag for B41 (Julien et al., 2015; Pugach et al., 2015; Sanders et al., 2013) . The unprocessed EM, DSC and glycan profiling data are shown in Fig. 1.S2. The DSC data were fitted using both two state and non-two state models (Fig. 1.S2D). The T_m values based on two-state model fitting are listed here, while values based on the alternative model fitting are in Table LSI . Binding of bNAbs and non-NAbs to PGT145-purified SOSIP.v4 and SOSIP.664 trimers was assessed by D7324-capture ELISA (Fig. 1.S2). Half maximal binding

concentrations (EC50, in μg/ml) or area under the curve (AUC) values are shown. AUC values were used for some bNAbs and non-NAb 17b because plateau values differed substantially (> 20%) between trimer variants, or because binding was too weak for a plateau to be reached (i.e., for 17b). Values determined with AUC values are in italic. Ab binding to each SOSIP.v4 trimer is expressed as a percentage of binding to the corresponding SOSIP.664 trimer (= 100%). Experimental Procedures

Construct design

The BG505, B41, and ZM197M SOSIP.664 constructs have been described elsewhere (Julien et al., 2015; Pugach et al., 2015; Sanders et al., 2013). The AMC008 env gene is derived from a subtype B virus, isolated 8 months post-seroconversion from a participant in the Amsterdam Cohort studies on HIV/AIDS that later developed a bNAb response (patient H18818 in reference (Euler et al., 2010)). The AMC008 SOSIP.664 gpl40 construct was designed as previously described (Julien et al., 2015; Pugach et al., 2015; Sanders et al., 2013), by introducing the following sequence changes: A501C and T605C (gpl20-gp4lECTO disulfide bond; (Binley et al., 2000)); I559P in gp41_ECTO (trimer-stabilizing; (Sanders et al., 2002)); REKR to R R R (SEQ ID NO: 186) in gpl20 (cleavage enhancement; (Binley et al., 2002)); a stop codon at gp4lECTO residue 664 (improvement of homogeneity and solubility; (Klasse et al., 2013)). SOSIP.664-D7324 trimers contain a D7324 epitope-tag sequence at the C-terminus of gp4lECTo; they were made by adding the amino-acid sequence GSAPTKAKRRWQREKR (SEQ ID NO: 187) after residue 664 in gp41_ECTO and preceding the stop codon (Sanders et al., 2013). Similarly, biotinylated SOSIP.664-aviB trimers were generated by adding the avindin-B (aviB) sequence GLNDIFEAQKIEWHE (SEQ ID NO: 188) after residue 664, followed by biotininylation as described (Sok et al., 2014). All experiments were performed with D7324-tagged trimers, except for DLS, SAXS, HD-X and ITC studies (untagged trimers) and Octet assays (aviB-tagged trimers). Point mutants were made by Quikchange site directed mutagenesis (Agilent, Stratagene), and verified by sequencing. Env protein expression Proteins encoded by the various env genes described above were expressed in adherent 293T cells, or the 293F or 293 S (GnT^/_) variants that are adapted for suspension cultures, essentially as described (Julien et al., 2013; Sanders et al., 2013). All experiments using purified trimers were performed with 293F cell-expressed proteins, except for ITC assays when the producer cell was 293 S. The 293T or 293 S cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin

(100 U/ml) and streptomycin (100 μg/ml). For trimer expression on a small-scale, 293T cells were seeded at a density of 5.5>< 10⁴/ml in a 6-well plate. The next day, when the cells had reached a density of 1.0>< 10⁶/ml, they were transfected using polyethyleneimine (PEI) as described elsewhere (Kirschner et al., 2006). Briefly, PEI- MAX (1.0 mg/ml) in water was mixed with expression plasmids for Env and Furin (Binley et al., 2000) in OPTI-MEM (Gibco). For one well, 3.75 μg of Env plasmid, 1.25 μg of Furin plasmid and 12.5 μΐ PEI-MAX (1 mg/ml) were added in 3 ml of growth media (DMEM +10% FCS + penicillin and streptomycin). Culture supernatants were harvested 72 h after transfection.

For larger-scale production, Env proteins were produced in 293F cells using a protocol similar to that described previously (Julien et al., 2013; Sanders et al., 2013). Briefly, PEI-MAX (1.0 mg/ml) in water was mixed with expression plasmids for Env and Furin in OPTI-MEM. For cultures in a 2L disposable Nalgene flask (VWR), 250 μg of Env plasmid, 62.5 μg of Furin plasmid and 0.94 mg of PEI-MAX were added to 1L of pre-warmed Free-style 293 expression medium (Life Technologies). 293F cells were cultured for 6-7 days at 37°C, in an atmosphere containing 8%> CO2 and at a rotation speed of 125 rpm. Trimer purification

Env proteins were purified from transfection supernatants by affinity chromatography using a PGT145- or 2G12-column, essentially as described (Julien et al., 2013; Pugach et al., 2015; Sanders et al., 2013). The columns were made by coupling PGT145 or 2G12 to CNBr-activated Sepharose 4B beads (GE Healthcare). Briefly, supernatants were vacuum filtered through 0.2-μιη filters and then passed (0.5-1 ml/min flow rate) through a column, which was then washed with 2 column volumes of buffer (0.5 M NaCl, 20 mM Tris, pH 8.0). Bound Env proteins were eluted using 1 column volume of 3 M MgCh. and then immediately buffer- exchanged into 75 mM NaCl, 10 mM Tris, pH 8.0, using Vivaspin 20 tubes. The proteins were then concentrated using Vivaspin columns with a 30-kDa cut off (GE Healthcare). 2G12-purified Env proteins were further fractionated by size exclusion chromatography (SEC) to obtain pure trimers, whereas the PGT145 column yielded pure trimers without the need for SEC. Protein concentrations were determined using UV280 absorbance and theoretical extinction coefficients via Expasy (ProtParam tool). Values were multiplied by a factor of 1.82 to account for Env glycosylation. All experiments were performed with PGT145-purified trimers except for ITC and Octet studies, which used 2G12/SEC-purified trimers.

SDS-PAGE

Env proteins were analyzed using SDS-PAGE followed by western blotting or Coomassie blue dye staining (Sanders et al., 2002; Schulke et al., 2002). The input material was mixed with loading dye (25 mM Tris, 192 mM Glycine, 20% v/v glycerol, 4% m/v SDS, 0.1 % v/v bromophenol blue in water) and incubated at 95°C for 5 min prior to loading on a 4-12%> or 8% Tris-Glycine gel (Invitrogen). For reducing SDS-PAGE, dithiothreitol (DTT; 100 mM) was included in the loading dye. The gels were run for 2h at 125 V (0.07 A) using 50 mM MOPS, 50 mM Tris, pH 7.7 as the running buffer (Invitrogen). Western blot analysis of SDS- PAGE gels using mouse MAb ARP3119 (1 :2,000 dilution, i.e. 0.2 μ^πιΐ), followed by HRP-labeled goat anti- mouse IgG (1 :5000; Jackson Immunoresearch, Suffolk, England), was performed as previously described (Sanders et al., 2002). The Western Lightning ECL system (PerkinElmer Life Sciences) was used for luminometric detection. Coomassie blue staining of SDS-PAGE gels was performed using the PageBlue Protein Staining Solution (Thermo Scientific).

BN-PAGE

For BN-PAGE (Sanders et al., 2002; Schiilke et al., 2002), the input Env proteins were mixed with loading dye (500μ1 20x MOPS Running Buffer + ΙΟΟΟμΙ 100% Ultrapure Glycerol (Invitrogen cat#15514-011) + 50μ1 5% Coomassie Brilliant Blue G-250 + 600μ1 ddH20) and directly loaded onto a 4-12% Bis-Tris NuPAGE gel. The gels were run for 1.5 h at 200 V (0.07 A) using Anode -Buffer (20x NativePAGE Running Buffer (Invitrogen) in dd H20) and Cathode-Buffer (1% NativePAGE Cathode-Buffer Additive in Anode-Buffer; both from Invitrogen). Western blot analysis of BN-PAGE gels was carried out using human MAb 2G12 (0.1 μg/ml), followed by HRP-labeled goat anti-human IgG (1 :5,000 dilution Jackson Immunoresearch, Suffolk, England) and the Western Lightning ECL system (PerkinElmer Life Sciences), essentially as described previously (Sanders et al., 2002). BN-PAGE gels were stained using the Colloidal Blue Staining Kit (Life Technologies).

Surface plasmon resonance (SPR)

SPR was performed on a Biacore 3000 instrument at 25°C, using HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.002% P20 surfactant) as the running buffer (GE Healthcare). The D7324 antibody

(Aalto BioReagents, Dublin, Eire) was coupled to the chip as previously described (Yasmeen et al., 2014), to an immobilization level of 9,000 resonance units (RU). The flow rate was adjusted to 10 μΐ/min. The ligands, i.e., D7324-tagged BG505 SOSIP.664, v4.1 and v4.2 trimers, diluted in running buffer to 20 μg/ml, were then captured, giving immobilization levels (RL values) of 300 RU. A control channel into which no trimers were injected was used for background subtraction. In the simple binding assessment, the analytes, i.e., the test

MAb or CD4-IgG2 (500 nM, diluted in HBS-EP), was allowed to associate with the trimers for 5 min before dissociation was recorded for 10 min. The flow rate was 50 μΐ/min throughout each run. After each cycle, the surface was regenerated by a 60 s injection of lOmM Glycine [pH 2.0], at a flow rate of 30 μΐ/min. Induction of conformational changes by CD4 was studied in the SPR format by injecting two analytes in a single cycle. The first analyte (CD4-IgG2; 500 nM) was injected for 200 s followed by the second (17b; 500 nM) for a further 200 s, both at a flow rate of 30 μΐ/min. After each cycle, the surface was regenerated as described above, before fresh trimer was captured for the next run. D7324-capture ELISA

The method has been described elsewhere (Derking et al., 2015; Sanders et al., 2013). Microlon-600 96-well, half-area plates (Greiner Bio-One, Alphen aan den Rijn, The Netherlands) were coated overnight with Ab D7324 (Aalto Bioreagents, Dublin, Ireland) at 10 μ^πιΐ in 0.1 M NaHCOs, pH 8.6 (50 μΐ/well). After washing and blocking steps, purified BG505 SOSIP.664-D7324 trimers were added at 2 μg/ml in TBS for 2 h.

Unbound trimers were removed by 2 wash steps with TBS before various concentrations of test Abs were added for 2 h. After 3 washes with TBS, HRP-labeled Goat anti-Human IgG (Jackson Immunoresearch, Suffolk, England) was added at a 1 :3000 dilution in TBS/2% skimmed milk for 1 h, followed by 5 washes with TBS/0.05% Tween-20. Colorimetric detection was performed using a solution containing 1% 3,3',5,5'- tetramethylbenzidine ( Sigma- Aldrich, Zwijndrecht, The Netherlands), 0.01% H2O2, 100 mM sodium acetate and 100 mM citric acid. Color development (absorption at 450 nm) was stopped using 0.8 M H2SO4 (25 μΐ) when a plateau value was reached in the two wells containing the highest Ab concentration. In some experiments (Figs. 1.SI, 1.S2), MAbs 17b or 412d were added to the immobilized Env proteins in the presence of sCD4 (1.0 μg/ml). In other experiments (Fig. 1.S1M), a fixed concentration of 14e (0.1 μg/ml), 19b (0.6 μg/ml) or 39F (1.0 μg/ml) was incubated in solution with escalating concentrations of V3 peptides (Genscript) before adding the mixture to the ELISA plate. The V3 peptides (wild-type:

CTPvPNNNTPvKSIRIGPQAFYATGDIIGDIRQAHC (SEQ ID NO: 182); A316W:

CTRPNNNTRKSIRIGPGQWFYATGDIIGDIRQAHC (SEQ ID NO: 183)) were cyclized by a disulfide bond between residues 1 and 35. To quantify antibody responses in immunized rabbits, sera (from week-22) were serially diluted in 3-fold steps from a 1 : 100 start point, using 40% sheep serum (Biotrading), 2% milk powder in TBS as the buffer. When V3 -directed Ab responses were analyzed, the sera were incubated in solution with a V3 peptide (1 μg/ml) for 1 h prior to adding the mixture to the test wells. The peptides were identical to the V3 region of the immunogen. As with the BG505 V3 peptides described above the peptides were cyclized by a disulfide bond between residues 1 and 35. The sequences of the AMC008 peptides were (wild-type:

CTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHC (SEQ ID NO: 184); A316W:

CTRPNNNTRKSINIGPGRWFYTTGEIIGDIRQAHC (SEQ ID NO: 185). The secondary antibody was Goat anti-Rabbit IgG (Jackson Immunoresearch, Suffolk, England), and the color development procedures were as described above.

ELISA-based thermostability assay

As a screening assay for trimer stability, we devised a new thermal melting assay suitable for use on culture supematants containing unpurified BG505 SOSIP.664-D7324 trimers (Fig. 1.S1D-G). Supematants (typically 60 μΐ containing -15 μg/ml of total Env protein) were incubated for 30 min in a temperature gradient ranging from 39-77°C using a G-storm PCR machine (GRI Lab Care). The supematants were then transferred to a 96- well plate and a D7324-capture ELISA was performed as described above. The 2G12 detection MAb was used at 0.1 μg/ml, a concentration that gives -75% of the maximal signal in this ELISA format and hence allows any temperature-dependent loss of 2G12 reactivity to be quantified. When multiple, unpurified BG505 SOSIP.664-D7324 trimer variants were tested, the results from this screening assay correlated well with the outcome of DSC experiments using the same, but purified, trimers (Fig. 1.S1G). However, the midpoints of thermal denaturation (T_m) in the present assay were consistently 3-5°C lower than obtained via DSC, probably because the 2G12 epitope unfolds or is perturbed at a lower temperature than the bulk of the trimer.

Differential scanning calorimetr (DSC)

Thermal denaturation was studied using a nano-DSC calorimeter (TA instruments). All Env protein samples were first extensively dialyzed against PBS, and the protein concentration then adjusted to 0.1-0.3 mg/ml. After loading the sample into the cell, thermal denaturation was probed at a scan rate of 60°C/h. Buffer correction, normalization and baseline subtraction procedures were applied before the data were analyzed using NanoAnalyze Software v.3.3.0 (TA Instruments). The data were fitted using a two-state model as well as a non-two-state model, as the asymmetry of some of the peaks suggested that unfolding intermediates were present. We report the T_m values derived from the two-state model in the main text, while the multiple T_m values based on the non-two-state models are in the SI section. The DSC experiments were all performed with SOSIP.664-D7324 trimers, but we have determined that the presence of the D7324-tag does not alter the T_m values compared to the corresponding non-tagged trimers (data not shown).

Dynamic light scattering (DLS)

DLS measurements were performed at 20°C using a Dynapro Nanostar instrument (Wyatt Technologies), with 40 acquisitions of 5 s each. Each sample was spun at 10000 x g for 10 min prior to the DLS measurement, to remove any trace aggregates or dust from the sample. The hydrodynamic radius (Rh) and the molecular weight (MW) were calculated using the Dynamics analysis software (Wyatt Technologies), assuming a spherical model. To determine the Rh of stabilized SOSIP.664 trimers more precisely, we opted for DLS instead of the SEC/quasi-elastic light scattering (QELS) method used previously (Julien et al., 2013). The reason is that the use of flow mode SEC coupled to static and QELS detectors is limited by fitting using a mono-modal model. Calculated Rh values derived from this method thus represent the mean and distribution of diffusion constants. For a protein sample containing a small amount of polydispersity that is attributable to the presence of larger species, the resulting Rh value can be artificially elevated. As some preparations of BG505

SOSIP.664 trimers can contain up to -5% of higher-order aggregates that were not completely separated from the trimer by SEC, their presence may account for the higher Rh values (i.e. 8.1 nm for the BG505 SOSIP.664 trimer (Julien et al., 2013) than are presented here. Overall, because DLS measurements allow for multi-modal fitting models, we considered them to be a better way to measure Rh values for the wild type and stabilized SOSIP.664 trimers.

Small angle X-ray scattering (SAXS) SAXS measurements were conducted on Beam Line 4-2 at the Stanford Synchrotron Radiation Laboratory (Smolsky et al., 2007). The focused 11 keV X-ray beam irradiated a thin-wall quartz capillary cell, placed 2.5 m upstream of the Rayonix MX 225HE detector (Evanston, IL). A 50 μΐ sample of various PGT145-purified BG505 SOSIP.664 trimer variants (1 - 2 mg/ml) were injected onto a high resolution Sepharose 200 column (GE Healthcare) with a flow rate of 50 μΐ/min in 20mM Na₃P0₄ pH 7.4, 150mM NaCl, 0.02% NaN₃, ImM EDTA. The column eluate passed through a UV detector cell and into the quartz capillary cell. 1 second X-ray exposures were collected every 5 s throughout the run, during which a circulating water bath maintained the capillary cell temperature at 8°C. The detector pixel numbers were converted to the momentum transfer using the formula: q = 4p*sinq/l, where 1 is the X-ray wavelength of 1.127 A and 2*q is the scattering angle calibrated using a silver behenate powder standard placed at the capillary position. A background scattering curve was obtained from the first 100 exposures (before the void volume), and was subtracted from all subsequent exposures during generation of the sample elution profile. The radius of gyration values (R_g) and 1(0) for each frame were batch-analyzed using AutoRg, and frames with stable R_g values were merged using Primus (Petoukhov et al., 2007) for the final scattering curve . The real space distance distribution functions were calculated from the merged data sets by indirect transformation using the program GNOM (Svergun, 1992).

Hydrogen/Deuterium exchange (HD-X)

Soluble two-domain CD4 (sCD4) (Garlick et al., 1990) was obtained from the NIH AIDS reagents program. Immediately before HDX-MS analysis, all proteins were SEC-purified using a Superdex S200 column (GE Healthcare) and a PBS-based elution buffer (20 mM sodium phosphate pH 7.4, 150 mM NaCl with 1 mM EDTA and 0.02 % sodium azide). Complexes were formed by overnight incubation at 4°C with a 3-fold molar excess of sCD4 (relative to each protomer). Native gels were run for each sample to assess complex formation (Fig. 1.S3C). Various PGT145-purified BG505 SOSIP.664 trimers (15 μg) were diluted 10-fold into deuterated PBS buffer and incubated at room temperature. The deuterium exchange reactions were quenched after 3 s, 1 min, 30 min and 20 h by mixing with an equal volume of cold 200 mM Tris-(2- Carboxyethyl)phosphine hydrochloride (TCEP), 0.2% formic acid (final pH 2.5). The samples were subsequently digested with pepsin (0.15 mg/ml) for 5 min on ice, flash frozen in liquid nitrogen and stored at - 80°C. Differences in exchange profiles that exceeded the error of the measurement were visualized on the trimer structure using custom macros in PyMOl (deLano WL, 2002).

Negative-stain electron microscopy (EM)

Purified BG505, B41, ZM197M and AMC008 SOSIP.664 trimers, either alone or as Fab complexes (with PGV04 and 35022), were analyzed by negative-stain EM. To form complexes, a 6-10 molar excess of each Fab was incubated with SOSIP overnight at room temperature. A 3 μΐ aliquot containing ~0.03 mg/ml of a trimer or Fab-trimer complex was applied for 5 s onto a carbon-coated 400 Cu mesh grid that had been glow discharged at 20 mA for 30 s, then negatively stained with 2%> (w/v) uranyl formate for 60 s. Data were collected on either an FEI Tecnai T12 electron microscope operating at 120 keV, with an electron dose of -25 e /A² and a magnification of 52,000x that resulted in a pixel size of 2.05A at the specimen plane, or an FEI Talos electron microscope operating at 200 keV, with an electron dose of -25 e /A² and a magnification of 92,000x that resulted in a pixel size of 1.57 A at the specimen plane. Images were acquired with a Tietz TemCam-F416 CMOS camera (FEI Tecnai T12) or FEI Ceta 16M camera (FEI Talos) using a nominal defocus range of 1000-1500.

Image processing and 3D reconstruction

Data processing methods are described elsewhere (Derking et al 2015). In total, 24,522 particles

were included in the final reconstruction for the 3D average of AMC008 SOSIP.v4.2 in complex with PGV04 and 35022 Fabs. The resolution of the final reconstruction is -15 A based on an Fourier shell correlation of 0.5.

Isothermal titration calorimetry (ITC)

ITC was performed using an Auto-iTC 200 instrument (GE Healthcare) using a protocol similar to one described previously (Julien et al., 2013; Sanders et al., 2013). Briefly, prior to conducting the titrations, proteins were dialyzed against Tris-saline buffer (150 mM NaCl, 20 mM Tris, pH 8.0). Absorbance at 280 nm using calculated extinction coefficients served to determine and adjust protein concentrations. The ligand present in the syringe was 19b Fab at a concentration of 10-20 μΜ, while the BG505, B41 or AMC008 SOSIP.664 trimer was present in the cell at a concentration of 4-6 μΜ. In each binding experiment, a 5 μcal reference power determination preceded the first injection of 0.5 μΐ, which was followed by 15 injections of 2.5 μΐ each at intervals of 180 s. Origin 7.0 software was used to derive the affinity constants (KD), the molar reaction enthalpy (ΔΗ) and the stoichiometry of binding (N), by fitting the integrated titration peaks via a single-site binding model or a two-site binding model, as appropriate. Glycan profiling

Env trimers (10 μg) were resolved by SDS-PAGE under non-reducing conditions, followed by staining with Coomassie blue. Bands corresponding to gpl40 were excised from the gels and washed alternately with acetonitrile and water, five times. N-linked glycans were then released by addition of protein N-glycosidase F (PNGase F) at 5000 U/ml and incubation at 37°C for 16 h, according to the manufacturer's instructions (NEB). The released glycans were subsequently eluted from gel bands by extensive washing with water, and then dried using a SpeedVac concentrator. Released glycans were labelled with 2-aminobenzoic acid (2-AA) as previously described (Neville et al., 2009). Briefly, glycans were resuspended in 30 μΐ of water followed by addition of 80 μΐ of labelling mixture (comprising 30 mg/ml 2-AA and 45 mg/ml sodium cyanoborohydride in a solution of sodium acetate trihydrate [4% w/v] and boric acid [2% w/v] in methanol). Samples were then incubated at 80°C for 1 h. Excess label was removed using Spe-ed Amide -2 cartridges, as previously described (Neville et al., 2009). Fluorescently labelled glycans were resolved by Hydrophilic interaction liquid chromatography-ultra performance liquid chromatography (HILIC-UPLC) using a 2.1 mm x 10 mm Acquity BEH Amide Column (1.7 μιη particle size) (Waters, Elstree, UK). The following gradient was run: time = 0 min (t = 0): 22.0% A, 78.0% B (flow rate of 0.5 ml/min); t = 38.5: 44.1% A, 55.9% B (0.5 ml/min); t = 39.5: 100% A, 0% B (0.25 ml/min); t = 44.5: 100% A, 0% B (0.25 ml/min); t = 46.5: 22.0% A, 78.0% B (0.5 ml/min), t = 48: 22.0%> A, 78.0%> B (0.5 ml/min), where solvent A was 50 mM ammonium formate, pH 4.4, and solvent B was acetonitrile. Fluorescence was measured using an excitation wavelength of 250 nm and a detection wavelength of 428 nm. Data processing was performed using Empower 3 software.

The percentage abundance of oligomannose-type glycans was calculated by integration of the relevant peak areas before and after Endoglycosidase H digestion, following normalization. Digestions were performed on free glycans at 37°C for 16 h. The digested glycans were purified using a PVDF protein-binding membrane plate (Millipore) prior to HILIC-UPLC analysis.

Rabbit immunizations

Rabbit immunizations and blood sampling were carried out under subcontract at Covance (Denver, PA) according to the schedule presented in fig. 1.4A. The protocol was broadly the same as previously described. Female New Zealand White rabbits (5 per group) were immunized intramuscularly with 40 μg of the various Env trimers. The proteins were formulated in 75 Units of ISCOMATRIX™, a saponin-based adjuvant obtained from CSL Ltd. (Parkville, Victoria, Australia) (Maraskovsky et al., 2009).

Neutralization assays

The TZM-bl reporter cell line, which stably expresses high levels of CD4 and the co-receptors CCR5 and

CXCR4 and contains the luciferase and β-galactosidase genes under the control of the HIV-1 long-terminal- repeat promoter, was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. Durham, NC).

TZM-bl cell neutralization assays using Env-pseudotyped viruses were performed at two different sites:

DUMC, Duke University Medical Center, Durham, NC; AMC, Academic Medical Center, Amsterdam

(methodology see (Sanders et al., 2013)). For additional information on the assay and all supporting protocols see: http://www.hiv.lanl.gov/content/nab-reference-strains/html/home.htm. The Env-pseudotyped viruses and their Tier classifications have been described elsewhere (deCamp et al., 2014; Goo et al., 2014; Li et al., 2006; Seaman et al., 2010; Simek et al., 2009). When the neutralization-sensitivity of the parental AMC008 virus was assessed using a panel of human sera and MAbs and a panel of test viruses at the Duke University

Medical Center, it was classified as a Tier IB virus (not shown), while the parental BG505.T332N virus has a Tier 2 phenotype (Sanders et al., 2015). To produce Env-pseudotyped viruses, HEK293T cells (2* 10⁵) were seeded in a 6-well tissue culture plate (Corning) in 3 ml DMEM (Gibco) containing 10%> FCS, penicillin (Sigma) and streptomycin (Sulphate-Gibco) (both at 100 U/ml) per well. The culture was refreshed after 1 d by adding 3 ml culture medium when the cells had reached a confluence of 90-95%>, and the cells were then transfected using Lipofectamine 2000 (Invitrogen). Per well, 1.6 μg of BG505.T332N plasmid and 2.4 μg of pSG3AEnv plasmid (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. Durham, NC)) were mixed in 250 μΐ of OPTI-MEM (Gibco). A 10-μ1 aliquot of lipofectamine 2000 was mixed with 240 μΐ of OPTI-MEM immediately before addition to the solution containing the expression plasmids. After incubation for 20 min at room temperature, the transfection mixture was added to the cells, and the culture supematants were harvested 48 h later as the source of Env-pseudotyped viruses for infection/neutralization experiments.

One day prior to virus infection, 1.7x l0⁴ TZM-bl cells per well were seeded in a 96-well plate in DMEM containing 10% FCS, l x MEM nonessential amino acids, penicillin and streptomycin (both at 100 U/ml), and incubated at 37°C for 24h in an atmosphere containing 5% CO2. To determine neutralization activity of rabbit sera, a fixed amount of virus (500pg of p24-antigen equivalent) was incubated for one hour at RT with 1 :3 serial diluted heat-inactivated sera, starting with a serum dilution of 1 :20. Afterwards the mix was added to the cells in the presence of 40 μg/ml DEAE-Dextran (Sigma) and Sanquinavir, in a total volume of 200 μΐ. Three days after infection was initiated, the medium was removed and the cells were washed once with PBS (150 mM NaCl, 50 mM sodium phosphate, pH 7.0) and lysed in Reporter Lysis Buffer (Promega, Madison, WI). Luciferase activity was measured using a Luciferase Assay kit (Promega, Madison, WI) and a Glomax Luminometer according to the manufacturer's instructions (Turner BioSystems, Sunnyvale, CA). All infections were performed in duplicate. Uninfected cells were used to correct for background luciferase activity. The infectivity of each mutant without inhibitor was set at 100%). Nonlinear regression curves were determined and 50% inhibitory serum dose (ID50) were calculated using a sigmoid function in Prism software version 5.0.

Biolayer interferometry

Biolayer interferometry (BLI) assays were performed using the Octet Red96 instrument (Pall ForteBio). C- terminal His-tagged sCD4 (domains 1 and 2, expressed in HEK293F and purified via Ni-NTA affinity and size-exclusion chromatography) was immobilized onto Ni-NTA probes for 120 s followed by a 60 s baseline calculation in IX kinetics buffer (phosphate-buffered saline pH 7.2 supplemented with 0.01% w/v bovine serum albumin and 0.002%> v/v Tween 20). The probes were then dipped for 300 s into wells containing 200 nM of BG505 SOSIP.664 WT or BG505 SOSIP.664 containing single or double stabilizing mutations (E64K, H66R, A316W, E64K/A316W, or H66R/A316W). After the association step the probes were placed into wells containing IX kinetics buffer for 600 s to measure dissociation. An inter-step correction was applied to align the end of the association curve to the begging of the dissociation curve, and a single binding site model was fit to the aligned curves to determine on- and off-rates.

References

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Sok, D., van Gils, M.J., Pauthner, M., Julien, J., Saye-Francisco, K.L., Hsueh, J., Briney, B., Lee, J.H., Le, K.M., Lee, P.S., et al. (2014). Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex. Proc. Natl. Acad. Sci. Ill, 17624-17629.

Svergun, D.I. (1992). Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495-503.

Yasmeen, A., Ringe, R., Derking, R., Cupo, A., Julien, J. -P., Burton, D.R., Ward, A.B., Wilson, I. a, Sanders, R.W., Moore, J.P., et al. (2014). Differential binding of neutralizing and non-neutralizing antibodies to native -like soluble HIV- 1 Env trimers, uncleaved Env proteins, and monomeric subunits. Retro virology 11, 4-1. Table 1.S1, related to Table 1.1: Detailed DSC analysis of PGT145-purified SOSIP.664 trimer variants.

In addition to the simplified two-state model analysis, the unfolding patterns were fitted using a non-two state model (see Fig. 1.S2D), revealing three individual unfolding peaks. The T_m values of the individual peaks are

Table 1.S2, related to Figure 1.1. Amino acids present at positions 535 and 543 of the <x6 helix in gp41.

Shown are the amino acids present in the wild-type sequences of clones JR-FL, K H1144, BG505, B41, ZM197M and AMC008 at positions 535 and 543, as well as the amino acids optimal for Env stability (in green; (Dey et al., 2008; Leaman and Zwick, 2013)). The right-most column specifies the amino acid changes that have now been incorporated into the various SOSIP.664 constructs, to optimize the efficient formation of trimer s.

Table 1.S3, related to Table 1.1 Nomenclature of new stabilized SOSIP.664 trimers. An overview of the modifications present in the stabilized SOSIP.664 trimer versions. Horizontal shading: present, vertical shading: not present, black: not systematically present.

Table 1.S6. Isothermal titration calorimetry (ITC) binding of 19b to various WT and stabilized SOSIP.664 trimers.

Antibody Binding partner (kcal (kcal (kcal Ni

(nM) mol ¹) mol ¹) mol ¹)

BG505 SOSIP.664 - WT -10.6 -41.4 30.8 21 0.1

73C-568C^* 0.1 73C-561C^* 0.1 73C-558C -11.4 -30.9 19.5 0.2 72C-564C^* 0.1

19b IgG

B41 SOSIP.664 - WT -39.5 27.6

L543N-A316W-E64K detectable bindin

AMC008 SOSIP.664 - WT -11.2 -29.6 18.4 11 0.3

I535M-L543N-A316W-E64K no detectable binding 0 I535M-L543N-A316W-H66 ^* <0.1

'Reported in Pugach et al. J Virol 2015.

Binding isotherms do not allow to confidently determining thermodynamic parameters of binding.

Table 1.S4, related to Figure 1.4. Midpoint neutralization titers for sera from macaques immunized with BG505 SOSIP.664 trimers or gpl20 monomer tested against a panel of Env-pseudotyped viruses. The TZM-bl cell assay was performed at Academic Medical Center, Amsterdam. ID50 values (serum diluti at which viral infectivity is inhibited by 50%). are given.

Table 1.S5, related to Figure 1.4. Midpoint neutralization titers for sera from macaques immunized with BG505 SOSIP.664 trimers or gpl20 monomer tested against a panel of Env-pseudotyped viruses. The TZM-bl cell assay was performed at Duke University Medical Center. ID50 values (serum dilution at which viral infectivity is inhibited by 50%) are given.

Example 2

We introduced everal mutations to increase the stability of the SOSIP.664 trimers. The L555K substitution was designed to stabilize the trimer by introducing a hydrogen bond between gp41 in one protomer with gpl20 from an adjacent protomer, and an L556K substitution was designed to create a hydrogen bond between HR1 in gp41 and a-helix 0 in gpl20 within a protomer. When co-expressed with furin under standard conditions, the resulting L555K and L556K trimers were fully cleaved as shown by the conversion of gpl40 bands to gpl20 and gp41 on a reducing SDS-PAGE gel. Hence the point substitutions do not adversely affect the overall trimer conformation. Although the melting temperatures were essentially unchanged, the yields of the L555K and L556K trimers were increased by ~3 and ~2- fold, respectively, compared to wild type, when purified on a PGT145 bnAb affinity column (Figure 2.3C).

A SOSIP.664-L555C/Q49C double mutant was then designed to introduce a new disulfide bond to covalently link gp41 with the gpl20 N-terminus of the adjacent protomer (Figure 2.3C).Its melting temperature of 75.2° is the highest that we have observed to date for a SOSIP.664 trimer variant. We used a panel of bnAbs, non-neutralizing antibodies (non-nAbs) and CD4 to probe the antigenic structure of the above trimer mutants by ELISA, and found bnAbs and CD4 bound equivalently to wild- type BG505 SOSIP.664. Importantly, for the double mutant L555C/Q49C, binding of the 17b non-bnAb to its CD4-induced epitope was decreased 5-fold.

We conclude that removal of the N137 glycan from the trimer has a profound effect on antibody binding affinity at all stages in the evolution of PGT121 lineage.

EXPERIMENTAL PROCEDURES

Expression and purification of proteins. BG505 SOSIP.664 wild-type and mutant trimers (SOSIP- 137A and SOSIP-N332A) cloned in a phMCV3 vector, were expressed in FreeStyle™ 293F or 293S cells and purified using a 2G12-coupled affinity matrix followed by SEC, with exception for the BG505 SOSIP.664 N332A mutant for which a GN lectin affinity column was used in the initial stage. The structure-based SOSIP.664 L555K, L556K and L555C-Q49C mutants were purified using a PGT145- coupled affinity matrix and then by SEC.

Antibody and envelope substitutions. Substitutions in the Env glycoprotein constructs were introduced using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA). Substitutions were verified by DNA sequencing (Retrogen, San Diego, CA).

SDS-PAGE and Blue Native-Page. BG505 SOSIP.664 and mutant trimers were analyzed using SDS- PAGE and BN-PAGE and bands were revealed after staining with Coomassie blue. Each sample was pre- mixed with loading buffer and shortly after loaded onto a 4-12% Bis-Tris NuPAGE gel (Invitrogen). Each run was for 1.5h at 200V. Isothermal titration calorimetry. ITC experiments were performed using a MicroCal Auto-iTC200 instrument (GE). All proteins were extensively dialyzed against a buffer containing 20 mM Tris, 150 mM NaCl, pH 7.4 before conducting the titrations. Subsequently, protein concentrations were adjusted and confirmed by using calculated extinction coefficients and absorbance at 280 nm. Ligands representing precursor and mature Abs of the PGT121 family were present in the syringe at concentrations ranging between 60-125 μΜ. The BG505 SOSIP.664 wild type and mutants were in the cell at concentrations ranging between 3.0-5.0 μΜ. Two-protein binding experiments were performed with the following parameters: cell at 25°C, 16 injections of 2.5 μΐ each, injection interval of 180 s, injection duration of 5 s, and reference power of 5 μcals. To calculate the dissociation constants (Kd), the molar reaction enthalpy (ΔΗ) and the stoichiometry of binding (N), Origin 7.0 software was used to fit and integrate the titration peaks using a single-site binding model.

Electron microscopy (EM). All complexes were analyzed by negative-stain EM. The 3H+3L Fab complex was formed with the BG505 SOSIP.664-N137A mutant, while 32H+3L Fab was in complex with the wild type BG505 SOSIP.664. Both Env proteins were produced in HEK 293S cells. For the 9H+3L complexes, the BG505 SOSIP.664 and BG505 SOSIP.664-N137A trimers were produced in HEK 293F cells. A 3 μΕ aliquot of 10 μg/ml of the complex was applied for 15s onto a glow discharged, carbon coated 400 Cu mesh grid and stained with 2% uranyl formate for 20s. Grids were imaged using a FEI Tecnai T12 electron microscope operating at 120 kV using 52,000 x magnification and electron dose of 25 e^"/A², resulting in a pixel size of 2.05 A at the specimen plane. Images were acquired with a Tietz 4k x 4k CCD camera in 5° tilt increments from 0° to 55° at a defocus of 1000 nm using LEGINON.

Image processing. Particles were picked automatically by using DoG Picker and put into a particle stack using the Appion software package. Initial reference-free 2D class averages were calculated using unbinned particles via the Xmipp Clustering 2D Alignment and sorted into 400 classes. Particles corresponding to the complexes were selected into a substack and another round of reference-free alignment was carried out using Xmipp Clustering 2D alignment and IMAGIC softwares. To generate an ab initio 3D starting model, a template stack of 120 images of 2D class averages was used with imposing C3 symmetry. This starting model was refined against 12,245 raw particles for 30 cycles using EMAN. The resolution of the final reconstruction for 3H+3L with BG505 SOSIP.664-N137A was calculated to be 17A, for 9H+3L with BG505 SOSIP.664 was 21 A, for 9H+3L with BG505 SOSIP-N137A was 19 A and for 32H+109L with BG505 SOSIP.664 was 22 A using an FSC cut-off of 0.5.

Differential scanning calorimetry (DSC). The thermal stabilities of BG505 SOSIP.664 and mutant trimers were analyzed on a VP -DSC calorimeter (GE Healthcare). Example 3

Addition of a second disulfide bond between gpl20 and gp41

To reduce flexibility and increase stability of BG505 SOSIP.664 trimers we screened for positions in gpl20 and gp41 that would accommodate the introduction of an alternative or additional intermolecular disulfide bond between the gpl20 and gp41 subunits. Since the original disulfide bond in SOSIP gpl40 between residues 501 and 605 is located at the membrane proximal side of the trimer, we focused on potential locations nearer to the trimer apex. Based on favorable biochemical and antigenic properties, we selected two variants, BG505 SOSIP.664 H72C-H564C and SOSIP.664 A73C-A561C, for further analyses (Fig. 3.1 A; 3. Figs 3. SI and 3.S2). To provide additional stability we combined the two new disulfide bonds with two substitutions that were previously shown to enhance BG505 SOSIP.664 stability: A316W, which fills a hydrophobic pocket and prevents the V3 from popping out of its hidden location underneath VI V2, and E64K, which prevents spontaneous sampling of the CD4-bound conformation (Fig. 3.1A;). From here on BG505 SOSIP.664 trimers containing the additional H72C- H564C or A73C-A561C are defined as BG505 SOSIP.v5.1 and BG505 SOSIP.v5.2 respectively (Fig. 3.1A).

Biochemical and biophysical properties of stabilized BG505 SOSIP.v5 trimers

The BG505 SOSIP.v5 trimers were purified via PGT145-affinity chromatography. PGT145-purification yielded higher amounts of pure BG505 SOSIP.v5 trimers compared to BG505 SOSIP.664 (2.9 and 2.9 mg/1 for SOSIP.v5.1 and SOSIP.v5.2 respectively, vs. 1.6 mg/1 for SOSIP.664; Table 3.1, Fig. 3.S3a, Fig 3. IB). Non-reducing SDS-PAGE analysis, i.e. conditions that retained the disulfide bonds in an oxidized state, showed that the mutants containing two disulfide bonds between gpl20 and gp41 had a slower migration pattern compared to the ones that had only one disulfide bond between gpl20 and gp41 (Fig. 3.S3a). The slower migration can be explained by decreased SDS uptake typically seen with more compact proteins. The data suggest that both disulfide bonds between gpl20 and gp41 form. Characterization of BG505 SOSIP.664 H72C-H564C and A73C-A561C by negative stain electron microscopy (EM) revealed that the PGT145-purified trimers retained a native-like conformation (>98 % native-like trimers; Table 3.1 ; Fig. 3.S3c). While BG505 SOSIP.664 was predominantly in a more open conformation after PGT145-purification (33% closed trimers), BG505 SOSIP.664 H72C-H564C and A73C-A561C proteins showed higher percentages of closed trimers (69%> and 67%>, respectively). The fraction of closed native-like trimers was further increased when the E64K and A316W substitutions were also present (BG505 SOSIP.v5.1 : 85% closed trimers; BG505 SOSIP.v5.2: 91% closed trimers; Table 3.1 Fig. 3.S3c).

Dynamic Light Scattering (DLS) experiments showed that the variant SOSIP.664 trimers that contained a new disulfide bond were monodisperse and had a hydrodynamic radius (¾) of ~69 A, similar to the original BG505 SOSIP.664 trimer (SI Table 1). When the A316W and E64K substitutions were also included the R_h dropped subtly (to 68 and 67 A for BG505 SOSIP.v5.1 and v.5.2, respectively; SI Table 1), pointing at an increase in compactness of the trimers. Furthermore, Small Angle X-ray Scattering (SAXS) experiments showed that the trimers were well folded and had a radius of gyration (R_g) of ~52 A (Table 3.1, Fig. 3.S3d). The Kratky plots showed a Gaussian- like peak at the low angle (low q) region that dips towards zero, indicating that these proteins are well folded globules.

We also characterized the glycan structures present on the different BG505 SOSIP.v5 trimer variants. The glycosylation profiles of the SOSIP.v5 were similar to BG505 SOSIP.664, with oligomannose glycoforms dominating (-64%), in particular MangGlcNAc2 and Man9GlcNAc2 glycans (18-23% for each)(Table 1, SI Fig XX). The high density of unprocessed oligomannose glycans is a distinctive feature of native-like Env trimers.

Next, we analyzed the thermostability of the mutants by differential scanning calorimetry (DSC). The midpoint of thermal denaturation T_m) of BG505 SOSIP.664 H72C-H564C and A73C-A561C were 72.0°C and 72.5°C, respectively, i.e. 4.4°C and 4.9°C higher than that of the original BG505 SOSIP.664 trimer (Table 1). The thermostability was further increased when the E64K and A316W substitutions were incorporated: The T_m values were 75.0°C and 75.3°C, for SOSIP.v5.1 and SOSIP.v.5.2, respectively, i.e. 7.4°C and 7.7°C higher than that of the original BG505 SOSIP.664 trimer; Table 3.1, Fig. 3.S3e).

Antigenic properties of stabilized BG505 SOSIP.664 trimers

We tested a panel of bNAbs to assess the antigenicity of the stabilized BG505 trimer variants by ELISA. All bNAbs bound equally well or better to the SOSIP.v5 variants compared to the parental SOSIP.664 proteins (Table 3.2a and table 3.2b, Fig. 3.S3f). Surface Plasmon Resonance (SPR) studies confirmed that the SOSIP.v5 trimers interacted strongly with the quanternary structure dependent epitopes at the trimer apex (PG16 and PGT145) and the gpl20/gp41 interface (35022 and PGT151) indicating that the substitutions did not affect the epitopes of these bNAbs (Fig. 3. IB). Next, we investigated the binding of non-NAbs against the V3 domain by ELISA. The addition of a new disulfide bond did not affect binding of V3 non-NAbs 19b and 14e but their binding was reduced when the E64K and A316W substitutions were also present (Table 3.2a and table 3.2b, Figure 3.S3f), consistent with previous findings described in example 1. BG505 SOSIP.664 shows minimal but detectable binding to non-NAbs against CD4-inducible (CD4i) epitopes in the absence of CD4. In the mutants this binding was eliminated, while in the presence of CD4 the binding was reduced (Table 3.2a and table 3.2b, Figure 3.S3f). These results were confirmed by SPR, showing undetectable binding of CD4i 17b non-NAb, and reduced binding when sCD4 was first boud to the trimer. These data indicated that the BG505 trimers containing the second disulfide bond alone or in combination with the E64K and A316W substitutions retained the capacity of presenting bNAb epitopes, while occluding non-NAb epitopes more efficiently compared to the original BG505 SOSIP.664 trimer. Binding of CD4-IgG2 to BG505 trimer variants was significantly reduced compared to its wild-type counterpart (Table 3.2a and table 3.2b). This finding is in line with the fact that SPR analysis showed a faster dissociation of CD4 IgG2 in the BG505 SOSIP.v5 variants compared to BG505 SOSIP.664 (Fig 3.1A). Structure of stabilized BG505 SOSIP trimers

The SOSIP.664 H72C-H564C trimer was expressed in 293 S cells and purified using 2G12-affmity chromatography followed by size exclusion chromatography (SEC) and its structure in complex with bNAbs PGT122, 35022 and NIH45-46 was solved by X-ray crystallography (Fig. 3.1). Overall, these data indicate that the new cysteines do not alter the overall quaternary structure of the BG505 SOSIP.664 trimer, and show that the new trimers bind efficiently to bNAbs to three different sites, including the gpl20-gp41 interface.

Dynamics of BG505 SOSIP.v5 trimers

The precise effect of the stabilizing mutations on protein dynamics and conformational flexibility was assessed by Hydrogen-Deuterium Exchange (HD-X) coupled with mass spectrometry (MS). Protein domains that adopt a stable secondary structure and/or are buried are protected from deuterium exchange, while exposed flexible domains undergo rapid deuterium exchange. We investigated the exchange profiles of BG505 SOSIP.664 A73C-A561C, SOSIP.v5.1 and SOSIP.v5.2 in comparison with the original BG505 SOSIP.664 trimer (Fig. 3.S4). All the mutants had similar exchange profiles as BG505 SOSIP.664 trimer with only few local changes. Similar to the wild-type BG505 protein, all the mutants showed a slow exchange in the gpl20 core, the stem in V1V2 and the N-terminal half of a9, indicative of buried or stable domains; and rapid exchange in the C-terminal portion of V2, the fusion peptide and the C-terminal half of a9, indicative of local flexibility. The BG505 SOSIP.v5.2 showed nearly identical exchange kinetics compared to the wild-type protein indicating that the structure and conformational dynamics were very similar. The other two variants showed subtly increased dynamics in regions close to the new disulfide bond, while the BG505 SOSIP.v5.1 variant also showed reduced dynamics in C- terminal half of al (Fig. 3.S4). Overall, the substitutions did not dramatically alter the conformational dynamics of the unliganded trimer.

Next, we investigated the propensity of the stabilized trimers to undergo CD4-induced conformational changes. The addition of sCD4 in BG505 SOSIP.664 resulted in less protection in the V2 and V3 loops and more protection in the CD4bs and layers 1-3 in gpl20, and al in gp41. Such changes were greatly diminished or completely abolished in the mutated proteins. Among all the mutants, BG505 SOSIP.v5.2 showed the least response to sCD4, entirely abrogating the CD4-induced exposure of the V2 and V3 domains (Fig. 3.2). These data showed that the presence of an extra disulfide bond prevented the apex of the trimer from opening when sCD4 was added, and this was further reinforced by adding the E64K and A316W substitutions. By all means these stabilized trimers appear trapped in the closed, unliganded state. Stabilizing SOSIP trimers from HIV-1 clades B and C

Next, we examined whether stabilization of the trimer through incorporation of an extra disulfide could be extrapolated to trimers from other clades. We introduced the H72C-H564C and A73C-A561C cysteine pairs into SOSIP.664 trimers from the clade B isolates AMC008 and B41, and the clade C isolate ZM197M. Based on the results of studies conducted in parallel we also introduced two amino acids that were already present in BG505 gp41 sequence and that we found to be beneficial in the context of these three isolates: I/V535M and/or L543N, as well as the substitutions E64K or H66R and A316W. E64K and H66R have similar effects on trimer stability and antigenicity.

The AMC008, B41 and ZM197M SOSIP.664 and SOSIP.v5 variants were purified using PGT145- affinity chromatography and analyzed by SDS-PAGE and BN-PAGE analysis. While AMC008 SOSIP.v5 yielded similar amounts of pure trimers compared to AMC008 SOSIP.664, ZM197M yielded considerably higher amounts of pure trimers compared to ZM197M SOSIP.664 (1.0 mg/1 for both SOSIP.V5.1 and SOSIP.v5.2, compared to 0.2 mg/1 for SOSIP.664; Table 3.1). Purification of B41 trimers yielded higher or lower amounts of pure trimers depending on the SOSIP.v5 used (0.2 mg/1 and 2.9 mg/1 for SOSIP.v5.1 and SOSIP.v5.2 respectively) compared to B41 SOSIP.664, which yielded XXX mg/1 (Table 3.1). All variants formed pure trimers that were cleaved efficiently (Fig. 3.S5). In non- reducing SDS-PAGE, the variants with the extra disulfide bonds ran slower, consistent with the results obtained with BG505 SOSIP.664 and in line with a more compact structure caused by the formation of the two disulfide bonds between gpl20 and gp41 (Fig. 3.S5a). Negative stain EM confirmed that these proteins formed native-like trimers, but the addition of the new disulfide bonds increased the percentage of trimers in the closed conformation (Table 3.3, Fig. 3.S5c). Thus, introduction of the disulfide bonds increased the proportion of closed trimers in AMC008

SOSIP.664 from 15% to 100% (SOSIP.v5.1 and SOSIP.v5.2), in B41 SOSIP.664 from 45% to 95% (SOSIP.V5.1) and XXX % (SOSIP.v5.2), and in ZM197M SOSIP.664 from 15% to 100% (SOSIP.v5.1 and SOSIP.v5.2)(Table 3.3, Fig. 3.S5c). The presence of fully closed trimers in ZM197M SOSIP.664 disulfide variants is remarkable, especially because ZM197M SOSIP.v4.2, which contains the mutations H66R and A316W, only increased the amount of closed native trimers up to 30%>.

We also characterized the glycan structures present AMC008 and B41 SOSIP variants compared to their wild-type counterparts. As seen for BG505 oligomannose glycoforms dominated (63-69%>), in particular Man₈GlcNAc2 and Man₉GlcNAc2 glycans (Table 3.1).

DSC experiments showed that the stability of the new AMC008, B41 and ZM197M SOSIP.664 proteins containing the new disulfide bonds was considerably enhanced. The new disulfide bonds increased the T_m of the AMC008 SOSIP.664 trimer from 60.2°C to 68.3°C (SOSIP.v5.1) and 68.5°C (SOSIP.v5.2), i.e. 8.PC and 8.3°C higher than that of the original unmodified AMC008 SOSIP.664 trimer (Table 3.3; Fig. 3.S5d). Similarly, the T_m of B41 SOSIP.664 was increased from 58.7°C to 63.0°C (SOSIP.v5.1) and 64.7°C (SOSIP.V5.2), and that of ZM197M SOSIP.664 from 62.2°C to 69.5°C (SOSIP.v5.1) and 69.2°C (SOSIP.v5.2) (Table 3.3; Fig. 3.S5d). Thus, the amino acid substitutions present in SOSIP.v5.1 and SOSIP.v5.2 trimers improve the stability of closed SOSIPtrimers from four different HIV-1 clades.

Antigenic properties of B41, AMC008 and ZM197M SOSIP.v5 trimers Overall the effects of the H72C-H564C and A73C-A561C disulfide bonds were similar for the AMC008 SOSIP.664 trimers as for the BG505 SOSIP.664 trimers. Thus, the trimers retained the ability to bind bNAbs while reducing binding to non-NAbs against the V3 and CD4i epitopes. Surprisingly, the introduction of the disulfide bonds in B41 SOSIP.664 and in ZM197M SOSIP.664 improved the binding of the quaternary structure dependent bNAbs PG16, PGT145 and PGT151 (Table 3.4, Fig. 3.S5D). This was particularly noticeable for B41 SOSIP.664, in which PGT151 -binding was dramatically changed from negligible binding in the absence of the new disulfide bond to strong binding in its presence (Table 3.4, Fig. 3.S5D), suggesting that the extra disulfide bond restores the local conformation of PGT151 epitope at the gpl20-gp41 interface. All B41 and ZM197M SOSIP.664 trimer variants containing the new disulfide bonds showed reduced binding to non-NAbs against the V3 and CD4i epitopes (Table 3.2a and table 3.2b, Fig. 3.S5D). Binding of CD4-IgG2 to AMC008, B41 and ZM197M SOSIP.664 disulfide trimer variants was markedly reduced compared to their wild-type counterpart, in line with what has been described for BG505 SOSIP.664 (Table 3.2a and table 3.2b).

Immunogenicity of BG505 SOSIP.v5 trimers in rabbits

We next immunized rabbits with PGT145-purified BG505 SOSIP.v5 and BG505 SOSIP.664 trimers. Rabbits were immunized at weeks 0, 4 and 20 as described herein. The immune responses were measured at week 22. i.e. 2 weeks after the third immunization. First, we quantified the antibody response by ELISA against the autologous envelope glycoprotein. All the animals that were immunized with BG505 SOSIP.664 and SOSIP.v5 exhibited high titers of binding antibodies in ELISA, (Fig. 3.3A). BG505 SOSIP.664 induces a substantial non-neutralizing (for Tier 2 viruses) anti-V3 response that might distract from neutralization relevant targets. We performed a V3 peptide competition ELISA in order to determine the proportion of the response directed to the V3. V3 responses accounted for -30% of the ELISA binding signal in BG505 SOSIP.664 immunized. Rabbits immunized with BG505 SOSIP.v5, in particular SOSIP.v5.2 trimers showed a significantly lower proportion of V3 directed response compared to BG505 SOSIP.664 immunized rabbits (Fig. 3.3B), consistent with the reduced presentation of V3-epitopes (Table 3.2a and table 3.2b).

BG505 SOSIP.664 induces NAbs against the autologous neutralization resistant (Tier 2) BG505.T332N virus, something that has not been achieved by other gpl40 immunogens. The sera from BG505

SOSIP.v5 recipient animals, in particular from those receiving SOSIP.v5.2, were at least as efficient at neutralizing BG505.T332N as the sera from BG505 SOSIP.664 immunized animals (Fig. 3.3C). Thus, the substitutions present in the SOSIP.v5 trimers do not compromise the autologous Tier 2 NAb response. NAbs against neutralization-sensitive (Tier 1) isolates are dominated by V3- directed antibodies that cannot neutralize Tier 2 viruses, and might potentially distract from bNAb responses. Since the percentage of the V3 response is lower in the animals that received SOSIP.v5 trimers. Neutralization of the ultrasensitive (Tier 1A) SF162 virus was significantly reduced, especially for BG505 SOSIP.v5.2 immunized rabbits (Fig. 3.3C). A similar trend was observed with the Tier 1A MN.3 (SI Table X). We assessed the neutralization of the rabbit sera against a panel of heterologous Tier 2 viruses. Cross- neutralization against heterologous Tier 2 viruses was sporadic and weak but more prevalent in the BG505 SOSIP.v5 immunized animals compared to BG505 SOSIP.664 immunized rabbits (Table 3. S3 and 3.S4). The autologous NAb responses (median IC50 values) elicited by the different trimers were generally comparable (SOSIP.664, 4432; SOSIP.v4.1, 4503; SOSIP.v5.1 + SOSIP.v5.2 pooled, 3457; SOSIP.v6, 7798), except for the SOSIP.v5.2 201C-433C hyper-stabilized trimer (median IC50, 226; not significant compared to SOSIP.664) (Figure 3 4!)). Thus, with the possible exception of the I201C- A433C change to the SOSIP.v5.2 construct, the modifications used to create new, more stable trimers do not impair the induction of the autologous BG505.T332N NAb response.

One of the goals of trimer-stabilization projects is to reduce the antigenicity and immunogenicity of epitopes for non-NAbs and Tier-1 NAbs, the latter dominated by V3 -directed antibodies, and thereby focus the immune response on more productive targets. Compared to the BG505 SOSIP.664 trimers and, more so, the gpl20 monomers, NAb titers against the Tier-1 A SF162 virus were reduced by 3 -fold for the combined SOSIP.v5.1 and SOSIP.v5.2 groups (p=0.0175 vs. SOSIP.664) and were also lower for

SOSIP.V5.2 201C-433C (not statistically significant vs. SOSIP.664). In contrast, SF162 NAb titers were higher in the SOSIP.v6 group compared to SOSIP.664 and the combined SOSIP.v5 groups (p=0.0031 ). to the same level as the SF162 NAb titers induced by gpl20, possibly associated with the high thermostability and longer half-life of the SOSIP.v6 trimer in vivo (Figure 3.4G ). Sera from the various BG505 trimer-immunized rabbits were generally only weakly and sporadically active against a panel of heterologous Tier 2 viruses. However, the clade A virus 92RW and the clade B virus SHIV162P3 were occasionally neutralized in the titer range 40-200 (Figures 3.41 and 3.4K). Here, the most consistently immunogenic trimer was SOSIP.v6 , with median IC50 values against 92RW and SHIV162P3 of 46 and 66, respectively (p=0.0142 and p=0.0021, respectively, for SOSIP.v6 compared with the combined SOSIP.664, SOSIP.v4.1 and SOSIP.v5 groups ). For each individual rabbit, we analyzed the number of heterologous Tier-2 viruses that were neutralized with IC50 values >40.

Neutralization at this titer level was only sporadic for the SOSIP.664, SOSIP.v4.1 or SOSIP.v5 groups; the majority of the sera were inactive against all heterologous Tier-2 viruses. In contrast, sera from the SOSIP.v6 group neutralized one (n=2), two (n=l) or three (n=2) of the viruses in the test panel at a titer >40 (Figure 3.4H). When the SOSIP.v6 group was compared with the SOSIP.664, SOSIP.v4.1,

SOSIP.v5.1 + SOSIP.v5.2 and SOSIP.5.2 I201C-A433C groups, the number of heterologous Tier-2 viruses neutralized with IC50 values >40 was significantly higher (n = 10 for SOSIP.V6 vs. n = 0, 4, 4 and 3, respectively; Kruskal-Wallis p<0.001 in each case) (Figure 3.4H ).

Comparison and combination with other trimer stabilization approaches

The introduction of a disulfide bind between gpl20 and gp41 of different protomers (E49C and L555C) efficiently cross-linked the trimers and increased the thermostability (T_m = 75.2°C) of the trimer while decreasing the binding of CD4i V3-directed non-NAbs.

To compare these strategies with the one described here, and to investigate whether these strategies were compatible with each other, we introduced these mutations in BG505 SOSIP.664 and SOSIP.v5.2 and determined the antigenic profile and thermal stability of the proteins. The 4 trimers were purified to high homogeneity using PGT145-affinity chromatography. However, the yields differed substantially. Thus, the BG505 SOSIP.664 I201C-A433C and E49C-L555C yielded 0.1 and 0.2 mg/1, substantially lower than BG505 SOSIP.664 (1.6 mg/1) and SOSIP.v5.2 (2.9 mg/1). However, when the strategies were combined, the yields improved (BG505 SOSIP.v5.2 I201C-A433C: 1.3 mg/1; BG505 SOSIP.v5.2 E49C-L555C: 0.8 mg/1). Reducing SDS-PAGE and NS-EM analyses showed that all proteins were efficiently cleaved and assumed a closed native-like conformation. However, the migration patterns on non-reducing SDS-PAGE gels differed substantially. Based on non-reduced SDS-PAGE, we observed that BG505 SOSIP.v5.2 I201C-A433C ran slower than BG505 SOSIP.664 and BG505 SOSIP.v5.2, indicative of higher compactness due to the presence of three non-native disulfide bonds. Since the E49C-L555C disulfide bond forms between protomers (i.e. between gpl20 from one protomer and gp41 from another protomer), we observed a trimer band in the non-reducing SDS-PAGE gels when we analyzed BG505 SOSIP.664 E49C-L555C trimers. When we analyzed the BG505 SOSIP.v5.2 E49C-L555C trimers, we observed the presence of two subpopulations. Based on the comparison with the migration patterns of a number of control proteins, we assume that the slower variant, accounting for -60% of the protein, contains all three non-native disulfide bonds (A501C-T605C, A73C-A561C, E49C-L555C), while the other population, running faster, lacks the A73C-A561C bond. Probably the presence of the E49C-L555C bond prevents the A73C-A561C bond from forming in a subpopulation of the trimers.

The addition of the I201C-A433C disulfide bond to SOSIP.v5.2 did not alter the binding of most bNAbs to the trimer, but completely abrogated the binding of the CD4i non-NAb 17b even in the presence of sCD4. The addition of the E49C-L555C interprotomer disulfide bond to BG505 SOSIP.v5.2 improved the binding of quaternary-dependent bNAbs, in particular of VRC026 (see Table 3.2a and table 3.2b), while maintaining the low binding of V3 and CD4i- directed antibodies,(Table 3.2a and table 3.2b and Figure 3.S3f).

DSC experiments showed that the stability of the BG505 trimers containing multiple stabilizing disulfide bonds improved significantly. The combination of I201C-A433C (T_m = XX°C) with SOSIP.v5.2 (T_m = 75.3°C) increased the thermostability substantially (T_m = 80.7°C for the combination mutant). The same was true for the mutant combining the E49C-L555C interprotomer disulfide bond (T_m = 75.2°C) with SOSIP.v5.2 (T_m = 92.2°C for the majority population of the combination mutant). We note that a a substantial unfolding event was detected at of 78.8°C, probably stemming from the subpopulation in which the 73-561 disulfide bond was not formed). The T_m of 92.2°C represents a dramatic improvement in stability (i.e. by 24.6°C) compared to the original BG505 SOSIP.664 (T_m = 67.6°C).

Table 3.1 shows the biophysical characterization of SOSIP.v5 trimers from isolates BG505, AMC008, B41 and ZM197M. The percentage of native-like and closed native-like trimers based negative stain EM are listed. The 2D class averages are shown in Supplementary Fig. 3.3c. The T_m values for each construct were obtained by DSC using a two state model. The percentages of MangGlcNAc2 and Man9GlcNAc2 glycans, as well as the total percentage of oligomannose glycans are given for each trimer.

Table 3.2a and table 3.2b show the antigenic characterization of SOSIP.v5 trimers from isolates BG505, AMC008, B41 and ZM197M. Half-maximal binding concentrations (EC50, in μg/ml) were derived from Ni-NTA ELISAs. The values are representative of at least 2 independent experiments.

Table 3.3 shows the yield of the different disulfide bonds. Conclusions

We have described the generation of new SOSIP.664 trimers that are stabilized and antigenically improved by the introduction of a second disulfide bond between gpl20 and gp41. Until very recently the only successful disulfide between the two subunits was the one between residues 501-605 ("SOS"), which we identified over 15 years ago without structural information on the gpl20-gp41 interface. Here we find that many potential positions that are compatible with structure do not allow the formation of well-behaved disulfide bonds. One explanation for this is that the introduction of new disulfide bonds interferes with and oxidative folding and disulfide isomerization in the endoplasmic reticulum. Ironically, the gp41 partner of the cysteine bonds that we describe here and that yield native-like trimers are located in the domain of gp41 between a6 and a7 that was not resolved in the initial trimer structures and newer structures reveal that this domain might be dynamic and adopt multiple conformations. The finding that multiple residues in this domain can form disulfide bonds with 72C or 73 C yielding well-behaved trimers are consistent with this. Thus, unexpectedly, a poorly-structured subdomain of the trimer is very amendable for structural stabilization by introduction of disulfide bonds.

The new trimers described here provide new insights into Env dynamics, biophysics and structure.

Furthermore, they are promising candidates as vaccines. The improved presentation of bNAb epitopes and reduced exposure of potentially interfering non-NAb epitopes should improve the induction of the desirable bNAbs. Furthermore, the improved stability and hyperstability might enhance the in vivo half- life and prolong the presentation of bNAb epitopes to naive B cells. While vaccination experiments with BG505 SOSIP.664 trimers have yielded consistent NAb responses against the autologous neutralization- resistant (Tier-2) virus BG505.T332N, something never achieved by other gpl40 immunogens, the availability of similar quality trimers based on isolates from clades A, B and C allows for the formulation of multivalent vaccines to generate Tier-2 level neutralization breadth.

Table 3.1

Residues added to SOSIP.664 SOSIP version Morphology Stability (DSC) Glycan composition

Yield (mg L) Native-like Closed T_m (°C) AT_m (°C)

(%) nativelike o (%)

SOSIP.664 +++ >98 35 67,6 0,0 23 64

72C-564C +++ >98 70 71,9 4,3

73C-561C +++ >98 70 72,5 4,9

64 316W SOSIP.v4.1 +++ >98 70 70,7 3, 1 21 23 64

64 316W 306L 308L* ++ >98 55 72,6*

64K 316W 72C-564C SOSIP.V5.1 +++ >98 85 75,0 7,4

64 316W 73C-561C SOSIP.V5.2 +++ >98 90 75,3 7,7 21 23 64

201C-433C ++ >98 65 74,5 6,9

64K 316W 73C-561C 201C-433C ++ >98 85 80,7 13,1 23 19

49C-555C 75,2 7,6

64 316W 73C-561C 306L 308L* >98 85 78,1* 11,5

o 64 316W 73C-561C 49C-555C SOSIP.V6 ++ >98 50 78.8/92.2 11.2/24.6 25 25 69

SOSIP.664 +++ >98 15 60,2 0,0 14 34 67

66R 316W SOSIP.v4.1 +++ >98 85 64,5 4,3 12 39 69

66R 316W 72C-564C SOSIP.V5.1 ++ >98 100 68,3

66R 316W 73C-561C SOSIP.V5.2 >98 100 8,3 14 30 65 o

o 64 316W 73C-561C 201C-433C +++ 72,6 12,4

O

64 316W 73C-561C 49C-557C SOSIP.v6 >98 100 76,7 16,5

Table 3.1 (continued)

Table 3.3

Disulfide bond Yield

501-605 +++

51-574 + 51-578 ++ 53-564

53-573

53-574 +++

72-554 ++ 72-555 +

72- 564 +++

73- 558 +++ 73-561 +++ 73-567 +++ 73-568 +++ 107-557

113-557

113-567 ++

220- 568 + 221-568 +

221- 571

221-578 +

221-579 +

221- 582 + 222-571

222- 579

492-574

492-585 ++ SI Table 1

DLS SAXS

introduced mutations/SOSIP version

Rg (A) Rg (A)

R_h (AT Pd (%)

(Guinier (GNOM)^b

SOSIP.664 69.2 2.6 52.8 52.5

SOSIP H72C-H564C ND ND ND ND

SOSIP A73C-A561 C 68.8 2.0 52.8 51.7

SOSIP. v4.1 68.7 3.3 53.0 51.9

SOSIP.V5.1 67.8 3.6 52.1 51.7

SOSIP.V5.2 67.2 4.4 54.6 51.8 a Standard deviation differs in +/- 0.3-0.5 A

b Standard deviation is +/- 0.2

Example 4

An HIV-1 subunit vaccine should induce a broad and potent neutralizing antibody (NAb) response against the envelope glycoprotein spike (Env) [1]. Soluble, stable mimics of the native spike, such as the BG505 SOSIP.664 gpl40 trimer, might be good starting points for such a vaccine [2-5]. These trimers bind virtually all known broadly neutralizing antibodies (bNAbs) but almost no non-neutralizing antibodies (non-NAbs), and adopt a native-like conformation with a well-defined structure [2, 6-8]. Furthermore, unlike other gpl40 proteins, soluble, adjuvanted BG505 SOSIP.664 trimers induce NAbs against the autologous, neutralization-resistant (tier 2) virus efficiently in animals [9] . Licensed subunit vaccines against viral pathogens, such as hepatitis B and human papillomavirus, are however particulate antigens [10].

The greater size and the capacity for multivalent antigen presentation and B cell receptor cross-linking provide such particulate vaccines with advantages over soluble proteins for inducing antibody responses [11]. We fused the ferritin (H. Pylori ferritin (GenBank accession no. NP 223316)) N-terminus, starting from Asp5, to the SOSIP.664 C-terminus, separated by a Gly-Ser-Gly (GSG) linker (Fig. 4.1a). The SOSIP.664-ferritin plasmid was co-transfected into 293F cells with a furin plasmid to maximize trimer cleavage and ensure it adopts a native conformation [14]. To select for antigenically and structurally well- folded Env proteins, the secreted nanoparticles and control trimers were purified using PGT145 bNAb- affinity chromatography [15]. Judged by BN-PAGE and SDS-PAGE analysis followed by Coomassie staining this purification method yielded highly pure (>95 % purity) SOSIP.664 trimer and SOSIP.664- ferritin protein preparations (Fig. 4.1b). SDSPAGE also confirmed that the SOSIP.664 component of the nanoparticles was cleaved efficiently between gpl20 and gp41 (Fig. 4.1b, left panel).

The antigenic structure of SOSIP.664 trimers and SOSIP.664-ferritin was compared using ELISA.

Proteins were captured using Galanthus nivalis lectin and probed with bNAbs and nonNAbs (Fig. 4.1c). Several bNAbs that bind to distinct Env epitopes (VRCOl, PGT121, PG9) showed similar binding to SOSIP.664 and SOSIP.664- ferritin, moreover non-NAbs (F105 and F240) displayed similarly poor reactivity with both proteins (Fig. 4.1c). We did observe lower affinity of gpl20/gp41 interface

(8ANC195, 35022 and PGT151) and gp41 (3BC315) bNAbs for SOSIP.664-ferritin, which might be explained by steric hindrance of neighboring trimers on the nanoparticle (Fig. 4.1c). The purified nanoparticles were analyzed by negative stain electron microscopy (NS-EM). More than 70 % of the particles on the EM grid resembled ferritin cages with protruding spikes that were 30-40 nm in diameter (Fig. 4. Id). When single particles were automatically picked and processed as described elsewhere [2], 2D class averages representing views along the three- and fourfold symmetry axes suggested that 65- 80 % of the SOSIP.664- ferritin particles were fully decorated with Env trimers (three and four spikes visible, respectively) (Fig. 4.1e). The lack of views along the twofold symmetry axis (i.e. six spikes visible) may be a result of the immobilization on the EM grid or flexibility of the GSG-linker that affects the alignment of the particles and visualization of each Env trimer. We first immunized mice (approved by the AMC animal ethics committee: DMB-102836; n = 8 mice per group) to compare the antibody response of SOSIP.664- ferritin nanoparticles with soluble (i.e.

monovalent) SOSIP.664 trimers. he anti-trimer binding responses were eightfold higher in mice vaccinated with nanoparticle-displayed trimers compared to soluble trimers (medians: 86 vs. 686;

P = 0.015) (Fig. 4.2a). We next immunized rabbits (approved by the Covance Institutional Animal Care and Use Committee (IACUC): 0082-14; n = 5 rabbits per group), using a triple DNA-prime, protein- boost regimen (Fig. 4.2b). Given the limited group sizes and the large spread in neutralization titers generally observed in other HIV-1 vaccination studies [9], we included historic control sera from four rabbits to increase the statistical power of this study. These rabbits were immunized with the soluble trimers in an independent experiment using the same DNA prime + protein boost protocol (approved by the Covance IACUC: 0001-14; n = 4 rabbits per group; unpublished results). As expected, the anti-trimer binding antibody responses rose and fell between immunizations, and were boosted by the protein-only immunization [9, 16]. The titers were two- to threefold higher at several time points for the rabbits given SOSIP.664-ferritin nanoparticles compared to the soluble trimers. This shows the benefits of particulate antigen presentation [12, 17, 18] (Fig. 4.2b).

We used the TZM-bl cell neutralization assay and viruses from different clades to assess the serum NAb titers 2 weeks after the protein boost in rabbits [19]. Sera from 4/5 rabbits given the SOSIP.664-ferritin nanoparticles neutralized the autologous BG505.T332 N tier 2 virus, and the median titer in this group was higher than in the soluble trimer group (603 vs. 186). However, because of the small group sizes, the difference was not statistically significant (P = 0.34) (Fig. 4.2c). The NAb titers against heterologous tier 1 viruses were also higher in the rabbits that received SOSIP.664-ferritin nanoparticles (Fig. 4.2d).

Median NAb titers against tier 1A viruses were 10- to 90-fold higher in the nanoparticle group: MN.3 (4,857 vs. 282; P = 0.019); SF162 (2,799 vs. 31 ; P = 0.004); MW.965 (18,563 vs. 1,127; P = 0.019). For the more resistant tier IB viruses the titers were also higher, although this did not reach statistical significance in all cases: 6535.3 (472 vs. 82; P = 0.029); BaL (171 vs. 71 ; P = 0.083); DJ286.3 (195 vs. 64; P = 0.1 l).The tier IB viruses HxB2, Q23envl7, ZM109F and ZM197M and the tier 2 viruses 94UG103, 92RW020, Q259.d2.17, Q769. d22, Q842.dl2 (all clade A), YU2 (clade B) and Cel l76_ A3 (clade C) were not neutralized by any rabbit sera.

Conclusions We conclude from this exploratory study that the nanoparticle display of SOSIP.664 trimers improves the magnitude of the overall antibody response and neutralization breadth at the tier 1 level.

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EXAMPLE 5

Introduction

The rationale for research on an Env-based immunogens to induce bNAbs is supported by two observations. First, 10-30% of the HIV-1 infected individuals develop bNAb responses. The top 1%, termed "elite neutralizes", develop exceptionally potent and broad neutralizing responses against multiple viruses from different subtypes, and studying them might teach us how to achieve this by vaccination. Second, passive immunizations with bNAbs in non-human primates can block SHIV infection, even in low doses and after repeated challenges, and can lower plasma viraemia in chronically infected individuals and macaques. Although these observations fuel hope that the induction of bNAbs by vaccination is possible, multiple obstacles need to be overcome.

Monomeric gpl20 immunogens and trimers that do not mimic the native Env protein have not been successful at inducing NAbs against relatively neutralization-resistant (Tier-2) primary viruses.

Therefore, proteins that are better structural and antigenic mimics of the native Env spike are being explored with the aim to induce bNAbs. One such strategy involves the stabilization of the intersubunit interactions by an intermolecular disulfide bond between gpl20 and gp41 (SOS) supplemented with a trimer stabilizing mutation in gp41 (I559P; IP) and the truncation at position 664 to prevent MPER- mediated aggregation (.664). The resulting proteins are termed SOSIP.664 gpl40 proteins and over the last few years, SOSIP.664 proteins from multiple virus isolates from different HIV-1 subtypes have been described. These proteins closely resemble the native viral spike when visualized by negative stain electron microscopy (NS-EM), cryo-EM and crystallography. In addition, multiple epitopes for bNAbs are expressed, while those for non-NAbs are generally occluded. The SOSIP.664 proteins based on the BG505 (subtype A) and B41 (subtype B) strains developed Tier-2 autologous NAbs in rabbits and macaques, something not achieved previously with other types of gpl40 proteins. However, only sporadic and very weak heterologous Tier-2 NAb responses were observed. Further modifications on the SOSIP design, yielding SOSIP version 4 (SOSIP.v4) proteins, improved trimerization and trimer stability, and reduced the exposure of non-NAb V3- and CD4i-epitopes. In introduction of a second dilsufide bond, connecting CI of gpl20 with the HR1 region of gp41, further increased trimer stability. Furthermore, these modifications allowed the generation of SOSIP proteins from virus strains that do not form well- behaved native-like trimers otherwise. Here we describe a new SOSIP proteins based on subtype B env genes of recently transmitted HIV-1 variant from an individual participating in the Amsterdam Cohort Studies on HIV-1 infection and AIDS (ACS), infected through male homosexual contact. The participant developed an very broad and potent antibody response early and was classified as elite neutralizer. The AMC009 SOSIP is based on HTV-1 variants isolated two month after seroconversion. The recombinant protein formed stable trimers with a native-like conformation, exposing bNAb epitopes while occluding most non-NAb epitopes. Rabbit immunizations with the monovalent AMC009 induced neither NAbs against Tier 1 viruses nor against heterologous Tier-2 viruses. The corresponding AMC009 virus was classified as Tier 2/3 virus. A sequential immunization using AMC008, AMC009 and AMCOl 1 resulted in weak and sporadic heterologous Tier-2 neutralisation. A trivalent cocktail of these proteins yielded similar results. In contrast a tetravalent cocktail consisting of AMC008, AMC009, AMCOl 1 and B41 SOSIP proteins, induced consistent heterologous responses against several Tier-2 viruses.

These findings show that heterologous intrasubtype Tier-2 NAb responses can be induced using patient- specific Env sequences.

Material and Methods

ACS participant The env gene used here is derived from early clonal viruses isolated from HIV-1 subtype B infected ACS participant, infected via male homosexual contact (men who have sex with men; MSM). The rationale to use early sequences is based on the observation that the development of bNAbs might be driven by epitopes exposed on early viruses The elite neutralizer HI 8877, the source of AMC009 SOSIP, entered the ACS while being HIV-1 negative and seroconverted during active follow-up. Serum obtained from 31 months post-seroconversion (post-SC) neutralized a panel of different viruses with very high breadth and potency (geometric mean neutralization titer: GM ID50; 782). Individual HI 8877 was under observation for more than five years and had constant CD4+ T-cell numbers, had a measurable viral load and did not receive highly active anti-retroviral therapy (HAART). In addition, this individual had no protective HLA-type and was homozygous for the CCR5 gene. Construct design

For individual HI 8877 env genes from five clonal viral isolates (HI 8877.2m.1B5 (SEQ ID NO: 53), HI 8877.2m.1C3 (SEQ ID NO: 54), HI 8877.2m.1F9 (SEQ ID NO: 55), HI 8877.2m.1G1 (SEQ ID NO: 56) and H18877.2m.2Bl (SEQ ID NO: 57)) from 2 months post-SC were used to obtain a consensus sequence, using a cutoff >60% for an amino acid at a specific position (i.e. only amino acids that occurred in 3 out of 5 sequences were retained in the consensus sequence). This consensus sequence was used to generate the expression vector to make the AMC009 SOSIP protein. The genes encoding the gpl40 SOSIP constructs were designed as described previously. In short, we introduced the A501C and T605C substitutions, resulting in a stabilizing intermolecular disulfide bond between gpl20 and gp41 ectodomain (gp41ECTO); the trimer-promoting I559P, V/T535M, L543Q and Q567K substitutions in gp41ECTO; the hexa-arginine furin cleavage site at the C-terminus of gpl20 to enhance cleavage ; a tissue plasminogen activator (tPA) signal peptide replacing the natural Env signal peptide to improve secretion; a stop codon at position 664 to prevent aggregation (HXB2 numbering system). The resulting gpl40 SOSIP proteins based on the env genes of individuals HI 8877 was designated AMC009 SOSIP version 3.1 (SOSIP.v3.1). The resulting codon-optimized env genes were obtained from Genscript (Piscataway, NJ) and cloned into the pPPI4 expression vector. During the course of this study the SOSIP design was further improved (SOSIP.v4 and SOSIP.v5). Accordingly, we further introduced A316W and H66R, resulting in SOSIP.v4.2 proteins , and subsequently A73C and A561C thereby introducing a second disulfide bridge between gpl20 and gp41ECTO, resulting in SOSIP.v5.2 . For some analyses we introduced a D7324 epitope-tag sequences or a His-tag at the C-terminus of gp41ECTO, by introducing the sequence GSAPTKAKRRVVQREKR (SEQ ID NO: 187) or GSGSGGSG-HHHHHHHH (SEQ ID NO:200), after residue 664 in gp41ECTO and before the stop codon, referred to as SOSIP-D7324 and SOSIP-His, respectively. In this study we describe the results obtained with the v4.2 and v5.2 proteins. All experiments were performed using the untagged SOSIP proteins, except for ELISA (SOSIP-D7324), DSC (SOSIP-D7324) and SPR (SOSIP-His). Env protein expression by transient transfection

The SOSIP protein was expressed as described earlier herein.

Negative stain electron microscopy (NS-EM) and image processing

Purified SOSIP protein was analysed by NS-EM. A 3 μΐ aliquot containing ~5.5 μg/ml of a protein was applied for 5 s onto a carbon-coated 400 Cu mesh grid that had been glow discharged at 20 mA for 30 s, then negatively stained with 2% (w/v) uranyl formate for 60 s. Images were collected on either a Tietz

TemCam-F416 CMOS camera using a FEI Tecnai T12 electron microscope operating at 120 keV, with an electron dose of -25 e-/A2 and a magnification of 52,000x that resulted in a pixel size of 2.05A at the specimen plane, or a FEI Ceta 16M camera using a FEI Talos electron microscope operating at 200 keV, with an electron dose of -25 e-/A2 and a magnification of 73,000x that resulted in a pixel size of 1.98 A at the specimen plane. The nominal defocus range for all data negative stain data sets was -1.5x10-6 to - 2.0xl0-6m..

Glycan profiling

Glycan profiling was done as described previously. Briefly, the Env protein (5.5 μg) was resolved by SDS-PAGE under non-reducing conditions, followed by Coomassie blue staining. Bands corresponding to gpl40 were excised from the gels, washed and N- linked glycans were then released according to the manufacturer's instructions (NEB). The released glycans were subsequently eluted from gel bands, then dried using a SpeedVac concentrator and labelled with 2-aminobenzoic acid (2-AA) as previously described. Fluorescently labelled glycans were resolved by Hydrophilic interaction liquid

chromatography-ultra performance liquid chromatography (HILIC-UPLC) using a 2.1 mm x 10 mm Acquity BEH Amide Column (1.7 μιη particle size) (Waters, Elstree, UK). The following gradient was run: time = 0 min (t = 0): 22.0% A, 78.0% B (flow rate of 0.5 ml/min); t = 38.5: 44.1% A, 55.9% B (0.5 ml/min); t = 39.5: 100% A, 0% B (0.25 ml/min); t = 44.5: 100% A, 0% B (0.25 ml/min); t = 46.5: 22.0% A, 78.0% B (0.5 ml/min), t = 48: 22.0% A, 78.0% B (0.5 ml/min), where solvent A was 50 mM ammonium formate, pH 4.4, and solvent B was acetonitrile. Fluorescence was measured using an excitation wavelength of 250 nm and a detection wavelength of 428 nm. Data processing was performed using Empower 3 software.

The percentage abundance of oligomannose-type glycans was calculated by integration of the relevant peak areas before and after Endoglycosidase H digestion, following normalization. Digestions were performed on free glycans at 37°C for 16 h. The digested glycans were purified using a PVDF protein- binding membrane plate (Millipore) prior to HILIC-UPLC analysis.

D7324-capture ELISA for gpl40 SOSIP-D7324 proteins

Briefly, Microlon 96-wells plates (Greiner Bio-One, Alphen aan den Rijn, The Netherlands) were coated overnight with mAb D7324 (Alto Bioreagents, Dublin, Ireland) at 10 μ^πιΐ in 0.1 M NaHC03, pH 8.6 (50 μΐ/well). After washing and blocking steps, purified D7324-tagged SOSIP protein (2.75 μg/ml) was added in TBS/10% FCS for 2 h. Unbound proteins were washed away by 2 wash steps, and TBS (150 mM NaCl, 20 mM Tris) plus 2% skimmed milk was added to further block non-specific protein-binding sites. Serially diluted mAbs or CD4-IgG2 in TBS/2% skimmed milk were then added for 2 h followed by 3 washes with TBS. In some cases, soluble CD4 (sCD4; 1 μg/ml) was added during the incubation with a mAb. Horseradish peroxidase labeled goat-anti-human immunoglobulin G (IgG) (Jackson

Immunoresearch, Suffolk, England) was added for 1 h at a 1 :3000 concentration in TBS/2%> skimmed milk, followed by 5 washes with TBS/0.05% Tween-20. Colorimetric detection was performed using a solution containing 1% 3,3',5,5'-tetramethylbenzidine ( Sigma- Aldrich, Zwijndrecht, The Netherlands), 0,01%) H202, 100 mM sodium acetate and 100 mM citric acid. Color development was stopped using 0.8 M H2S04 after 5 min, and absorption was measured at 450 nm .

Surface Plasmon Resonance (SPR)

SPR analyses were performed on a Biacore 3000 instrument. Ab binding to immobilized proteins was performed as previously described with some modifications. All experiments were performed at 25oC. In all experiments HBS-EP (10 mM HEPES [pH7.4], 150 mM NaCl, 3 mM EDTA, 0.002% P20 surfactant) was used as running buffer (GE Healthcare) at maximum flow rate (50 μΐ/min) analyte injections to avoid mass transport limitation. At the end of each cycle, the sensor surface was regenerated by a single pulse with lOmM glycine (pH 2.0) for 120s. For the binding of a panel of Abs to BG505 SOSIP.v3.1, AMC009 SOSIP.v4.2, His-tagged proteins were captured at RL ~ 500-530 RU on anti-His CM5 sensor chips. IgGs of VRCOl, 3BNC60, PG16, PGT145, PGT121, PGT151, 35022, F105 and 19b were injected individually at 500 nM for 300 s and allowed to dissociate for 600s.

Rabbit immunizations

Rabbit immunizations with AMC009 SOSIP protein and blood sampling were carried out under subcontract at Covance (Denver, PA). Female New Zealand White rabbits (n=5 per group), were immunized intramuscularly with 22 μg of the AMC009 SOSIP protein, formulated in 75 Units of ISCOMATRIXTM. In addition, rabbit immunizations were performed with a cocktail of AMC008, AMC009 and AMCOl 1 SOSIP proteins. One group of animals was immunized with a total of 22 μg of the SOSIP proteins per animal per dose (low dose), 7.3 μg per individual SOSIP protein, while the other group received a total of 66 μg of the SOSIP proteins per animal per dose (high dose), 22 μg per SOSIP protein, formulated in 75 Units of ISCOMATRIXTM. Each group was immunized four times (at week 0, 4, 20 and 36) with the corresponding SOSIP.v4.2 protein, except the first, fourth and fifth groups which received the corresponding SOSIP.v5.2 proteins during the fourth immunization.

Another study assessed the immunogenicity of a tetravalent cocktail and a sequential immunization. For the later AMC008 v5.2, AMC009 v5.2, AMCOl 1 v5.2 and B41 v4.1 were administered sequentially at week 0, 4, 20 and 36, each of the five animals received 22 μg of protein. The five animals of the tetravalent cocktail group received a cocktail of the above mentioned proteins and week 0, 4, 20 and 38, with a total of 22 μg of SOSIP protein per animal.

TZM-bl neutralization assay and neutralization depletion experiments

The TZM-bl reporter cell line, stably expresses high levels of CD4 and the co-receptors CCR5 and CXCR4 and contains the luciferase and β-galactosidase genes under the control of the HIV-1 long- terminal-repeat promoter, and was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (John C. Kappes, Xiaoyun Wu, and Tranzyme Inc., Durham, NC). TZM-bl cell neutralization assays using viruses were performed as described previously. For additional information on the assay and all supporting protocols see: http://www.hiv.lanl.gov/content/nab- reference-strains/html/home.htm. A full-length molecular clone (pLAI) was used as the basis for the introduction of the parental AMC009 and AMCOl 1 Envs. To produce the viruses, HEK293T cells (2x 105) were seeded in a 6-well tissue culture plate (Corning) in 3 ml DMEM (Gibco) containing 10% FCS, penicillin (Sigma) and streptomycin (Sulphate-Gibco) (both at 100 U/ml) per well. After 1 day when the cells reached a confluence of 90-95% the cells were transfected using Lipofectamine 2000 (Invitrogen). Per well, 4 μg of molecular clone was mixed in 250 μΐ of OPTI-MEM (Gibco). 10-μ1

Lipofectamine 2000 was mixed with 240 μΐ of OPTI-MEM immediately before addition to the solution containing the expression plasmids and incubated for 20 min at RT. Then the transfection mixture was added to the cells, and the culture supematants were harvested after 48 h for infection and neutralization experiments. The molecular clone based on the AMC009 Env was not infectious and could not be used for neutralization experiments.

For the infection and neutralization experiments, TZM-bl cells were seeded in a 96-well plate in DMEM containing 10%> FCS, l x MEM nonessential amino acids, penicillin and streptomycin (both at 100 U/ml), and incubated at 37°C for 24 h in an atmosphere containing 5% C02. To determine neutralization activity of rabbit sera, a fixed amount of virus was incubated for one hour at RT with 1 :3 serial diluted heat- inactivated sera, starting with a serum dilution of 1 :20. Afterwards, the mix was added to the cells in the presence of 40 μg/ml DEAE-Dextran (Sigma) and 400 nM Sanquinavir, in a total volume of 200 μΐ. Three days after infection was initiated, the medium was removed and the cells were washed once with PBS and lysed in Reporter Lysis Buffer (Promega, Madison, WI). Luciferase activity was measured using a the Bright-Glo Luciferase Assay System (Promega, Madison, WI) and a Glomax Luminometer according to the manufacturer's instructions (Turner BioSystems, Sunnyvale, CA). Uninfected cells were used to correct for background luciferase activity. The infectivity of each mutant without inhibitor was set at 100%. Nonlinear regression curves were determined and the 50% inhibitory serum dose (ID50) was calculated using a sigmoid function in Prism software version 5.0.

Results

Design and biochemical characterization of the subtype B SOSIP protein Here, we describe the production and properties of a stable and homogenous SOSIP gpl40 protein that express multiple bNAb epitopes. This protein is based on subtype B env genes from recently transmitted clonal virus isolates from a MSM infected ACS participant. This participant is an elite neutralizer (HI 8877) and AMC009 SOSIP was derived from this individual. Various modifications were introduced in the sequence to initially create the AMC009 SOSIP.v3.1. During the course of this study several SOSIP design improvements became available. First, an A316W substitution in combination with either E64K or H664 (SOSIP.v4.1 and SOSIP.v4.2 respectively) improved trimerization and trimer stability, and reduced exposure and immunogenicity of non-NAb epitopes. Second, an additional disulfide bond between gpl20 and gp41 residues A73C and A561C provided additional stability. This disulfide bond was added to AMC009 SOSIP to create AMC009 SOSIP.v5.2. The same mutations were applied to AMCOl 1 v4.2 SOSIP, creating AMCOl 1 v5.2. We also prepared proteins containing a D7324- or His-tag immediately downstream from residue 664, permitting the oriented immobilization of the proteins on ELISA plates or on SPR chips. The presence of the tag had no influence on the properties of the AMC009 proteins.

The modified env genes were transiently transfected into 293F cells, together with the furin gene to enhance the cleavage between gpl20 and gp41ECTO. The secreted Env proteins were affinity-purified using bNAb PGT145. SDS-PAGE analyses performed under reducing and non-reducing conditions and stained with Coomassie blue showed that the purified SOSIP proteins were fully and efficiently cleaved into their gpl20 and gp41ECTO subunits (+DTT vs -DTT). Single gpl40 bands were observed under non-reducing conditions, showing that there were no inter-protomer disulfide -bonds. A Coomassie blue stained BN-PAGE showed trimers only, without any amounts of monomers, dimers or higher aggregates.

AMC009 SOSIP protein resembles the native Env trimer

We used negative stain electron microscopy (NS-EM) to study the morphology of the PGT145-purified SOSIP.v4.2 and v5.2 proteins. The reference-free 2D class averages revealed that the proteins adopted a native-like morphology. Using NS-EM, we have previously identified closed and partially open native- like trimers, reflecting conformational flexibility and heterogeneity within the native-like trimer populations. The partial open conformation, distinguishably different from nonnative-like trimers or the open CD4-bound conformation, is characterized by the presence of density at the distal end of one or more protomers and is probably an intrinsic feature of SOSIP proteins, as it is also observed for other SOSIP proteins. While the AMC009 SOSIP.v3.1 protein revealed a high proportion of native-like trimers in the partially open conformation (20 %) the SOSIP.v4 protein was predominantly in the closed conformation (SOSIP.v4.1 : 67%; SOSIP.v4.2: 87%), confirming that the A316W and E64K/H66R substitutions reduce spontaneous trimer opening.

To further study the 3D-dimensional structure we analyzed the AMC009 SOSIP.v4.2 in complex with bNAb PGV04 (added as Fab) by cryo-EM. We obtained 3D reconstructions at a resolution of 4.3 A (Fig. 3). Overlaying the structures with each other and that of BG505 SOSIP.664 (PDB: 4TVP) showed that the AMC009 SOSIP.v4.2 trimer assumed a native like conformation very similar to that of BG505 SOSIP.664. A notable difference was observed in the VI region. The AMC009 SOSIP.v4.2 protein has a longer VI region which assumed an helical conformation. However, we note that model building in this part of the protein was difficult, probably because of conformational flexibility. The glycan profile of AMC009 SOSIP protein is dominated by oligomannose glycans

To acquire information on the glycosylation profile of the AMC009 SOSIP.v4.2 protein we made use of HILIC-UPLC. Native virion-derived Env contains a large number of underprocessed oligomannose glycans. While uncleaved gpl40 proteins have a high percentage of processed glycans, native-like SOSIP trimers usually have a high oligomannose content, resembling the glycan composition of native Env. The underprocessed glycans on native virion-derived Envs are probably a consequence of the protection from processing enzymes, partially dependent on its tight and compact conformation. The percentages of underprocessed glycans, Man5-9GlcNAc2, were 72%> for v4.2 and 77%> for v5.2 . Notably, both versions Man8GlcNAc2 and Man9GlcNAc2 structures accounted for around half of the glycan population. These results show that the SOSIP proteins have a glycan profile that is comparable with virion-derived Envs. AMC009 SOSIP protein is thermostable

The differential scanning calorimetry (DSC) was used to measure the thermal stability of the PGT145- purified SOSIP proteins. For AMC009 SOSIP.v4.2 the DSC profile, obtained with a two-state model, showed a thermal denaturation midpoint (Tm) of 67.6°C. The proteins were also analyzed using a non- two state model, revealing three individual unfolding peaks. We obtained similar Tm values with a subset of PGT145-purified SOSIP-D7324 proteins. The Tm value of AMC009 is comparable with the BG505 SOSIP protein. The additional disulfide bridge between gpl20 and gp41ECTO did increase the thermostability of AMC009 SOSIP.v5.2 with 3.9°C (Tm = 70.3°C).

AMC009 SOSIP proteins react with multiple bNAbs To study the antigenicity of the different proteins, we conducted ELISA and SPR experiments with SOSIP.v4.2 protein and bNAbs and non-NAbs. We also performed ELISAs with SOSIP.v3.1,

SOSIP.v4.1 and SOSIP.v5.2 variants and the results obtained were comparable with those derived from SOSIP.v4.2 proteins. If they were not, they were consistent with the effect of the amino acid

modifications present in the different versions. An Env protein that resembles the native-like spike should bind most bNAbs, but not non-NAbs. First, we tested the binding of bNAbs that recognize the apex of the trimer and depend on the proper quaternary conformation of the protein. PG16 and PGT145 bound relatively weakly to AMC009 SOSIP. Next we tested the binding of bNAbs that target the CD4bs, the site at which the virus attaches to CD4+ target cells. CD4bs bNAbs VRCOl and 3BNC117 interacted similarly efficiently with AMC009 SOSIP protein. CD4 IgG2 bound strongly AMC009 SOSIP. PGT 121 and PGT128 are antibodies that bind to the N332-glycan V3 cluster. Binding of PGT128 was the same for all the SOSIP proteins, but the binding affinity of PGT121 to AMC009 SOSIP was higher compared to AMC016 and AMC018. Next we tested the binding affinity of 2G12 and PGT135, two antibodies that bind to the glycan outer domain cluster, also involving the N332 glycan. The binding affinity for 2G12 was almost the same for the SOSIP proteins. The binding affinity of PGT135 was the highest for

AMC009 SOSIP, substantially decreased for AMC016 SOSIP and more so for AMC018 SOSIP. bNAbs PGT151 and 35022, bind to the interface of gpl20 and gp41 and require proper quaternary structure. The AMC016 and AMC018 SOSIP proteins bound similarly to PGT151, while the binding of AMC009 SOSIP was decreased. The maximum ELISA signal obtained with PGT151 and AMC009 was low, probably related to fast dissociation (see below), and loss of signal during washing steps. We also observed differences in the binding affinity of 35022 to the different SOSIP proteins. AMC018 SOSIP bound relatively weak compared to the AMC009 and AMC016 SOSIP proteins.

In general, the SOSIP.v3.1 and v4.1 proteins bound bNAbs with similar affinities to their SOSIP.4.2, as observed previously for the comparison of SOSIP.v3.1 and SOSIP.v4 proteins based on other virus isolates. A notable exceptions was the improved binding of PGT 145 to AMC016 SOSIP.v4.2 vs.

SOSIP.v3.1 and the improved binding of PG16 to AMC018 SOSIP.4.2 vs. AMC018.v3.1. AMC009 SOSIP.V5.2, showed higher binding affinity with PGT145 and PGT151 compared to its SOSIP.v4.2 counterparts, suggesting that this version better recapitulates the epitopes for these trimer-preferring bNAbs at the apex of the trimer. We also tested the binding affinity of non-NAbs to the three SOSIP proteins. Non-NAbs cannot neutralize viruses because their epitopes are absent or not accessible on the native Env trimer. On native-like trimers non-NAb epitopes should be minimally exposed. In ITC, NS-EM and SPR assays, non-NAbs against the CD4bs and V3 do not interact efficiently with SOSIP trimers. However, binding of these non-NAbs to D7324-tagged SOSIP trimers can be readily detected in ELISA, although the reason remains to be understood. It is probable that the antigen capture in ELISA induces some local protein unfolding, resulting in the presentation of on a subset of proteins of some non-NAb epitopes. Alternatively, proteins captured on an ELISA plate might be more or flexible, allowing different conformations to be sampled over time in a way that registers more strongly in an ELISA than in other binding assays.

In agreement with previous studies on SOSIP.v4 modifications, we found that the non-NAbs b6 and F105 against the CD4bs, and 14e and 19b against the V3 epitope bound substantially less efficiently to the SOSIP.4.1 and SOSIP.v4.2 proteins compared to the SOSIP.v3.1 proteins. We observed binding of the AMC016 and AMC018 SOSIP.v3.1 proteins to the CD4i non-NAbs 17b and 412d in the absence of sCD4, but not to the SOSIP.v4 proteins, confirming the role of the v4 modifications in reducing spontaneous exposure of the CD4i-epitopes. Addition of sCD4 facilitated binding of both 17b and 412d to all trimers with one exception: 412d did not bind to AMC009, neither in the absence nor in the presence of sCD4, suggesting that the epitope for 412d is not available or present on the AMC009 SOSIP proteins. Binding of the non-NAbs b6 and 14e was weaker for the AMC009 SOSIP.v5.2 protein compared to the v4.2, which indicate that v5.2 occludes these epitopes more efficiently than v4.2.

To confirm the data obtained by ELISA using D7324-tagged AMC009 SOSIP.v4.2 trimers, we performed binding experiments with His-tagged AMC009 SOSIP.v4.2 trimers and a subset of bNAbs and non-NAbs using SPR, and compared them with BG505 SOSIP.664. The SPR data generally agreed well with the data obtained via ELISA (Fig. 4). Thus, PG16 and PGT145, the V1V2 quaternary-dependent bNAbs, bound with low and intermediate levels, respectively, with a slow dissociation rate. PGT121, a bNAbs against the V3 and outer domain glycan cluster, and VRCOl and 3BNC60, two CD4bs-directed bNAbs, bound very efficiently. The bNAbs PGT151 and 35022, both directed against the gpl20-gp41 interface and dependent on proper quaternary structure, both reacted very efficiently with AMC009 SOSIP.v4.2, although both the association and dissociation kinetics for PGT151 were rather high. The non-NAbs F105 and 19b were not reactive with AMC009 SOSIP.v4.2 in SPR assays.

Overall, we conclude that AMC009, AMC016 and AMC018 SOSIP.v4.2 proteins display most bNAb epitopes. The clonal AMC009 viruses are relatively neutralization-resistant (Tier-2)

To test the sensitivity for bNAbs of the parental autologous viruses, we constructed molecular clones, using the pLAI backbone, and incorporated the Env of AMC009. First, we tested the infectivity of the viruses. AMC009 proved not to be infectious. The latter observation is probably a consequence of the loss of amino acids (or a combination of amino acids) which happened during construction of a consensus sequence based on five clonal viral isolates. This might have been vital for the virus' infectivity. Instead the five clonal AMC009 env isolates from the earliest time point were introduced into pLAI backbone. Clone 1 and 4 were infections. Based on neutralization results clone 1 was further used in the

neutralization assays.

Next, we established the overall neutralization sensitivity of the AMC009 clone 1 virus. Tier

categorization, was based on the neutralization sensitivity to a panel of mAbs directed against various epitopes, as well as a panel of sera from HIV-1 infected humans. AMC009 was classified as relatively neutralization-resistant (Tier-2) viruses .

Monovalent AMC009 SOSIP trimers induce no autologous nor heterologous Tier-2 NAbs in rabbits

We performed four immunization studies to assess to immunogenicity of AMC009 SOSIP protein in rabbits (n=5 per group). The first study was performed to test whether monovalent AMC009 SOSIP would induce a heterologous Tier-2 response. The rabbits received four immunizations, at weeks 0, 4, 20, and 36, and the NAb responses were measured at weeks 22 and 38 (for immunization). SOSIP.v4.2 proteins were used for all immunizations with the exception of the fourth immunization, for which the SOSIP.v5.2 protein was used. One animal showed weak autologous response at week 38, another one neutralized with low titers REJO, a heterologous Tier-2 virus.

Heterologous NAbs responses were observed in almost all animals against the four easy-to-neutralize (Tier-1) viruses tested: namely SF162, BaL, AMC008 and ZM197M . Median NAb titers against the subtype B viruses SF162 and BaL (Tier-IA and Tier-IB, respectively) were lower for AMC016 SOSIP - immunized animals (median ID50 values of 73 and 80) compared to AMC009 and AMCOl 8 SOSIP recipients (median ID50 values of 364 and 135 for AMC009, and 998 and 136 for AMC018, for SF162 and BaL, respectively). We also observed high NAb titers against AMC008 (Tier-IB), a virus based on the Env sequence of another subtype B infected participant from the ACS. The NAb responses against AMC008 were generally higher for animals receiving AMC009 and AMCOl 6 immunogens (median ID50 values of 122 and 57) compared to AMCOl 8 SOSIP vaccinees (median ID50 of 47). Intersubtype neutralization of ZM109f (Tier-IB; subtype C) was weak in animals immunized with AMC016 and AMCOl 8 immunogens and virtually absent in AMC009 SOSIP recipients.

Next, we tested NAb responses against heterologous Tier-2 viruses. Most circulating primary HIV-1 isolates have a Tier-2 neutralization phenotype. An HIV-1 vaccine should ideally induce NAbs against such viruses, but no Env-based vaccine has been able to do so. We observed weak and sporadic heterologous NAb responses against REJO, WITO and SHIV162p3, three Tier-2 subtype B viruses, but one AMCOl 6 SOSIP recipient animal (UA0075) developed strong neutralization against REJO and SHIV162p3 (ID50 values of 386 and 357). We observed no heterologous NAb responses against three other Tier-2 subtype B viruses, AMCOl 1, 92BR020 and B41.

A trivalent cocktail of AMC008, AMC009 and AMCOl 1 SOSIP trimers induces Tier-2 NAbs in rabbits

The second rabbit immunization study was performed to assess whether a cocktail of SOSIP proteins (AMC008, AMC009, and AMCOl 1) would induce better antibody response compared to monovalent immunizations. For this study we only used SOSIP proteins derived from ACS individuals who developed elite bNAbs (AMC009 and AMCOl 1) and bNAbs (AMC008). The immunization regimen was similar as for the first study, but SOSIP.v5.2 proteins were used for all three strains in the fourth immunization. Furthermore, the first group of animals (n=5) received 22 μg protein in total per immunization (i.e. 7.3 μg of each component) while the second group (n=5) received 22 μg of each protein (i.e. 66 μg in total). All 10 animals developed NAb responses against the autologous Tier- IB AMC008 virus (median ID50 of 154), but fewer animals developed NAb responses against the autologous Tier-2 AMCOl 1 virus (5/10 responders; median ID50 of 74 for the responders). All animals induced strong NAb responses against SF162 (median ID50 of 770) and BaL (median ID50 of 283), and weaker responses against ZM109f (median ID50 of 56), and heterologous Tier-1 NAb responses were slightly higher in the high dose group.

Remarkably, we observed weak but consistent neutralization of the heterologous Tier-2 viruses REJO (8/10 animals; median ID50 for the responders: 55) and WITO (9/10 animals; median ID50 values for the responders: 52). Furthermore, 2 animals developed potent NAb responses against SHIV162p3 (UA0083: ID50 = 2847, UA0088: ID50 = 210), while another animal neutralized the subtype A virus BG505.T332N (UA0090: ID50 = 116). Although the heterologous Tier-2 NAb responses reported here remain relatively weak and sporadic, they are stronger and more frequent than those induced by monovalent vaccination using BG505, B41, AMC008, AMC009, AMC016, or AMC018 SOSIP proteins.

Discussion

One approach to induce bNAbs is based on the hypothesis that an HIV-1 immunogen that is a stable antigenic mimic of the native trimeric envelope spike, is better at inducing NAbs than other Env forms. Native-like trimers present most bNAb epitopes, and few potentially distractive non-NAb epitopes.

Furthermore, native-like trimers pose the appropriate constraints on the NAb approach angles. Over the last two years, multiple native-like proteins based upon different subtypes have been described, and here we report on three novel SOSIP trimers derived from early clade B isolates to add to this arsenal.

We described the design, structures, biochemical and biophysical properties, antigenicity and

immunogenicity of stable native-like SOSIP trimers derived from three subtype B infected ACS participants. We initially constructed SOSIP.v3.1 protein, but as soon as they became available we introduced the E64K and A316W or H66R and A316W substitutions, creating SOSIP.v4.1 and

SOSIP.v4.2 proteins, and subsequently the A73C and A561C substitutions, creating SOSIP.v5.2. In line with earlier studies using AMC008, BG505, B41 and ZM197M SOSIP proteins, these substitutions stabilized the proteins in their closed conformation, further occluding non-NAb epitopes and increasing their thermostability. The proteins showed a high percentage of underprocessed oligomannose glycans, which is typical of virus-derived Envs and soluble native-like Env trimers.

NAb responses against Tier-1 A viruses are dominated by V3 specificities and are not effective against Tier-2 viruses. It is possible that responses against the highly immunodominant V3 might distract from bNAb responses. The responses against such immunological decoys can be reduced by using SOSIP.v4 proteins instead of SOSIP.664 proteins. Here, we used SOSIP.v4.2 immunogens (or SOSIP.v5.2 in one case), which indeed minimized the exposure of the V3 epitopes in vitro.

To see whether the NAb response would increase and broaden when using a cocktail of immunogens, we immunized animals with AMC008, AMC009 and AMCOl 1 SOSIP proteins. AMC008 and AMCOl 1 are immunogens also derived from subtype B ACS participants, who developed bNAbs and elite bNAbs, respectively. The trivalent vaccine induced heterologous NAb responses against the Tier-2 subtype B viruses REJO, SHIV162p3 and WITO. These responses were significantly stronger than those induced by monovalent AMC008 or AMC009 SOSIP vaccination, although we note that monovalent AMCOl 1 SOSIP vaccination also resulted in some heterologous Tier-2 NAb induction. Our results therefore might suggest, that heterologous Tier-2 NAb response can be increased by using cocktail immunogens. We choose to formulate a trivalent cocktail based on three subtype B Env proteins and we observed cross- neutralization of subtype B viruses. In conclusion, by using stabilized immunogens derived from subtype B HIV-1 infected individuals from the ACS, we induced heterologous Tier-2 NAb responses. These responses were more frequent and stronger than those induced by BG505 and B41 SOSIP trimers. These results further validate the native-like trimer concept and encourage efforts to improve native-like trimers so that they can be tested in human trials.

HIV-1 Envelope sequences (SEQ IDN NO: 10-99) from elite neutralizers aligned to the HxB2 reference sequence (SEQ ID NO: l). "*" = Stop, and "X" = unknown amino acid residue

1 10 20 30 40 50

SEQ ID NO: :1 MRVKEKYQHL WRWGWRWGTM LLGMLMICSA TEKLWVTVYY GVPVWKEATT

SEQ ID NO: :10 MRVKEIRKNY QHF- -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :11 MRVKEIRKNY QHL- -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :12 MRVKEIRKNY QHL- -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :13 -XVMMY*EIX XHL- -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: 14 -XVXRXKEIX XHL- -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :15 RKIX XHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :16 -XSVT*RKIX XHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :17 -XAXKY*EIX XHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :18 -XAXKHTEIX XHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :19 MRVKEIRKNY QHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :20 MRVKEIRKNY QHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :21 XYX XHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :22 MRVKEIRKNY QHL' -WKGGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :23 MRVKEIRKNY QHL' -WKWGIL LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: 24 MRVKEIRKNY QHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :25 MRVKEIRKNY QHL' -WKWGIL LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :26 MRVKEIRKNY QHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :27 MRVKEIRKNY QHL' -WKWGIM LLGILMICSD AEQLWVTVYY GVPVWKEATT

SEQ ID NO: 28 MRVKEIRKNY QHL' -WKGGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :29 MRVKEIRKNY QHL' -WKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

51 60 70 80 90 100

SEQ ID NO: :1 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLVNVT ENFNMWKNDM

SEQ ID NO: :10 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLKNVT ENFNMWKNNM

SEQ ID NO: :11 TLFCASDARA YNTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNNM

SEQ ID NO: :12 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKSNM

SEQ ID NO: :13 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 14 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :15 TLFCASDARA HDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :16 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLKNVT ENFNMWKNDM

SEQ ID NO: :17 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :18 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :19 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :20 TLFCASDARA YDKEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :21 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :22 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEEVLKNVT ENFNMWKNDM

SEQ ID NO: :23 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEIVLKNVT ENFNMWKNDM

SEQ ID NO: 24 TLFCASDARA SDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :25 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEIVLENVT ENFNMWKNDM

SEQ ID NO: :26 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEIVLKNVT ENFNMWKNDM

SEQ ID NO: :27 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEIVLKNVT ENFNMWKNDM

SEQ ID NO: 28 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEEVLKNVT ENFNMWKNDM

SEQ ID NO: :29 TLFCASDARA YDTEIHNVWA THACVPTDPN PQEVVLKNVT ENFNMWKNNM 101 110 120 130 140 143

SEQ ID NO: :1 VEQMHEDIIS LWDQSLKPCV KLTPLCVSLK CTDLKNDTNT NSS

SEQ ID NO: :10 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLGNATNT NATNTTN

SEQ ID NO: :11 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLRNTTNT TSGNATNNNS

SEQ ID NO: :12 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLK CTDLRNATNT NATNTTS

SEQ ID NO: :13 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NATNTTS

SEQ ID NO: 14 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLGNATNT NATNTTS

SEQ ID NO: :15 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLGNATNT NATNATS

SEQ ID NO: :16 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NTTDTTS

SEQ ID NO: :17 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLGNATNT NTTNTNS

SEQ ID NO: :18 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NATNTTS

SEQ ID NO: :19 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLKNATNT NVTSTTS

SEQ ID NO: :20 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NATNTTS

SEQ ID NO: :21 VEQMHEDIIS LWDQSLKPCV KLTPLCVILN CTDWKNATNT NATKTTS

SEQ ID NO: :22 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTEWKNATNT NDTKTTS

SEQ ID NO: :23 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLGNATNT NATKTNS

SEQ ID NO: 24 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLGNATNT NATKTNS

SEQ ID NO: :25 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDLGNATNT NATKTNS

SEQ ID NO: :26 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NATKATS

SEQ ID NO: :27 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NATKTTS

SEQ ID NO: 28 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NDTKTTS

SEQ ID NO: :29 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLK CTDLRNATNT NATNTTR

144 153 163 173 183 191

SEQ ID NO: :1 SGRMIMEKGE IKNCSFNIST SIRGKVQKEY AFFYKLDI IP IDNDT- -TSY

SEQ ID NO: :10 SSRGTMEGGE IKNCSFNITT SMRDKVQKEY ALFYKLDVVP IKNDN- -TSY

SEQ ID NO: :11 NSGGIMEGGE MKNCSFNITT SMRDKVQKEY ALFYKLDVVP IEDDN- -TSY

SEQ ID NO: :12 SSRGTMEGGE IKNCSFNITT RIRDKVQKEY ALFYKLDVVP IGKDN- -TSY

SEQ ID NO: :13 SSMGTMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IEDDN- -TSY

SEQ ID NO: 14 SSWGTMEGGE IKNCSFNITT SMRDKVQKEY ALFYKLDVVP IDNDN- -TSY

SEQ ID NO: :15 NSGGTMEGGE IKNCSFNITT KMRDKVQKEY ALFYKLDVVP IGNDN- -TSY

SEQ ID NO: :16 SSRGTIEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IEEDN- -TSY

SEQ ID NO: :17 SSRGTMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IKDDN- -TSY

SEQ ID NO: :18 SSMGTMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IKDDN- -TSY

SEQ ID NO: :19 NSGGTMERGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP MEDDN- -TSY

SEQ ID NO: :20 SSMGTMEGGE IKNCSFNITT NIRDKVQKEY ALFYKLDVVP IGNDN- -TSY

SEQ ID NO: :21 SSMGTMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IEDDN- -TSY

SEQ ID NO: :22 SSMGTMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP MEDDN- -TSY

SEQ ID NO: :23 SSMGMMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IEDNN- -TSY

SEQ ID NO: 24 SSMGMMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IETDN- -TSY

SEQ ID NO: :25 SSMGMMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IEDNN- -TSY

SEQ ID NO: :26 SSMGTMEGGE IKNCSFNITT NMRDKVQKEY ALFYKLDVVP IDNDNDNTSY

SEQ ID NO: :27 SSMGTMEGGE IKNCSFNITT KMRDKVQKEY ALFYKLDVVP IDNDNDNTSY

SEQ ID NO: 28 SSMGTMEGGE IKNCSFNITT NMKDKVQKEY ALFYKLDVVP IEGEN- -TSY

SEQ ID NO: :29 SNRGTMEGGE IKNCSFNITT RIRDKVQKEY ALFYKLDVVP IEKDN- -TSY 192 201 211 221 23: 241

• I I I • I • · I I

SEQ ID NO I KLT SCNTSVI TQACPKVS FE PI PIHYCAPA GFAILKCNNK TFNGTGPCTN SEQ ID NO 10 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCNDK KFNGTGPCTN SEQ ID NO II RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 12 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCNDK KFNGTGPCTN SEQ ID NO 13 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 14 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCNDK KFNGTGPCTN SEQ ID NO 15 RLISCNTSVV TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 16 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 17 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 18 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 19 RLISCNTSVI TQACPKISFE PIPIHYCAPA GFAILKCKDK KFHGTGPCTN SEQ ID NO 20 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKNK KFNGTGPCTN SEQ ID NO 21 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 22 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 23 RLISCNTSVI TQACPKISFE PIPIHYCAPA GFAILKCNNK KFNGTGPCTN SEQ ID NO 24 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 25 RLISCNTSVV TQACPKISFE PIPIHYCAPA GFAILKCKDK KFNGAGPCTK SEQ ID NO 26 RLISCNTSVV TQACPKVSFE PIPIHYCAPA GFAILKCNDK KFNGTGPCTN SEQ ID NO 27 RLISCNTSVV TQACPKISFE PIPIHYCAPA GFAILKCNNK KFNGTGPCTN SEQ ID NO 28 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN SEQ ID NO 29 RLISCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCKDK KFNGTGPCTN

242 25: 26: 271 281 291

I I I I • · I I

SEQ ID NO: :1 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSVNFTDNA KT IIVQLNTS

SEQ ID NO: :10 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :11 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :12 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :13 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: 14 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :15 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :16 VSTVQCTHGI RPWSTQLLL NGSLAEEEIV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :17 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: :18 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :19 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :20 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNES

SEQ ID NO: :21 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: :22 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS

SEQ ID NO: :23 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: 24 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: :25 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: :26 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: :27 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: 28 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTNNA KI IIVQLNKS

SEQ ID NO: :29 VSTVQCTHGI RPWSTQLLL NGSLAEEEVV IRSANFTDNA KI IIVQLNKS 2 92 301 311 321 330 340

SEQ ID NO: :1 VEINCTRPNN NTRKRIRIQR GPGRAFVTIG K-IGNMRQAH CNISRAKWNN

SEQ ID NO: :10 VEINCTRPNN NTRKSIHI— GPGRAFYTTG EIIGDIRQAH CNISGTKWND

SEQ ID NO: :11 VEINCTRPNN NTRKSIHI— GPGRAFYTTG GIIGDIRQAH CNISGTEWND

SEQ ID NO: :12 VEINCTRPNN NTRKSIHI— GPGRAFYTTG EIIGDIRQAH CNISGTKWND

SEQ ID NO: :13 VEINCTRPNN NTRKSINI— GPGRAFWTTG EIIGDIRQAH CNISGTQWND

SEQ ID NO: 14 VEINCTRPNN NTRKSINI— GPGRAFWTTG EIIGDIRQAH CNISGTKWND

SEQ ID NO: :15 VEINCTRPNN NTRKSINI— GPGRAFWTTG GIIGDIRQAH CNISGPKWND

SEQ ID NO: :16 VEINCTRPNN NTRKSINM— GPGRAFWTTG DIIGDIRQAH CNISGTQWND

SEQ ID NO: :17 VEINCTRPNN NTRRSINI— GPGRAFWTTG EIIGDIRQAH CNISGTEWND

SEQ ID NO: :18 VEINCTRPNN NTRRSINI— GPGRAFWTTG EIIGDIRQAH CNISGTEWND

SEQ ID NO: :19 VEINCTRPNN NTRKSINM— GPGRAFWTTG DIIGDIRQAH CNISGTQWND

SEQ ID NO: :20 VEINCTRPNN NTRRSINI— GPGRAFWTTG EIIGDIRQAH CNISGTKWND

SEQ ID NO: :21 VEINCTRPNN NTKRSINI— GPGRAFWTTG DIIGDIRQAH CNISGPKWND

SEQ ID NO: :22 VEINCTRPNN NTRKSINI— GPGRAFWTTG EIIGDIRQAH CNISGPKWND

SEQ ID NO: :23 VEINCTRPNN NTRKSINI— GPGRAFWTTG GIIGDIRQAH CNISGPKWND

SEQ ID NO: 24 VEINCTRPNN NTKRSINI— GPGRAFWTTG DIIGDIRQAH CNISGPKWND

SEQ ID NO: :25 VEINCTRPNN NTRKSINI— GPGRAFWTTG DIIGDIRQAH CNISGPKWND

SEQ ID NO: :26 VEINCTRPNN NTRKSINI— GPGRAFWTTG DIIGDIRQAH CNISGTQWND

SEQ ID NO: :27 VEINCTRPNN NTRKSINI— GPGRAFWTTG DIIGDIRQAH CNISGTQWND

SEQ ID NO: 28 VEINCTRPNN NTRKSINI— GPGRAFWTTG EIIGDIRQAH CNISGPKWND

SEQ ID NO: :29 VEINCTRPNN NTRKSINM— GPGRAFWTTG GIIGDIRQAH CNISGPKWND

341 350 360 370 380 390

SEQ ID NO: :1 TLKQIASKLR EQFGNNKTII FKQSSGGDPE IVTHSFNCGG EFFYCNSTQL

SEQ ID NO: :10 TLKQIFVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :11 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :12 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCDSTPL

SEQ ID NO: :13 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: 14 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :15 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTPL

SEQ ID NO: :16 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :17 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :18 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTPL

SEQ ID NO: :19 TLKQIVVKLR EQFGN- ^■KTIV FNHSSGGDPE IVRHSFNCGG EFFYCDSTQL

SEQ ID NO: :20 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :21 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :22 TLKQIVVKLR EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :23 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: 24 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :25 TLKQIVVKLR EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :26 TLKQIVVKLR EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :27 TLKQIVVKLR EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: 28 TLKQIVVKLR EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL

SEQ ID NO: :29 TLKQIVVKLK EQFGN- ^■KTIV FNHSSGGDPE IVMHSFNCGG EFFYCNSTQL 391 400 410 418 428 438

SEQ ID NO: :1 FNSTWFNSTW STEGSNNTEG S—DTITLPC RIKQIINMWQ KVGKAMYAPP

SEQ ID NO: :10 FNSTW— — DTTGSNYTGT IVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :11 FNSTWTW — DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :12 FNSTW— — DTTGSNYTGT IVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :13 FNSTW— — DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: 14 FNSTW— — DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :15 FNSTW— — DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :16 FNSTW—N— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :17 FNSTW—N— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :18 FNSTW—N— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :19 FNSTW—N— DATGSNYTGT NYNGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :20 FNSTW—N— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :21 FNSTW—N— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :22 FNSTWTWN— DTTGSNYTGT NYTGTIVLPC RIKQFVNMWQ EVGKAMYAPP

SEQ ID NO: :23 FNSTWTWN— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: 24 FNSTW—N— DTTGSNYTGT NYTGTIVLPC RIKQFVNMWQ EVGKAMYAPP

SEQ ID NO: :25 FNSTWTWN— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: :26 FNSTWTWN— DTTGSNYTGT NYTGTIVLPC RIKQFVNMWQ EVGKAMYAPP

SEQ ID NO: :27 FNSTWTWN— DTTGSNYTGT NYNGTIVLPC RIKQIVNMWQ EVGKAMYAPP

SEQ ID NO: 28 FNSTWTWN— DTTGSNYTGT NYTGTIVLPC RIKQFVNMWQ EVGKAMYAPP

SEQ ID NO: :29 FNSTW—N— DTTGSNYTGT NYTGTIVLPC RIKQIVNMWQ EVGKAMYAPP

439 44£ ] 464 474 484 I I I I

SEQ ID NO I ISGQ IRCSSN ITGLLLTRDG SEI FRPGGGD MRDNWRSELY SEQ ID NO 10 IKGQIRCSSN ITGLILIRDG GTNRSE N TEIFRPGGGD MRDNWRSELY SEQ ID NO II IKGQIRCSSN ITGLILVRDG GKNESE N -EI FRPGGGD MRDNWRSELY SEQ ID NO 12 IRGQIRCSSN ITGLILIRDG GKNRSE-D— TEIFRPGGGD MRDNWRSELY SEQ ID NO 13 IKGQIRCSSN ITGLILIRDG GTNRSES—N TEIFRPGGGD MRDNWRSELY SEQ ID NO 14 IKGQIRCSSN ITGLILIRDG GMNRSES TEIFRPGGGD MRDNWRSELY SEQ ID NO 15 IKGQIRCSSN ITGLILIRDG GTNRSES—N TEIFRPGGGD MRDNWRSELY SEQ ID NO 16 IKGQIRCSSN ITGLILIRDG GRNRSESENN TEIFRPGGGD MRDNWRSELY SEQ ID NO 17 IKGQIRCSSN ITGLILIRDG GTNRSE-D-N TEIFRPGGGD MRDNWRSELY SEQ ID NO 18 IKGQIRCSSN ITGLILIRDG GRNRSE—NN TEIFRPGGGD MRDNWRSELY SEQ ID NO 19 IQGLIRCSSN ITGLILIRDG GRNRSESENN TEIFRPGGGD MRDNWRSELY SEQ ID NO 20 IKGQIRCSSN ITGLILIRDG GTNRSES TEIFRPGGGD MRDNWRSELY SEQ ID NO 21 IKGQIRCSSN ITGLILIRDG GRNRSESENN TEIFRPGGGD MRDNWRSELY SEQ ID NO 22 IQGLIRCSSN ITGLILIRDG GTNRSESENN TEIFRPGGGD MRDNWRSELY SEQ ID NO 23 IQGLIRCSSN ITGLILIRDG GTNRSES—S TEIFRPGGGD MRDNWRSELY SEQ ID NO 24 IKGQIRCSSN ITGLILIRDG GTNRSESENN TEIFRPGGGD MRDNWRSELY SEQ ID NO 25 IQGLIRCSSN ITGLILIRDG GTNRSES—N TEIFRPGGGD MRDNWRSELY SEQ ID NO 26 IQGLIRCSSN ITGLILIRDG GNNSSE—NN TEIFRPGGGD MRDNWRSELY SEQ ID NO 27 IQGLIRCSSN ITGLILIRDG GTNSSE—NN TEIFRPGGGD MRDNWRSELY SEQ ID NO 28 IKGQIRCSSN ITGLILIRDG GTNRSESENN TEIFRPGGGD MRDNWRSELY SEQ ID NO 29 IQGLIRCSSN ITGLILIRDG GRNRSESENN TEIFRPGGGD MRDNWRSELY 4 85 4 94 50 ^ 514 52 534

I • I I I

SEQ ID NO: :1 KYKVVKIEPL VVQREKRAVG IGALFLGFLG

SEQ ID NO: :10 KYKVVKIEPL GIAPTKAKRR VVQREKRAVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :11 KYKVVKIEPL GIAPTKAKRR VVQREKRAVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :12 KYKVVKIEPL GIAPTKAKRR VVQREKRAVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :13 KYKVVKIEPL GIAPTKAKRR VVQREKRTVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: 14 KYKVVKIEPL GIAPTKAKRR VVQREKRAAG IGAVFLGFLG AAGSTMGAVS

SEQ ID NO: :15 KYKVVKIEPL GIAPTKAKRR VVQREKRTVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :16 KYKVVKIEPL GIAPTKAKRR VVQREKRTVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :17 KYKVVKIEPL GIAPTKAKRR VVQREKRTVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :18 KYKVVKIEPL GIAPTKAKRR VVQREKRTVG IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :19 KYKVVKIEPL GIAPTKAKRR VVQREKRAA- IGALFLGFLG AAGSTMGAAS

SEQ ID NO: :20 KYKVVKIEPL GIAPTKAKRR VVQREKRAGG IGALFLGFLG AAGSTMGAAS

SEQ ID NO: :21 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :22 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :23 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: 24 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :25 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :26 KYKVVQIEPL GIAPTKAKRR VVQREKRA-G IGALFLGFLG AAGSTMGAAS

SEQ ID NO: :27 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: 28 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

SEQ ID NO: :29 KYKVVKIEPL GIAPTKAKRR VVQREKR—G IGAVFLGFLG AAGSTMGAAS

535 54 ^ 554 5 64 57 4 584

• · I I I I . I I I

SEQ ID NO: :1 MT LTVQARQL LSGIVQQQNN LLRAI EAQQH LLQLTVWG IK QLQARILAVE

SEQ ID NO: :10 LTVQARLL LSGIVQQQNN LLRAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :11 MTLTVQARLL LSGIVQQQNN LLRAIKAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :12 LTVQARLL LSGIVQQQNN LLKAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :13 LTVQARQL LSGIVQQQNN LLKAIEAQQN LLQLTVWGIK QLQARVLAVE

SEQ ID NO: 14 MTLTVQARLL LSGIVQQQNN LLRAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :15 LTVQARQL LSGIVQQQNN LLKAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :16 LTVQARQL LSGIVQQQNN LLRAIETQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :17 LTVQARQL LSGIVQQQNN LLKAIKAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :18 LTVQARQL LSGIVQQQNN LLKAIEAQQN LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :19 LTVQARLL LSGIVQQQNN LLKAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :20 LTVQARQL LSGIVQQQNN LLKAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :21 LTVQARQL LSGIVQQQNN LLKAIETQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :22 LTVQARQL LSGIVQQQNN LLKAIEAQQH MLQLTVWGIK QLQARVLAVE

SEQ ID NO: :23 LTVQARQL LSGIVQQQNN LLKAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: 24 LTVQARQL LSGIVQQQNN LLKAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :25 LTVQARQL LSGIVQQQNN LLKAIEAQQH MLQLTVWGIK QLQARVLAVE

SEQ ID NO: :26 LTVQARLL LSGIVQQQNN LLRAIEAQQH LLQLTVWGIK QLQARVLAVE

SEQ ID NO: :27 LTVQARQL LSGIVQQQNN LLKAIEAQQH LMQLTVWGIK QLQARVLAVE

SEQ ID NO: 28 LTVQARQL LSGIVQQQNN LLKAIEAQQH MLQLTVWGIK QLQARVLAVE

SEQ ID NO: :29 LTVQARQL LSGIVQQQNN LLKAI DAQQH LLQLTVWGIK QLQARVLAVE 585 594 604 614 62 4 634

SEQ ID NO: :1 RYLKDQQLLG IWGCSGKLIC TTAVPW ASW SNKSLEQIWN HTTWMEWDRE

SEQ ID NO: :10 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWN NMTWMEWERE

SEQ ID NO: :11 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWN NMTWMEWERE

SEQ ID NO: :12 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWN NMTWMEWERE

SEQ ID NO: :13 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: 14 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :15 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :16 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :17 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :18 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :19 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :20 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :21 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :22 RYLKDQQLLG ILGCSGKLIC TTAVPWNTSW SNKSYNQIWD SMTWMEWERE

SEQ ID NO: :23 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYDQIWD NMTWMEWERE

SEQ ID NO: 24 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

SEQ ID NO: :25 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYDQIWD NMTWMEWERE

SEQ ID NO: :26 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYDQIWD NMTWMEWERE

SEQ ID NO: :27 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYDQIWD NMTWMEWERE

SEQ ID NO: 28 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD SMTWMEWERE

SEQ ID NO: :29 RYLKDQQLLG IWGCSGKLIC TTAVPWNTSW SNKSYNQIWD NMTWMEWERE

635 64 4 65 664 67 4 684

I • I I I I I

SEQ ID NO 1 INNYTSLIHS LIEESQNQQE KNEQELLEL D KWAS LWNWFN ITNWLWYIKL

SEQ ID NO 10 IDNYTSLIYT LIEDSQNQQE KNEQELLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 11 IDNYTSLIYT LIEDSQNQQE KNEQELLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 12 IDNYTTLIYT LIEESQNQQE KNEQELLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 13 IDNYTSLIYT LIEKSQNQQE KNEQDLLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 14 IDNYTSLIYT LIEKSQNQQE KNEQELLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 15 IDNYTSLIYN LIEKSQNQQE KNEQDLLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 16 IDNYTSLIYT LIEDSQNQQE KNEQDLLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 17 IDNYTSLIYT LIEDSQNQQE KNEQDLLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 18 IDNYTSLIYT LIEKSQNQQE KNEQDLLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 19 IDNYTSLIYN LIEKSQNQQE KNEQDLLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 20 IDNYTSLIYT LIEKSQNQQE KNEQELLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 21 IDNYTNLIYT LIEDSQNQQE KNEQDLLELD KWASLWNWFD ITNWLWYIKI

SEQ ID NO 22 IDNYTSLIYT LIEDSQNQQE KNEQDLLELD KWASLWNWFD ITNWLWYIKI

SEQ ID NO 23 IDNYTNLIYN LIEKSQNQQE KNEQDLLELD KWASLWNWFD ITNWLWYIKI

SEQ ID NO 24 IDNYTSLIYT LIEDSQNQQE KNEQDLLELD KWASLWNWFD ITNWLWYIKI

SEQ ID NO 25 IDNYTNLIYN LIEKSQNQQE KNEQDLLELD KWASLWNWFN ITNWLWYIKI

SEQ ID NO 26 IDNYTSLIYN LIEKSQNQQE KNEQDLLELD KWASLWNWFD ITNWLWYIKI

SEQ ID NO 27 IDNYTSLIYN LIEKSQNQQE KNEQDLLELD KWASLWNWFD ITKWLWYIKI

SEQ ID NO 28 IDNYTSLIYT LIEDSQNQQE KNEQDLLELD KWASLWNWFD ITNWLWYIKI

SEQ ID NO 29 IDNYTSLIYT LIEDSQNQQE KNEQDLLELD KWASLWNWFD ITNWLWYIKI 685 694 704 714 724 734

• · I I . I I

SEQ ID NO: :1 FI MIVGGLVG LRIVFAVL SI VNRVRQGYS P LSFQTHLPT P RGPDRPEGIE

SEQ ID NO: :10 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTPLPT RGPDRPEGIE

SEQ ID NO: :11 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :12 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTPLPT RGPDRPEGIE

SEQ ID NO: :13 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: 14 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :15 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :16 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :17 MIVGGLIG LRIVFTVL SV VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :18 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :19 MIVGGLIG LRIVFAVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :20 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :21 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :22 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :23 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: 24 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :25 MIVGGLIG LRIVFAVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :26 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :27 MIVGGLIG LRIVFAVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: 28 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

SEQ ID NO: :29 MIVGGLIG LRIVFTVL SI VNRIRQGYS LSFQTLLPT RGPDRPEGIE

735 744 754 764 774 784

SEQ ID NO: :1 EEGGERDRDR SIRLVNGSLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :10 EEGGERDRDR SDRLVTGLLA LIWVDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :11 EEGGERDRDR SDRLVTGFLA LIWADLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :12 EEGGERDRDR SDRLVTGFLA LIWVDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :13 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: 14 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :15 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRNL LLIVTRIVEL

SEQ ID NO: :16 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :17 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :18 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :19 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :20 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :21 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :22 EEGGERDRDR SDRLVTGFLA LIWANLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :23 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: 24 EEGGERDRDR SDRLVTGFLA LIWNDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :25 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :26 EEGGERDRDR SDRLVTGFLA LIWADLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :27 EEGGERDRDR SDRLVTGFLA LIWDDLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: 28 EEGGERDRDR SDRLVTGFLA LIWADLRSLC LFSYHRLRDL LLIVTRIVEL

SEQ ID NO: :29 EEGGERDRDR SDRLVTGFLA LIWVDLRSLC LFSYHRLRDL LLIVTRIVEL 785 794 804 814 824 834

SEQ ID NO: :1 LGRRGWEALK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEWQ

SEQ ID NO: :10 LGRRGWGVLK YWWNLLQYWS QELRNSAVSL LNATAIAVAE GTDRAIEVLQ

SEQ ID NO: :11 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :12 LGRRGWGVLK YWWNLLQYWS QELRNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :13 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: 14 LGRRGWGVLK YWWNLLQYWS QELRNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :15 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVLQ

SEQ ID NO: :16 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :17 LGRRGWGVLK YWWNLLQYWS QELRNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :18 LGRRGWGVLK YWWNLLQYWS QELRNSAISL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :19 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL FNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :20 LGRRGWGVLK YWWNLLQYWS QELRNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :21 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :22 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :23 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL FNATAIAVAE GTDRVIEVSQ

SEQ ID NO: 24 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :25 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL FNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :26 LGRRGWGVLK YWWNLLQYWS QELRNSAISL FNATAIAVAE GTDRVIEVLQ

SEQ ID NO: :27 LGRRGWGVLK YWWNLLQYWS QELRNSAISL LNATAIAVAE GTDRVIEVLQ

SEQ ID NO: 28 LGRRGWGVLK YWWNLLQYWS QELKNSAVSL LNATAIAVAE GTDRVIEVSQ

SEQ ID NO: :29 LGRRGWGVLK YWWNLLQYWS QELRNSAVSL LNATAIAVAE GTDRVIEVSQ

835 844 854 857

SEQ ID NO I GACRAIRHIP RRIRQGLERI LL*

SEQ ID NO 10 RAFRAILHIP VRIRQGLERA LV*

SEQ ID NO II RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 12 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 13 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 14 RAFRAILHIP VRIRQGLERA LV*

SEQ ID NO 15 RTFRAILHVP VRIRQGLERA LV*

SEQ ID NO 16 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 17 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 18 RAFRAILHIP VRIRQGLERA LV*

SEQ ID NO 19 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 20 RAFRAILHIP VRIRQGLERA LV*

SEQ ID NO 21 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 22 RAFRAILHIP VRIRQGLERA LV*

SEQ ID NO 23 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 24 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 25 RAFRAILHVP VRIRQGLERA LV*

SEQ ID NO 26 RIFRAILHVP VRIRQGLERA LV*

SEQ ID NO 27 RTFRAILHVP VRIRQGLERA LV*

SEQ ID NO 28 RAFRAILHIP VRIRQGLERA LV*

SEQ ID NO 29 RAFRAILHVP VRIRQGLERA LV* 1 10 2 0 30 4 0 50

SEQ ID NO: :1 MRVKEKYQHL WRWGWRWGTM LLGMLMICSA TEKLWVTVYY GVPVWKEATT

SEQ ID NO: :30. . -MRVKGIRKN YQHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :31. . -MRVKGIRKN YQHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :32 -MRVKGIRKN YQHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :33 -MRVKGIRKN YQHLWKWGIM LLGILMICSA VEQLWVTVYY GVPVWKEATT

SEQ ID NO: :34 -MRVKGIRKN YQHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :35 -MRVKGIRKN YQ WGMM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :36 -MRVKGIRKN YQHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :37 -MRVKGIRKN YQHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :38 -MRVKGIRKN YQHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :39 -MRVKGIRKN YQHWWKWGMM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: 40 -XSDDGXXXI -SHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: 41 -XGDERXXEI -SHLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: 42 -XSDAXXXEI -SHLWKWGIM LLGILMICSA VEQLWVTVYY GVPVWKEATT

SEQ ID NO: :43 —EDEGIXKI -*HLWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

SEQ ID NO: :44 -XKNEGXXXI -SHWWKWGMM LLGILMICSA AERLWVTVYY GVPVWKEATT

SEQ ID NO: :45 -XSXEGIXXI -*HLWKWGIM LLGILMICSA VEQLWVTVYY GVPVWKEATT

SEQ ID NO: 46 —XSVKY*EI -*HLWKWGIM LLGILMICSA AEQLWVTVYY GVPVWKEATT

SEQ ID NO: :47 -XAMKGXXKI -SHLWKWGIM LLGILMICSA AERLWVTVYY GVPVWKEATT

SEQ ID NO: 48 —ECEGXXEI -SHLWKWGIM LLGILMICSA AGQLWVTVYY GVPVWKEATT

SEQ ID NO: 49 -XRNEGXXEI -*HLWKWGIM LLGILMICSA AERLWVTVYY GVPVWKEATT

SEQ ID NO: :50 -MRVKGIRKN YQHLWKWGIM LLGILMICSA AERLWVTVYY GVPVWKEATT

SEQ ID NO: :51 —XWWKWGMM LLGILMICSA AERLWVTVYY GVPVWKEATT

SEQ ID NO: :52 -MRVKGIRKN YQHWWKWGIM LLGILMICSA TEQLWVTVYY GVPVWKEATT

51 6 C ⁾ 7 0 80 9 C ⁾ 1 01 0

. . . . 1 . . . . 1 . . . . 1 . . . . 1

SEQ ID NO: :1 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLVNVT ENFNMWKNDM

SEQ ID NO: :30 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :31 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :32 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :33 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :34 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :35 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :36 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :37 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :38 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :39 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: 40 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: 41 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: 42 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :43 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :44 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :45 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: 46 TLFCASDARA YDTEVHNVWA THACVPTDPN PQEVVLKNVT ENFNMWKNDM

SEQ ID NO: :47 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: 48 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: 49 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :50 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :51 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM

SEQ ID NO: :52 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT EKFNMWKNNM 101 110 120 130 135 137

I I I I I I I I I I

SEQ ID NO:l VEQMHEDIIS LWDQSLKPCV KLTPLCVSLK CTDLK— — D

SEQ ID NO: 30 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNC- SEQ ID NO: 31 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT D

SEQ ID NO: 32 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT D

SEQ ID NO: 33 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT D

SEQ ID NO: 34 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT D

SEQ ID NO: 35 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT D R SEQ ID NO: 36 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTD

SEQ ID NO: 37 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT D G

SEQ ID NO: 38 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT D

SEQ ID NO: 39 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT E G

SEQ ID NO: 40 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT DGG—NTTDG SEQ ID NO: 41 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT DCVTSNCTDW

SEQ ID NO: 42 VEQMQEDI IS LWDQSLKPCV KLTPLCVTLN CTDCVTSNCT N G

SEQ ID NO: 43 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT DGG—NTTDG

SEQ ID NO: 44 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT DGG—NTTDG

SEQ ID NO: 45 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTLNCT DGG

SEQ ID NO: 46 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDWKNATNT NTTD-

SEQ ID NO: 47 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTSNCT D

SEQ ID NO: 48 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDRVTLNCT DRVTLNCTDQ

SEQ ID NO: 49 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTSNCT DCVTLNCTDR

SEQ ID NO: 50 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCVTSNCT DCVTSNCTDG SEQ ID NO: 51 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCAT-NCT D G

SEQ ID NO: 52 VEQMHEDIIS LWDQSLKPCV KLTPLCVTLN CTDCGT-NCT D G

138 144 154 164 174 184

SEQ ID NO: :1 TNTNSSS GRMIMEKGEI KNCSFNISTS IRGKVQKEYA FFYKLDI IPI

SEQ ID NO: :30 KNTTNTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA ILYKLDLVPI

SEQ ID NO: :31 NTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :32 NTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :33 NTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :34 NTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :35 GNTTNTTSSS WGK- ^■MEGGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :36 NTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :37 GNTTNTTSSS WGK- ^■MEGGEI KKCSFNVTTN IRDKVQKEYA FFYKLDLVPI

SEQ ID NO: :38 NTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :39 GNTTNTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: 40 GNTTNVTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: 41 GNTTNTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: 42 GNTTNTTSSS WGK- ^■MEGGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :43 GNTTNITSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :44 GNTTNITSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :45 -NTTNTTSSS WGK- ^■MEGGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: 46 TTSSS RGTI :-EGGEI KNCSFNITTN MRDKVQKEYA LFYKLDVVPI

SEQ ID NO: :47 NTTSSS WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: 48 GNTTNTTSSS WGK- ^■MERGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: 49 GNTTNTTSSI WGK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :50 GNTTNTTSSS WGK- ^■MEEGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :51 GNTTNTNSSS WEK- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA VFYKLDLVPI

SEQ ID NO: :52 GNTTNTNSSS WGE- ^■MEKGEI KNCSFNVTTN IRDKVQKEYA TFYKLDLVPI 185 191 192 197 207 217 227

SEQ ID NO: :1 DNDTTSY KLTSCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :30 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :31 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :32 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :33 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :34 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :35 KNDTASY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :36 KNDTDSY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :37 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :38 KNDTASY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :39 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: 40 KNDTDSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: 41 KNDTASY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: 42 KNDTASY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :43 KNDTASY RIINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :44 QNDTDSYKND NASYRLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :45 KNDTASY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: 46 EEDNTSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :47 KNDTASY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: 48 KNDTNSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: 49 KNDTASY RLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :50 KNDTASY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :51 KNDTNSY RLISCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

SEQ ID NO: :52 KNDNGSYKNN TASYRLINCN TSVITQACPK VSFEPIPIHY CAPAGFAILK

228 237 247 257 267 277

SEQ ID NO: :1 CNNKTFNGTG PCTNVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSVNF

SEQ ID NO: :30 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :31 CKDKKFNGTG PCTNVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :32 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :33 CKDKKFNGTG PCTNVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :34 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :35 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :36 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :37 CKDKKFNGTG PCTNVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :38 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :39 CKDKKFNGTG PCAKVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: 40 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: 41 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: 42 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :43 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :44 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :45 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: 46 CKDKKFNGTG PCTNVSTVQC THGIRPVVST QLLLNGSLAE EEIVIRSANF

SEQ ID NO: :47 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: 48 CKDKKFNGTG PCTNVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: 49 CKDKKFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :50 CKDKKFNGTG PCAHVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :51 CKDKKFNGTG PCTNVSTVQC THGIRPVVST QLLLNGSLAE EEVVIRSENF

SEQ ID NO: :52 CNDKEFNGTG PCANVSTVQC THGIRPVVST QLLLNGSLAE EEWTRSENF 2 78 28 7 2 97 307 31 7 32 6

SEQ ID NO: :1 TDNAKTIIVQ LNTSVEINCT RPNNNTRKRI RIQRGPGRAF VTIG-KIGNM

SEQ ID NO: :30 TNNAKIIIVQ LNESVKINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: :31 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: :32 TNNAKIIIVQ LNESVKINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: :33 TNNAKIIIVQ LNGSVEINCT RPNNNTRKGI HI- -GPGRAF YTTGGIIGDI

SEQ ID NO: :34 TNNAKIIIVQ LNESVKINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: :35 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: :36 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: :37 TNNAKIIIVQ LNEYVEINCT RPNNNTRKGI HI- -GPGRAV YTTGAIIGDI

SEQ ID NO: :38 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAI YTTGKIIGDI

SEQ ID NO: :39 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: 40 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAI YTTGKIIGDI

SEQ ID NO: 41 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAI YTTGKIIGDI

SEQ ID NO: 42 TNNAKIIIVQ LNKSVEINCT RPNNNTRKGI HI- -GPGRAI YTTGKIIGDI

SEQ ID NO: :43 TNNAKIIIVQ LNEPVEINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: :44 TNNAKVIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAF YTTGAIIGNI

SEQ ID NO: :45 TNNAKIIIVQ LNGSVEINCT RPNNNTRKGI HI- -GPGRAF YTTG-IIGDI

SEQ ID NO: 46 TDNAKIIIVQ LNKSVEINCT RPNNNTRKSI NM- -GPGRAF WTTGDIIGDI

SEQ ID NO: :47 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAI YTTGKIIGDI

SEQ ID NO: 48 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAF YTTGDIIGDI

SEQ ID NO: 49 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAI YTTGKIIGDI

SEQ ID NO: :50 TNNAKIIIVQ LNESVEINCT RPNNNTRKGI HI- -GPGRAF YTTG-IIGDI

SEQ ID NO: :51 TNNAKIIIVQ LKKSVEINCT RPNNNTRKGI HI- -GPGRAF YTTG-IIGDI

SEQ ID NO: :52 TNNAKIIIVQ LNGSVEINCT RPNNNTRKGI HI- -GPGRAF YTTG-IIGDI

327 33 6 34 6 356 366 37 6

SEQ ID NO: :1 RQAHCNISRA KWNNTLKQIA SKLREQFGNN KTIIFKQSSG GDPEIVTHSF

SEQ ID NO: :30 RQAHCNLSRT KWNDTLKRW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: :31 RQAHCNLSRT KWNVTLKRW VKLREQFRN- KTIVFKQSSG GDPEIVMHNF

SEQ ID NO: :32 RQAHCNLSRT KWNDTLKRW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: :33 RQAHCNLSRT KWNDTLKQVA VKLREQFRN- KTIVFKQSSG GDPEIVMHNF

SEQ ID NO: :34 RQAHCNLSRT KWNDTLKRW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: :35 RQAHCNLSRT KWNDTLKQW VKLREQFRN- KTIVFKQSSG GDPEIVMHNF

SEQ ID NO: :36 RQAHCNLSRT KWNNTLKRW VKLREQFRN- KTIVFNQSSG GDPEIVMHNF

SEQ ID NO: :37 RQAHCNLSRT KWNDTLKRW VKLREQFRN- KTIVFKQSSG GDPEIVMHNF

SEQ ID NO: :38 RQAHCNLSRT KWNDTLKQW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: :39 RQAHCNLSRT KWNDTLKRW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: 40 RQAHCNLSRT KWNDTLKQW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: 41 RQAHCNLSRT KWNDTLKQW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: 42 RQAHCNLSRT KWNDTLKQW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: :43 RQAHCNLSRT KWNDTLKGW VKLREQFRN- KTIVFKQSSG GDPEIVMHNF

SEQ ID NO: :44 RQAHCNLSRT KWNDTLKQVA VKLREQFRN- KTIVFKQSSG GDPEIVMHSF

SEQ ID NO: :45 RQAHCNLSRT KWNDTLKRW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: 46 RQAHCNISGT QWNDTLKQIV VKLKEQFGN- KTIVFNHSSG GDPEIVMHSF

SEQ ID NO: :47 RQAHCNLSRT KWNDTLKQW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: 48 RQAHCNLSRT KWNNTLKRW VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: 49 RQAHCNLSRT KWNDTLKQVA VKLREQFRN- KTIVFNQSSG GDPEIVMHSF

SEQ ID NO: :50 RQAHCNLSRT KWNDTLKQVA VKLREQFRN- KTIVFKQSSG GDPEIVMHSF

SEQ ID NO: :51 RQAHCNLSRT KWNDTLKQVA VKLRKQFRN- KTIVFKQSSG GDPEIVMHSF

SEQ ID NO: :52 RQAHCNLSRT KWDDTLKQVA VKLREQFRD- KTIVFKQSSG GDPEIVMHSF 377 386 396 406 414 424

SEQ ID NO: :1 NCGGEFFYCN STQLFNSTWF NSTWSTEGSN NTEGS- -DTI TLPCRIKQII

SEQ ID NO: :30 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :31 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :32 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :33 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :34 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :35 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :36 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :37 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :38 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :39 NCGGEFFYCN TTQLFNSTW- NGI-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: 40 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: 41 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: 42 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :43 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :44 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :45 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: 46 NCGGEFFYCN STQLFNSTW- NDT-- --TGSN YTGTNYTGTI VLPCRIKQIV

SEQ ID NO: :47 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: 48 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: 49 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :50 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :51 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

SEQ ID NO: :52 NCGGEFFYCN TTQLFNSTW- NGT-- --EVLN NTGGN- -DTI VLPCRIKQII

425 434 444 454 462 470

SEQ ID NO: :1 NMWQKVGKAM YAPPISGQIR CSSNITGLLL TRDGGNS-N- —NESEIFRP

SEQ ID NO: :30 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNS--G TKNETEIFRP

SEQ ID NO: :31 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNNRNG TENETEIFRP

SEQ ID NO: :32 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNS--G TKNETEIFRP

SEQ ID NO: :33 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNNRNG TENETEIFRP

SEQ ID NO: :34 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNS--G TKNETEIFRP

SEQ ID NO: :35 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNNRNG TENETEIFRP

SEQ ID NO: :36 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNNRNG TENETEIFRP

SEQ ID NO: :37 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNNRNG TENETEIFRP

SEQ ID NO: :38 NMWQEVGKAM YAPPISGKIR CSSNITGIIL TRDGGNNQS- ETEIFRP

SEQ ID NO: :39 NMWQEVGKAM YAPPISGKIR CSSNITGIIL TRDGGNNQS- ETEIFRP

SEQ ID NO: 40 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNG TENETEIFRP

SEQ ID NO: 41 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNG TENETEIFRP

SEQ ID NO: 42 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNG TENETEIFRP

SEQ ID NO: :43 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNG TENETEIFRP

SEQ ID NO: :44 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNG TENETEIFRP

SEQ ID NO: :45 NMWQEVGKAM YAPPISGKIR CSSNITGIIL TRDGGNNQS- ETEIFRP

SEQ ID NO: 46 NMWQEVGKAM YAPPIKGQIR CSSNITGLIL IRDGGRNRSE SENNTEI FRP

SEQ ID NO: :47 NMWQEVGKAM YAPPISGQIR CSSNITGIIL TRDGGNNRNG TENETEIFRP

SEQ ID NO: 48 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNG TENETEIFRP

SEQ ID NO: 49 NMWQEVGKAM YAPPISGKIR CSSNITGIIL TRDGGNNQS- ETEIFRP

SEQ ID NO: :50 NMWQEVGKAM YAPPISGLIR CSSNITGIIL TRDGGNNES- ETEIFRP

SEQ ID NO: :51 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNG TGNETEI FRP

SEQ ID NO: :52 NMWQEVGKAM YAPPISGIIR CSSNITGIIL TRDGGNNQNE TKNETEIFRP 4 71 48 0 4 90 500 51 0 520

SEQ ID NO: :1 GGGDMRDNWR SELYKYKVVK IEPLGVAPTK AKRRWQREK RAVGIGALFL

SEQ ID NO: :30 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :31 GGGDIRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :32 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :33 GGGDIRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :34 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :35 GGGDIRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :36 GGGDIRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :37 GGGDIRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :38 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :39 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: 40 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: 41 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: 42 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :43 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :44 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :45 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: 46 GGGDMRDNWR SELYKYKVVK IEPLGIAPTK AKRRWQREK RTVGIGAVFL

SEQ ID NO: :47 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: 48 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: 49 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :50 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAVFL

SEQ ID NO: :51 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

SEQ ID NO: :52 GGGDMRDNWR SELYKYKVVQ IEPLGVAPTK AKRRWQREK RAVGLGAMFL

521 53 C ⁾ 54 0 550 560 571 0

SEQ ID NO: :1 GFLGAAGSTM GAASMTLTVQ ARQLLSGIVQ QQNNLLRAIE AQQHLLQLTV

SEQ ID NO: :30 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :31 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :32 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :33 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :34 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :35 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :36 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVR QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :37 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :38 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :39 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: 40 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: 41 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: 42 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :43 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :44 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :45 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: 46 GFLGAAGSTM GAASMTLTVQ ARQLLSGIVQ QQNNLLRAIE TQQHLLQLTV

SEQ ID NO: :47 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: 48 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: 49 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :50 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :51 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVQ QQNNLLKAIE AQQHLLQLTV

SEQ ID NO: :52 GFLGAAGSTM GAASVTLTVQ ARQLLSGIVR QQNNLLKAIE AQQHLLQLTV 571 580 590 600 610 620

• · I I I I I I • · I I

SEQ ID NO: :1 WG IKQLQARI LAVERYLKDQ QLLGIWGCSG KL ICTTAVPW NASWSNKSLE

SEQ ID NO: :30 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :31 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :32 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :33 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :34 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :35 IKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :36 IKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :37 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :38 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :39 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: 40 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: 41 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: 42 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :43 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :44 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :45 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: 46 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NTSWSNKSYN

SEQ ID NO: :47 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: 48 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: 49 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :50 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :51 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLN

SEQ ID NO: :52 WGIKQLQARV LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLD

621 630 640 650 660 670

SEQ ID NO: :1 QIWNHTTWME WDREINNYTS LIHSLIEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :30 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :31 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :32 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :33 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :34 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :35 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :36 KIWDNMTWME WEREINNYTD LIYTLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :37 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :38 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :39 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: 40 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: 41 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: 42 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :43 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :44 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :45 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: 46 QIWDNMTWME WEREIDNYTS LIYTLIEDSQ NQQEKNEQDL LELDKWASLW

SEQ ID NO: :47 KIWDNMTWME WEREINNYTD LIYTLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: 48 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: 49 KIWDNMTWME WEREINNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :50 KIWDNMTWME WEREIDNYTD LIYNLLEESQ NQQEKNEQEL LELDKWASLW

SEQ ID NO: :51 KIWENMTWME WEREINNYTD LIYNLLEESQ TQQEKNEQEL LELDKWASLW

SEQ ID NO: :52 QIWDNMTWME WEREINNYTG FIYTLLEESQ NQQEKNEQEL LELDKWASLW 671 68 0 690 7 00 71 0 720

I I I I I I I I I

SEQ ID NO 1 NWFN ITNWLW YIKLFIMIVG GLVGLRIVFA VLSIVNRVRQ GYSPLSFQTH SEQ ID NO 30 NWFDITNWLW YIKIFIMIVG GLVGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 31 NWFDITNWLW YIKIFIMIVG GLVGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 32 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 33 NWFDITNWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 34 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 35 NWFDITNWLW YIKIFIMIVG GLVGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 36 NWFDITNWLW YIKIFIMIVG GLVGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 37 NWFDITKWLW YIKIFVMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 38 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 39 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 40 NWFDITNWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 41 NWFDITNWLW YIKIFIMVVG GLVGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 42 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 43 NWFDITNWLW YIKIFIMIVG GWVGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 44 NWFDITNWLW YIKIFIMIVG GLVGLRIVFT VLSIVNRVRQ GYSPLPFQTP SEQ ID NO 45 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 46 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRIRQ GYSPLSFQTL SEQ ID NO 47 NWFDITNWLW YIKIFIMIVG GLVGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 48 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 49 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 50 NWFDITNWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 51 NWFDITKWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP SEQ ID NO 52 TWFDITNWLW YIKIFIMIVG GLIGLRIVFT VLSIVNRVRQ GYSPLSFQTP

721 730 74 0 750 7 60 77 0

SEQ ID NO: :1 LPTPRGPDRP EGIEEEGGER DRDRS IRLVN GSLALIWDDL RSLCLFSYHR

SEQ ID NO: :30 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :31 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :32 RPAPRGPDRP EGTEEEGGER DKDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :33 RPAPRGPDRP EGTEEEGGER DRDTSSQLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :34 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :35 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :36 RPAPRGPDRP EGIEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :37 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :38 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :39 RPAPRGPDRP EGTEEEGGER DRDTSSQLVD GFLAIIWVDL RSLCLFGYHR

SEQ ID NO: 40 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: 41 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: 42 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :43 RPAPRGPDRP EGTEEEGGER DRDTSSQLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :44 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :45 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: 46 LPTPRGPDRP EGIEEEGGER DRDRSDRLVT GFLALIWDDL RSLCLFSYHR

SEQ ID NO: :47 RPAPRGPDRP EGTEEEGGER DRDTSSQLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: 48 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: 49 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :50 RPAPRGPDRP EGTEEEGGER DRDTSSQLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :51 RPAPRGPDRP EGTEEEGGER DRDTSSQLVD GFLAIIWVDL RSLCLFSYHR

SEQ ID NO: :52 RPAPRGPDRP EGTEEEGGEK DRDTSSHLVD GFLAIIWVDL RSLCLFSYHR 771 780 790 800 810 820

SEQ ID NO: :1 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVSLLNATAI

SEQ ID NO: :30 LRDLLLIVTR IVELLGRRGW EALRYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :31 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :32 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :33 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :34 LRDLLLIVTR IVELLGRRGW EALRYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :35 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :36 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :37 LRDLLLIVTR IVELLGRRGW EALRYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :38 LRDLLLIVTR IVELLGRRGW EALKYWGNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :39 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: 40 LRDLLLIVTR IVELLGRRGW EALKYWGNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: 41 LRDLLLIVTR IVGLLGRRGW EALKYWWNLL QYWGQELKNS AVNLLNTIAI

SEQ ID NO: 42 LRDLLLIVTR IVGLLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :43 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :44 LRDLLLIVTR IVGLLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :45 LRDLLLIVTR IVELLGRRGW EALKYWGNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: 46 LRDLLLIVTR IVELLGRRGW GVLKYWWNLL QYWSQELKNS AVSLLNATAI

SEQ ID NO: :47 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: 48 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: 49 LRDLLLIVTR IVEFLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :50 LRDLLLIVTR IVELLGRRGW EALKYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :51 LRDLLLIVTR IVELLGRRGW EALRYWWNLL QYWSQELKNS AVNLLNTIAI

SEQ ID NO: :52 LRDLLLIVTR IVELLGRRGW EALKYWGNLL QYWSQELKNS AVNLLNTIAI

821 830 840 850 857

SEQ ID NO: :1 AVAEGTDRVI EWQGACRAI RHIPRRIRQG LERILL

SEQ ID NO: :30 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :31 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :32 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :33 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :34 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :35 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :36 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :37 AVAEGTDRI I EVLQRAYRAI LHIPTRIRQG AERALL

SEQ ID NO: :38 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :39 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: 40 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: 41 AVAEGTDRW EVLQRAYRAI LHIPTRIRQG AERALL

SEQ ID NO: 42 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :43 AVAEGTDRI I EWQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :44 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :45 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: 46 AVAEGTDRVI EVSQRAFRAI LHVPVRIRQG LERALV

SEQ ID NO: :47 AVAEGTDRI I EVLQRICRAI LHIPARIRQG AERALL

SEQ ID NO: 48 AVAEGTDRI I EVLQRAYRAI IHIPTRIRQG AERALL

SEQ ID NO: 49 AVAEGTDRI I EVLQRICRAV LHIPTRIRQG AERALL

SEQ ID NO: :50 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :51 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL

SEQ ID NO: :52 AVAEGTDRI I EVLQRICRAI LHIPTRIRQG AERALL 1 10 20 30 40 50

SEQ ID NO: :1 MRVKEKYQHL WRWGWRWGTM LLGMLMICSA TEKLWVTVYY GVPVWKEATT

SEQ ID NO: :53 CSA AGKLWVTVYY GVPVWKEATT

SEQ ID NO: :54 CSA ADQLWVTVYY GVPVWKEATT

SEQ ID NO: :55 CSA ADQLWVTVYY GVPVWKEATT

SEQ ID NO: :56 CSA ADKSWVTVYY GVPVWKEATT

SEQ ID NO: :57 CSA TDRLWVTVYY GVPVWKEATT

SEQ ID NO: :58 CSA AGNLWVTVYY GVPVWKEATT

SEQ ID NO: :59 CSA AGQLWVTVYY GVPVWKEATT

SEQ ID NO: 60 CSA TDRLWVTVYY GVPVWKEATT

SEQ ID NO: :61 CSA AGNLWVTVYY GVPVWKEATT

SEQ ID NO: :62 CSA TDQLWVTVYY GVPVWKEATT

SEQ ID NO: :63 MKVKEIMKHL WRWG -TM LLGILMICSA VDQLWVTVYY GVPVWKEATT

SEQ ID NO: 64 MKVKEIMKHL WRWG -TM LLGILMICSA ADQLWVTVYY GVPVWKEATT

SEQ ID NO: :65 MKVKEIMKHL WRWG -TM LLGILMICSA ADQLWVTVYY GVPVWKEATT

SEQ ID NO: 66 MKVKEIRKHL WRWG -TM LLGILMICSA TDRLWVTVYY GVPVWKEATT

SEQ ID NO: :67 MKVKEIMKHL WRWG -TM LLGILMICRA ADQLWVTVYY GVPVWKEATT

SEQ ID NO: 68 MKVKEIMKHL WRWG -TM LLGILMICSA AGQLWVTVYY GVPVWKEATT

SEQ ID NO: 69 MKVKEIMKHL WRWG -TM LLGILMICSA AGNLWVTVYY GVPVWKEATT

SEQ ID NO: :70 CSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :71 CSA ADQLWVTVYY GVPVWKEETT

SEQ ID NO: :72 CSA ADQLWVTVYY GVPVWKEATT

SEQ ID NO: :73 CSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :74 CRA ADQLWVTVYY GVPVWKEATT

SEQ ID NO: :75 XL WRWG -TM LLGILMICSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :76 MKVKEIMKHL WRWG -TM LLGILMICSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :77 MKVKEIMKHL WRWG -TM LLGILMICSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: :78 MKVKEIMKHL WRWG -TM LLGILMICSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: :79 MKVKEIMKHL WRWG -TM LLGILMICSA TDQLWVTVYY GVPVWKEATT

SEQ ID NO: 80 MKVKEIMKHL WRWG -TM ILGILMICSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: :81 MKVKEIMKHL WRWG -TM LLGILMICSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: 82 MKVKEIMKHL WRWG -TM LLGILMICSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :83 MKVKEIMKHL WRWG -TM LLGILMICSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: 84 CSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: :85 CSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: 86 CSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: :87 CSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: 88 CSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: 89 MKVKEIMKHL WRWG -TM LLGILMICSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :90 MKVKEIMKHL WRWG -TM LLGILMICSA DDQLWVTVYY GVPVWKEATT

SEQ ID NO: :91 MKVKEIMKHL WRWG -TM LLGILMICSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: :92 MKVKEIMKHL WRWG -TM LLGILMICSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :93 MKVKEIMKHL WRWG -TM LLGILMICSA DDQLWVTVYY GVPVWKEATT

SEQ ID NO: 94 MKVKEIMRHL WRWG -TM LLGILMICSA EDKLWVTVYY GVPVWKEATT

SEQ ID NO: :95 CSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: 96 CSA DDQLWVTVYY GVPVWKEATT

SEQ ID NO: :97 CSA DDQLWVTVYY GVPVWKEATT

SEQ ID NO: 98 CSA EDQLWVTVYY GVPVWKEATT

SEQ ID NO: 99 CRA DDQLWVTVYY GVPVWKEATT 51 60 70 80 90 100

SEQ ID NO: :1 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLVNVT ENFNMWKNDM

SEQ ID NO: :53 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :54 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :55 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :56 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :57 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :58 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :59 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: 60 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :61 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: :62 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :63 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 64 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :65 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: 66 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :67 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 68 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLENVT ENFNMWKNDM

SEQ ID NO: 69 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :70 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :71 TLFCASDAKA YDTKVHNVWA THACVPTDPN PQEVVLDNVT ENFNMWKNDM

SEQ ID NO: :72 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :73 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :74 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :75 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :76 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :77 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :78 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :79 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 80 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :81 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 82 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :83 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 84 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLDNVT ENFNMWKNDM

SEQ ID NO: :85 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 86 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :87 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 88 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 89 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLDNVT ENFNMWKNDM

SEQ ID NO: :90 TLFCASDAKA YDTEVHNVWA THACVPTDPD PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :91 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :92 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :93 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 94 TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: :95 TLFCASDAKA YKTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 96 TLFCASDAKA YETEVHNVWA THACVPTDPN PQEVVLDNVT ENFNMWKNDM

SEQ ID NO: :97 TLFCASDAKA YEKEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWENDM

SEQ ID NO: 98 TLFCASDAKA YKTEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM

SEQ ID NO: 99 TLFCASDAKA YEKEVHNVWA THACVPTDPN PQEVVLGNVT ENFNMWKNDM 101 110 120 130 139 145 I I I I I I I I I I

SEQ ID NO:l VEQMHEDIIS LWDQSLKPCV KLTPLCVSLK CTD-LKNDTN TNSSSG

SEQ ID NO: 53 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN AS

SEQ ID NO: 54 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNAT

SEQ ID NO: 55 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN AS

SEQ ID NO: 56 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN AN

SEQ ID NO: 57 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ANATNA

SEQ ID NO: 58 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 59 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 60 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ANATNASA—

SEQ ID NO: 61 VEQMHEDIIS LWDQSLKPCV ELTPLCITLN CTDYVGNAAN ASATNA

SEQ ID NO: 62 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ANTTNAT

SEQ ID NO: 63 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 64 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 65 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 66 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGKATN ASATNA

SEQ ID NO: 67 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 68 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 69 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ASATNA

SEQ ID NO: 70 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CNDYVGNATN ASATNA

SEQ ID NO: 71 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CNDYVGNATN ASATNA

SEQ ID NO: 72 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNATN ANTTNANDTN

SEQ ID NO: 73 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGKATN AS

SEQ ID NO: 74 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CNDYVGNATN ASATNA

SEQ ID NO: 75 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CIDYVGNATN ANATNA

SEQ ID NO: 76 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CIDYVGNATN A-TTNA

SEQ ID NO: 77 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGNANN ASATNA

SEQ ID NO: 78 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CADYVGNATN TNTTNTNATD SEQ ID NO: 79 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGSATN VSATNAT

SEQ ID NO: 80 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CADYVGNTTN TNTTNANATD

SEQ ID NO: 81 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYVGKATN ASDTNAT

SEQ ID NO: 82 VEQMHEDIIS LWDQSLKRCV ELTPLCVTLD CIDYVGNATN ANTTKVNAIK

SEQ ID NO: 83 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CIDYVGNATN A-TTNA

SEQ ID NO: 84 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CI DYVRNATN ASATNA

SEQ ID NO: 85 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CNDYGGNATN ASATNA

SEQ ID NO: 86 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CINYVGNATN ANATNA

SEQ ID NO: 87 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CINYVGNATN ANAINATT—

SEQ ID NO: 88 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CIGYVGNATK ANATNTT SEQ ID NO: 89 VEQMHEDIIS LWDESLKPCV ELTPLCVTLN CINYVGNVTN ANAINATT—

SEQ ID NO: 90 VEQMHEDIIS LWEESLKPCV ELTPLCVTLN CINYEGNATN ANTTNATT—

SEQ ID NO: 91 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CTDYMGKATN VSATNTTR-N

SEQ ID NO: 92 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CINYVGNATN A

SEQ ID NO: 93 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CIDYVGNATN ASATTAP SEQ ID NO: 94 VEQMHEDIIS LWDQSLKPCV ELTPLCVTLN CIDYVGNATN ASATTAP

SEQ ID NO: 95 VEQMHEDIIS LWDESLKPCV ELTPLCVTLN CTDYVGNATS ANAT

SEQ ID NO: 96 VEQMHEDIIS LWDESLKPCV ELTPLCVTLN CTDYVGNASA TXATSG

SEQ ID NO: 97 VEQMHEDIIS LWDESLKPCV ELTPLCVTLN CTDYVGNATS ANAT

SEQ ID NO: 98 VEQMHEDIIS LWDESLKPCV ELTPLCVTLN CTDYVGNATS ANAT

SEQ ID NO: 99 VEQMHEDIIS LWDESLKPCV ELTPLCVTLN CTDYMGKATN VSANNT 146 153 163 173 183

SEQ ID NO: :1 RM IMERGE IRNCSFNIST SIRGRVQREY AFFYRLDI I P

SEQ ID NO: :53 —TTNATG— GMGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :54 TGTTNATG— GIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :55 —TTNATG— GIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :56 —TTNATG— GIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :57 -RTTNATG— GIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :58 TNSSNATG— SIGGTVEREE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :59 TNTTS— DIGGTVEREE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: 60 TNATNATG— GIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :61 TS-TNATD— GIGGTVERGE IRNCSFNITT SIRDRVQREH ALFYRLDIVP

SEQ ID NO: :62 -IGGTVEKGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :63 TNTTG— SIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: 64 —TTNVTG— SIGGTVEKGE IRNCSFNITP SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :65 —TTNAT -IGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: 66 —TNTTD SIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :67 —TTNAT -IGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: 68 TNTTG— SIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: 69 -TSTNATG— DIGGTVERGE IRNCSFNITP SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :70 —TTNATD— SIGGTAERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :71 —TTNATD— SIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :72 ANTTNATG— SIGGTVERGE IRNCSFNITP SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :73 —ATNAT TVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :74 —TTNATD— SIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :75 —TTKATG— SIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: :76 —TTKATG— GIGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: :77 —TNNAT -IGGTVERGE IRNCSFNITT SIRDRVQEEY ALFYRLDIVP

SEQ ID NO: :78 ANTINATG— SIGGRVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :79 TVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: 80 ANTINATG— SIGGRVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :81 —SNNATG— STGGTVERGE IRNCSFNITT SIRDRVQREN ALFYRLDIVP

SEQ ID NO: 82 ANTTNATG— -IGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :83 —TTKATGG- -IGGTVERGE IRNCSFNITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: 84 ANATDG- -IGGRVERGE INNCSFNITT SIRDRVQREY ALFYRLDIVP

SEQ ID NO: :85 —TINATDS- -IGGTVERGE IRNCSFNITT SIRDRVQREH ALFYRLDIVQ

SEQ ID NO: 86 —TTSAPTNA TIGGTVDRGE IRNCSFNITT SIRDRVQREH ALFYRLDIVQ

SEQ ID NO: :87 SAPTNATIG- GTVDRGE IRNCSFNITT SIRDRVQREH ALFYRLDIVQ

SEQ ID NO: 88 NTTTSAPTNA TIGGTVDGGE IRNCSFNITT SIRDRVQREY ALFYRLDIVQ

SEQ ID NO: 89 SAPTSATIG- GTVDRGE IRNCSFNITT SIRDRVQREH ALFYRLDIVQ

SEQ ID NO: :90 SAPTSATIG- GTVDGGE IRNCSFNITT SIRDRVQREH ALFYNLDIVQ

SEQ ID NO: :91 TTSNNATGN- -IGGTVERGE IRNCSFDITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: :92 —TINATIG- GTVEGGE IRNCSFNITT SIRDRVQREY ALFYRLDIVQ

SEQ ID NO: :93 —TNNATIG- GTVDREE IRNCSFNITT SIRDRVQREY ALFYRLDIVQ

SEQ ID NO: 94 —TNNATIG- GTVDREE IRNCSFNITT SIRDRVQREY ALFYRLDIVQ

SEQ ID NO: :95 GE IRNCSFDITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: 96 GE IRNCSFDITT SIRDRVQREY ALFYNLDIVR

SEQ ID NO: :97 GE IRNCSFDITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: 98 GE IRNCSFDITT SIRDRVQREY ALFYRLDIVS

SEQ ID NO: 99 -SANNTTSK- —NATGNIGE IRNCSFDITT SIRDRVQREY ALFYRLDIVS 184 191 201 211 221 231

SEQ ID NO: :1 IDND—TTSY KLTSCNTSVI TQACPKVSFE PIPIHYCAPA GFAILKCNNK

SEQ ID NO: :53 IDNDNTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :54 IDNDNTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :55 IDNDNTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :56 IDNDNTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :57 IDNDNTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :58 IDNDNTNDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNNK

SEQ ID NO: :59 IDNDNTSNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 60 IDNDNTSDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :61 IDNDNTNDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :62 IDNDNTSNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :63 IDNDNTSDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 64 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :65 IDND—NDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 66 IDNDNTSDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :67 IDND—NDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 68 IDNDNTSNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 69 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :70 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :71 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :72 IDNDNTSNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :73 IDNDNTSDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :74 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :75 IDND—NDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :76 IDND—NNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDN

SEQ ID NO: :77 IDND—NDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDN

SEQ ID NO: :78 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :79 IDNDNTSDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 80 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :81 IDND—NDSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 82 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :83 IDND—NNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 84 IDNGDTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :85 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 86 IDKENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :87 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 88 IDNENTNNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNEK

SEQ ID NO: 89 IDKENTNNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDN

SEQ ID NO: :90 IDKENTNNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :91 IDND—NNSY RLINCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :92 IDKENTNNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :93 IDKENTNNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 94 IDKENTNNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :95 IDND—NNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 96 IDNENSNNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: :97 IDND—NNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 98 IDND—NNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK

SEQ ID NO: 99 IDND—NNSY RLISCNTSVI KQACPKVSFE PIPIHYCAPA GFAILKCNDK 232 24: 251 261 271 281

I I

SEQ ID NO: :1 TFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSVNFTDNA

SEQ ID NO: :53 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: :54 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: :55 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFMNNA

SEQ ID NO: :56 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: :57 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: :58 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTDNA

SEQ ID NO: :59 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: 60 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: :61 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: :62 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: :63 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNA

SEQ ID NO: 64 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTDNA

SEQ ID NO: :65 KFNGTGPCTK VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTNNA

SEQ ID NO: 66 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTDNA

SEQ ID NO: :67 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTNNV

SEQ ID NO: 68 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTDNA

SEQ ID NO: 69 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNA

SEQ ID NO: :70 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNT

SEQ ID NO: :71 EFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEKEW IRSQNFTDNT

SEQ ID NO: :72 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTNNV

SEQ ID NO: :73 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNA

SEQ ID NO: :74 EFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNT

SEQ ID NO: :75 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNA

SEQ ID NO: :76 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNT

SEQ ID NO: :77 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNT

SEQ ID NO: :78 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNA

SEQ ID NO: :79 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTNNV

SEQ ID NO: 80 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNA

SEQ ID NO: :81 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNV

SEQ ID NO: 82 EFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNT

SEQ ID NO: :83 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTDNT

SEQ ID NO: 84 KFNGTGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: :85 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: 86 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: :87 KFNGTGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: 88 KFNGTGPCTN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: 89 KFNGTGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFKKND

SEQ ID NO: :90 KFNGKGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: :91 KFNGTGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTKNT

SEQ ID NO: :92 KFNGTGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: :93 KFNGKGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFMEND

SEQ ID NO: 94 KFNGTGPCTN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTKND

SEQ ID NO: :95 KFKGTGPCKN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTEND

SEQ ID NO: 96 KFNGTGPCKN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRFQNFTNNN

SEQ ID NO: :97 KFNGTGPCKN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTKNT

SEQ ID NO: 98 KFKGTGPCKN VSTVQCTHGI KPVVS TQLLL NGSLAEEEVV IRSQNFTEND

SEQ ID NO: 99 KFNGTGPCKN VSTVQCTHGI RPVVS TQLLL NGSLAEEEVV IRFQNFTDNN 282 29: 30: 311 321 330

• I I I • · · I I I • · I I

SEQ ID NO 1 KTI IVQLNTS VEINCTRPNN RIQR GPGRAFVTIG -K IGNMRQAH SEQ ID NO 53 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 54 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 55 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 56 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 57 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 58 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 59 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 60 KVI IVQLNES WINCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 61 KVI IVQLNES WINCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 62 KVI IVQLNES WIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 63 KVI IVQLNES WINCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 64 KVI IVQLNES WIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 65 KVI IVQLNES WIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 66 KVI IVQLNES WIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 67 KVI IVQLNES WINCIRPNN APGRAFHTTG AI IGDIRQAH SEQ ID NO 68 KVI IVQLNES VEIDCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 69 KVI IVQLKES WIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 70 KVI IVQLNES WITCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 71 KVI IVQLNES WITCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 72 KVI IVQLNES WIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 73 KVI IVQLNES WIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 74 KVI IVQLNES WITCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 75 KVI IVQLNES WITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 76 KVI IVQLKES WITCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 77 KVI IVQLNES VEIDCTRPNN APGRAFYTTG AI IGDIRQAH SEQ ID NO 78 KVI IVQLKES VEITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 79 RVI IVQLNES VEIYCTRPNN APGRSFYTTG EI IGDIRQAH SEQ ID NO 80 KVI IVQLNKS WITCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 81 KVI IVQLNES VEIYCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 82 KVI IVQLNGS VEIDCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 83 KII IVQLNES VEIDCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 84 KVI IVQLNES VEITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 85 KVI IVQLNES VEIYCIRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 86 KVI IVQLNES VEIYCIRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 87 KVI IVQLNES VEITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 88 KVI IVQLNES VEINCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 89 RVI IVQLKES VEITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 90 RVI IVQLKES VEIICTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 91 KVI IVQLNKP VEITCTRPNN APGRSFYTTG EI IGDIRQAH SEQ ID NO 92 RVI IVQLKES VEITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 93 GVI IVQLKES VKITCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 94 GVI IVQLKES VEITCTRPNN APGRSFYTTG AI IGDIRQAH SEQ ID NO 95 GVI IVQLKES VDITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 96 KII IVQLNNP VDITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 97 KVI IVQLNKP VDITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 98 GVI IVQLKES VDITCTRPNN APGRAFYTTG EI IGDIRQAH SEQ ID NO 99 KII IVQLKES VEITCTRPNN APGRAFYTTG EI IGDIRQAH 331 340 350 360 370 380

SEQ ID NO: :1 C ISRAKWNN TLKQIASKLR EQFGNNKTI I FKQSSGGDPE IVTHSFNCGG

SEQ ID NO: :53 C ISRVKWEN TLKQIATKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :54 C ISRVKWEN TLKQIATKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :55 CNISRVKWEN TLKQIATKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :56 CNISRVKWEN TLKQIATKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :57 CNISRVKWEN TLKQIATKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :58 CNISKEKWEN TLKQIATKLR EQFK- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :59 CNISKEKWEN TLKQIVTKLR EQFE- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 60 CNISKEKWEN TLKQIATKLR EQFK- -NKTIT FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :61 CNISKEKWEN TLKQIVTKLR EQFK- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :62 CNISREKWKN TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :63 CNISKGKWEN TLKQIVTKLR EQFK- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 64 CNISREKWKN TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :65 CNISREKWKN TLKQIVTKLR EQFK- -NKTIT FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 66 CNISKGKWEN TLKQIVTKLR EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :67 CNISREKWKN TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 68 CNISKGKWEN TLKQIATKLR EQFE- -NKTIV FNQSSGGDPE IVMHSXNCGG

SEQ ID NO: 69 CNISREKWKN TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :70 CNISKGKWEN TLKQIVTKLR EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :71 CNISRENWKN TLKQIVTKLS EQFK- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :72 CNISREKWKN TLKQIVTKLR EQFE- -NKTIT FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :73 CNISKGKWEN TLKQIVTKLR EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :74 CNISGKKWGN TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :75 CNISGEKWEK TLKQIVTKLK EQFK- -NKTIA FNQSSGGDPE IEMHSFNCGG

SEQ ID NO: :76 CNISKGKWKN TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :77 CNISGKKWGN TLKQIVTKLR EQFK- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :78 CNISRAQWKN TLKQIVTKLG EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :79 CNISKGKWEN TLKQIATKLK EQF-- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 80 CNISRAEWKN TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :81 CNISRKNWKN TLKQIVTKLR EQFK- -NRTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 82 CNISREKWKN TLKQIVTKLR EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :83 CNISKGKWEN TVKQIVTKLR EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 84 CNISGGKWEK TLKQIVTKLK EQFK- -NKTIA FTQSSGGDPE IEMHSFNCGG

SEQ ID NO: :85 CNISKGKWQN TLKQIVTKLR EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 86 CNISKGKWQN TLKQIVTKLR EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :87 CNISGKKWEK TLKQTVTKLK EQFK- -NKTIA FTQSSGGDPE IEMHSFNCGG

SEQ ID NO: 88 CNISKGKWKK TLKQIVTKLR EQFK- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 89 CNINGKKWEK TLNQTVTKLK EQFK- -NKTIA FTQSSGGDPE IEMHSFNCGG

SEQ ID NO: :90 CNISGGEWDK TLKQIVTKLR EQFE- -NKTIV FNQLSGGDPE IVMHSFNCGG

SEQ ID NO: :91 CNISRENWGK TLKQIVTKLG EQFK- -NKTIA FNQSPGGDPE IVMHSFNCGG

SEQ ID NO: :92 CNISGKKWEK TLNQTVTKLK EQFK- -NKTIA FTQSSGGDPE IEMHSFNCGG

SEQ ID NO: :93 CNISSANWTN TLKQIVTKLG EQFE- -NKTIA FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: 94 CNISSANWTN TLKQIVTKLG EQFG- -NKTIV FNQSSGGDPE IVMHSFNCGG

SEQ ID NO: :95 CNISRANWTS ILKQIVPKLG EQFG- -NKTIV FNQSTGGDPE IVMHSFNCRG

SEQ ID NO: 96 CNISRANWTS TIKQIVTKLG EQFE- -NKTIV FNQSSGGDPE IVMHSFNCRG

SEQ ID NO: :97 CNISRANWTD TLKQIVPKLG EQFG- -NKTIV FNQSTGGDPE IVMHSFNCRG

SEQ ID NO: 98 CNISRANWTS ILKQIVPKLG EQFG- -NKTIV FNQSTGGDPE IVMHSFNCRG

SEQ ID NO: 99 CNISRANWTS TIKQILTKLG EQFE- -NKTIV FNQSSGGDPE IVMHSFNCGG 381 390 398 4 07 413 423

SEQ ID NO: :1 EFFYCNSTQL FNSTWF—NS TWSTEGS -NN TEG--- --SDT ITLPCRIKQI

SEQ ID NO: :53 EFFYCNTTQL FNSTWN —DTEVS -NY N --DTH ITLPCRIKQI

SEQ ID NO: :54 EFFYCNTTQL FNSTWN —DTEGS -NY NDKN-- --ITH ITLPCRIKQI

SEQ ID NO: :55 EFFYCNTTQL FNSTWN —DTEVS -NH N --DEN ITLPCRIKQI

SEQ ID NO: :56 EFFYCNTTQL FNSTWN —DTEGS -NY NDKN-- --ITH ITLPCRIKQI

SEQ ID NO: :57 EFFYCNTTQL FNSTWN —DTEVS -NY N --ITH ITLPCRIKQI

SEQ ID NO: :58 EFFYCNTTQL FNSTWN —GTEVS -NY T --NEN ITLPCRIKQI

SEQ ID NO: :59 EFFYCNTTQL FNSTWN —GTEVS -NY T --NEN ITLPCRIKQI

SEQ ID NO: 60 EFFYCNTTQL FNSTWN —GTEVS -NY T --NEN ITLPCRIKQI

SEQ ID NO: :61 EFFYCNTTQL FNSTWN --GTEVS -NY T --NEN ITLPCRIKQI

SEQ ID NO: :62 EFFYCNTTQL FNSTWN --DTEVS -NY NDNN-- --ITN ITLPCRIKQI

SEQ ID NO: :63 EFFYCNTTQL FNSTWN --GTEVS -NY T --NEN ITLPCRIKQI

SEQ ID NO: 64 EFFYCNTTQL FNSTWNFNST WNDTEVS -NY T --VEN ITLPCRIKQI

SEQ ID NO: :65 EFFYCNTTQL FNSTWN —DTGVS -NY DDKN-- --ITN ITLPCRIKQI

SEQ ID NO: 66 EFFYCNTTQL FNSTWKFNST WNGTEVS -NY T --NEN ITLPCRIKQI

SEQ ID NO: :67 EFFYCNTTQL FNSTWNFNST WNDTEVS -NY T --VEN ITLPCRIKQI

SEQ ID NO: 68 EFFYCNTTQL FNSTWN --GTEVS -NY T --NEN ITLPCRIKQI

SEQ ID NO: 69 EFFYCNTTQL FNSTWNFNST WNDTEVS -NY T --VEN ITLPCRIKQI

SEQ ID NO: :70 EFFYCNTTQL FNSTWN —GTEAS -NY T --NEN ITLPCRIKQI

SEQ ID NO: :71 EFFYCNTTQL FNSTWNFNST WNDTKAS -NY T --NEN ITLPCRIKQI

SEQ ID NO: :72 EFFYCNTTQL FNSTWN —DTGVS -NY DDRN-- --ITN ITLPCRIKQI

SEQ ID NO: :73 EFFYCNTTQL FNSTWN —GTEGS -NY T --NEN ITLPCRIKQI

SEQ ID NO: :74 EFFYCNTTQL FNSTWNFNST WNDTEVS -NY T --VEN ITLPCRIKQI

SEQ ID NO: :75 EFFYCNTAQL FNSTWN —DTEVP -HY N --NET ITLPCRIKQI

SEQ ID NO: :76 EFFYCDTTQL FNSTWN —DTEVS -NY TVENITLHEN ITLPCRIKQI

SEQ ID NO: :77 EFFYCNTTQL FDSTWNFNST WDDTKVS -NY T --NET ITLPCRIKQI

SEQ ID NO: :78 EFFYCNTTQL FHSTWNFNNT WNDTEGS -NY NDNN-- --ITN ITLPCRIKQI

SEQ ID NO: :79 EFFYCNTTQL FNSTWNFNST WNDIKVS -NY T ITLPCRIKQI

SEQ ID NO: 80 EFFYCDTTQL FNSTWN --DTEVS -NY TVENVTLHEN ITLPCRIKQI

SEQ ID NO: :81 EFFYCNTTQL FNSTWN --DTEVS -NY DDRN-- --ITN ITLPCRIKQI

SEQ ID NO: 82 EFFYCNTTQL FNSTWN —DTERS -NY NDNN-- --ITN ITLPCRIKQI

SEQ ID NO: :83 EFFYCDTTQL FNSTWN —DTEVS -NY TVENITLHEN ITLPCRIKQI

SEQ ID NO: 84 EFFYCNTTQL FNSTWN —DTKVP -HY N --NET ITLPCRIKQI

SEQ ID NO: :85 EFFYCDTTQL FNSTWN —DTAVS -NY TVKNTTLHEN ITLPCRIKQI

SEQ ID NO: 86 EFFYCDTTQL FNSTWN —DTAVS -NY TVKNTTLHEN ITLPCRIKQI

SEQ ID NO: :87 EFFYCNTTQL FNSTWN —DTEVQ -HY N --NET ITLPCRIKQI

SEQ ID NO: 88 EFFYCDTTQL FNSTWN WNDTEVS -NY TVENATLHEN ITLPCRIKQI

SEQ ID NO: 89 EFFYCNTTQL FNSTWN --DTEVQ -HY N --NET ITLPCRIKQI

SEQ ID NO: :90 EFFYCNTTQL FNSTWN --DTEGS -RY N --NKT ITLPCRIKQI

SEQ ID NO: :91 EFFYCNTTQL FNSTWNFNST WNATEGT -NY NDNN-- --ITN ITLPCRIKQI

SEQ ID NO: :92 EFFYCNTTQL FNSTWN —DTEVQ -HY N --NET ITLPCRIKQI

SEQ ID NO: :93 EFFYCNTTQL FSSTWNFNGT WNSTEV- —Y T --DET ITLPCRIKQI

SEQ ID NO: 94 EFFYCNTTQL FSSTWNFNGT WNSTEVS -NY I --DGT ITLPCRIKQI

SEQ ID NO: :95 EFFYCDTTQL FNSTW T RNETGGWSNY NDNN-- --ITN ITLPCRIKQI

SEQ ID NO: 96 EFFYCDTTQL FNSTW T RNETEGWSNY NDNN-- --TTN ITLPCRIKQI

SEQ ID NO: :97 EFFYCDTTQL FNSTW T WNKTGGWSNY NDNN-- --ITN ITLPCRIKQI

SEQ ID NO: 98 EFFYCDTTQL FNSTW T RNETGGWSNY NDNN-- --ITN ITLPCRIKQI

SEQ ID NO: 99 EFFYCDTTQL FNSTWNFNGT WNEAEG- SNY KDNN-- --ITN ITLPCRIKQI 424 433 443 453 463 469

SEQ ID NO: :1 INMWQKVGKA MYAPPISGQI RCSSNITGLL LTRDGGNSNN ES-- —EIFR

SEQ ID NO: :53 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSSES KTSE -TETFR

SEQ ID NO: :54 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSNEN KTSE -TETFR

SEQ ID NO: :55 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSNEN KTSE -TETFR

SEQ ID NO: :56 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSNES KT— —ETFR

SEQ ID NO: :57 INMWQKVGKA MYAPPIRGQI RCSSNITGLL ITRDGGSNES KTSE -TETFR

SEQ ID NO: :58 INMWQKVGKA MYAPPIRGRI RCSSNITGLL LTRDGGSNEN ET— —ETFR

SEQ ID NO: :59 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSNET -T— —ETFR

SEQ ID NO: 60 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSDES ETNETTETFR

SEQ ID NO: :61 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGG-NES KTSE -TETFR

SEQ ID NO: :62 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSDES ETNETTETFR

SEQ ID NO: :63 INMWQKVGKA MYAPPIRGRI RCSSNITGLL LTRDGGSNET -TETFR

SEQ ID NO: 64 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LARDGGSNET -TETFR

SEQ ID NO: :65 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGNESN SE— -TETFR

SEQ ID NO: 66 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSDES ETNETTETFR

SEQ ID NO: :67 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LARDGGSNET -TETFR

SEQ ID NO: 68 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LTRDGGSNEN E -TETFR

SEQ ID NO: 69 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LARDGGS—N ET— -TETFR

SEQ ID NO: :70 INMWQKVGKA MYAPPIRGQI SCSSNITGLL LTRDGGNETS E -TETFR

SEQ ID NO: :71 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LARDGGSNEN E -TETFR

SEQ ID NO: :72 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LARDGGSNES --SE -TETFR

SEQ ID NO: :73 INMWQKVGKA MYAPPIRGRI RCSSNITGLL LTRDGGNESS E -TETFR

SEQ ID NO: :74 INMWQKVGKA MYAPPIRGQI KCSSNITGLL LTRDGGNETN E -TETFR

SEQ ID NO: :75 INMWQKVGKA MYAPPIRGQI KCSSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: :76 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LARDGGNKTN ET— -TETFR

SEQ ID NO: :77 INMWQKVGKA MYAPPIRGQI KCSSNITGLL LTRDGGNKNN ET— -TETFR

SEQ ID NO: :78 INMWQKVGKA MYAPPIRGQI KCSSNITGLL LARDGGSNES DT— —ETFR

SEQ ID NO: :79 INMWQKVGKA MYAPPIRGKI RCSSNITGLL LTRDGGSDES EA— —ETFR

SEQ ID NO: 80 INMWQKVGKA MYAPPIRGQI RCSSNITGLL LARDGGNKTN ET— -TETFR

SEQ ID NO: :81 INMWQKVGKA MYAPPIRGQI KCSSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: 82 INMWQKVGKA MYAPPIRGQI KCSSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: :83 INMWQKVGKA MYAPPIRGQI KCSSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: 84 INMWQKVGKA MYAPPIRGQI NCTSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: :85 INMWQKVGKA MYAPPIRGQI KCTSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: 86 INMWQKVGKA MYAPPIRGQI KCTSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: :87 INMWQKVGKA MYAPPIRGQI NCTSNITGLL LTRDGGNKTN ET— —ETFR

SEQ ID NO: 88 INMWQKVGKA MYAPPIRGQI SCSSNITGLL LARDGGNKTN ET— -TETFR

SEQ ID NO: 89 INMWQKVGKA MYAPPIRGQI NCTSKITGLL LTRDGGNKTN E -TETFR

SEQ ID NO: :90 INMWQKVGKA MYAPPIRGQI NCTSTITGLL LTRDGGNKTN ET-ETTETFR

SEQ ID NO: :91 INMWQKVGKA MYAPPIRGQI ECSSNITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: :92 INMWQKVGKA MYAPPIRGQI NCTSTITGLL LTRDGGNKTN ET— —ETFR

SEQ ID NO: :93 INMWQKVGKA MYAPPIRGQI NCTSTITGLL LTRDGGNKTN ET-ETTETFR

SEQ ID NO: 94 INMWQKVGKA MYAPPIRGQI QCSSKITGLL LTRDGGNKTN ET— -TETFR

SEQ ID NO: :95 INMWQKVGKA MYAPPIRGHI QCTSKITGLL LTRDGGNTTN ET— —ETFR

SEQ ID NO: 96 INMWQKVGKA MYAPPIRGHI QCTSKITGLL LTRDGGNTTN ET— —ETFR

SEQ ID NO: :97 INMWQKVGKA MYAPPIRGHI QCTSKITGLL LTRDGGNTTN ET— —ETFR

SEQ ID NO: 98 INMWQKVGKA MYAPPIRGHI QCTSKITGLL LTRDGGNTTN ET— —ETFR

SEQ ID NO: 99 INMWQKVGKA MYAPPIRGQI NCTSKITGLL LTRDGGNTTN KT— —ETFR 470 479 489 499 509 518

SEQ ID NO: :1 PGGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVG-IGAL

SEQ ID NO: :53 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :54 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :55 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :56 PAGGDMRDNW RSEVYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :57 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :58 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :59 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: 60 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :61 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :62 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :63 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: 64 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :65 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: 66 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :67 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGALGAV

SEQ ID NO: 68 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: 69 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :70 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :71 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :72 PAGGDMRDNW RSELYKYKW EIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :73 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAM

SEQ ID NO: :74 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :75 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAV

SEQ ID NO: :76 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGALGAV

SEQ ID NO: :77 PAGGNMRDNW RSELYKYKW KIEPLGVAPT KAKRRAVQRE KRAVGVIGAV

SEQ ID NO: :78 PAGGNMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGALGAV

SEQ ID NO: :79 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRTVGVIGAM

SEQ ID NO: 80 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KARRRWQRE KRAVGAIGAV

SEQ ID NO: :81 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAM

SEQ ID NO: 82 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGALGAV

SEQ ID NO: :83 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KARRRWQRE KRAVGAIGAM

SEQ ID NO: 84 PEGGDMRDNW RSELYKYKW KIEPLGVAPT RAKRRWQRE KRAVGAIGAM

SEQ ID NO: :85 PEGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGALGAV

SEQ ID NO: 86 PEGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGALGAV

SEQ ID NO: :87 PAGGEMRDNW RSELYKYKW KIEPLGVAPT KARRRWQRE KRAVGAIGAM

SEQ ID NO: 88 PAGGDMRDNW RSELYKYKW KIEPLGVAPT RAKRRWQRE KRAVGAIGAM

SEQ ID NO: 89 PAGGEMRDNW RSELYKYKW KIEPLGVAPT KARRRWQRE KRAVGALGAV

SEQ ID NO: :90 PAGGEMRDNW RSELYKYKW KIEPLGVAPT KARRRWQRE KRAVGAIGAM

SEQ ID NO: :91 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KARRRWQRE KRAVGALGAV

SEQ ID NO: :92 PAGGEMRDNW RSELYKYKW KIEPLGVAPT RAKRKWQRE KRAVGVIGAM

SEQ ID NO: :93 PAGGEMRDNW RSELYKYKW KIEPLGVAPT KARRRWQRE KRAVGAIGAM

SEQ ID NO: 94 PEGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAM

SEQ ID NO: :95 PIGGEMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGTIGAM

SEQ ID NO: 96 PVGGEMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGTIGAM

SEQ ID NO: :97 PVGGNMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGTIGAM

SEQ ID NO: 98 PIGGEMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGAIGAM

SEQ ID NO: 99 PAGGDMRDNW RSELYKYKW KIEPLGVAPT KAKRRWQRE KRAVGTIGAM 519 52 i3 53f3 54f3 55i 568

. . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1

SEQ ID NO: :1 FLGFLGAAGS TMGAASMTLT VQARQLLSGI VQQQNNLLRA IEAQQHLLQL

SEQ ID NO: :53 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :54 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: :55 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :56 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :57 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: :58 FLGFLGAAGS TMGAASVTLT VQAILLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :59 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 60 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :61 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: :62 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :63 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: 64 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: :65 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 66 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :67 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 68 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: 69 FLGFLGAAGS TMGAAS IMLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :70 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :71 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :72 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :73 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :74 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :75 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :76 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: :77 FLGFLGAAGS TMGAASITLT VQARLLLFGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :78 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :79 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 80 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: :81 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 82 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :83 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 84 FLGFLGAAGS TMGAAAVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :85 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 86 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :87 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 88 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 89 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :90 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :91 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNYLLRA IKAQQHMLQL

SEQ ID NO: :92 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :93 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 94 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: :95 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 96 FLGFLGAAGS TMGAASVTLT VQARLLLSGI VQQQNNLLRA IKAQQHMLQL

SEQ ID NO: :97 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 98 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL

SEQ ID NO: 99 FLGFLGAAGS TMGAASITLT VQARLLLSGI VQQQNNLLRA lEAQQHMLQL 569 57 ί] 58£ 59£ 60£ 61^'

. .. . 1 1 . .. . 1 1 .. .. 1 1 . .. . 1 .. • · 1

SEQ ID NO: :1 TVWGIKQLQA RILAVERYLK DQQLLGIWGC SGKLICTTAV PWNASWS -NK

SEQ ID NO: :53 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :54 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :55 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :56 TVWGIKQLQA RVLAVERYLG DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :57 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :58 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :59 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 60 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :61 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :62 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :63 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 64 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :65 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 66 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :67 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 68 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 69 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :70 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :71 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :72 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNSSWS -NR

SEQ ID NO: :73 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :74 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK

SEQ ID NO: :75 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNSSWS -NR

SEQ ID NO: :76 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :77 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK

SEQ ID NO: :78 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK

SEQ ID NO: :79 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 80 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :81 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNSSWS -NR

SEQ ID NO: 82 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: :83 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 84 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSSNR

SEQ ID NO: :85 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSSNR

SEQ ID NO: 86 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSSNR

SEQ ID NO: :87 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSN-K

SEQ ID NO: 88 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NR

SEQ ID NO: 89 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSSNR

SEQ ID NO: :90 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSN-K

SEQ ID NO: :91 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK

SEQ ID NO: :92 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSN-K

SEQ ID NO: :93 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSN-K

SEQ ID NO: 94 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSN-K

SEQ ID NO: :95 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWSRNR

SEQ ID NO: 96 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK

SEQ ID NO: :97 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK

SEQ ID NO: 98 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK

SEQ ID NO: 99 TVWGIKQLQA RVLAVERYLR DQQLLGIWGC SGKLICTTAV PWNNSWS -NK 618 62 7 637 647 657 667

I • · I I I I I

SEQ ID NO: :1 SLEQIWNHTT WMEWDRE INN YTSL IHSLIE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :53 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQDKNE QELLELDKWA

SEQ ID NO: :54 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QKLLELDKWA

SEQ ID NO: :55 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :56 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :57 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :58 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :59 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 60 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :61 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :62 SLDMIWNNMT WIEWEREIDN YTGLIYTLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :63 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 64 SLDRIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :65 SLDRIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 66 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :67 SLDRIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 68 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 69 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :70 SLDMIWNNMT WIEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :71 SLDRIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :72 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :73 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :74 SLDRIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :75 SLEMIWNNMT WIEWEKEIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :76 SLDMIWDNMT WIEWEREIDN YTDLIYSLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :77 SLEMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :78 SLEMIWNNMT WIEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :79 SLDMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 80 SLEMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :81 SLEMIWNNMT WIEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 82 SLEMIWNNMT WIEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :83 SLDMIWDNMT WIEWEREIDN YTDLIYSLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 84 SLDRIWDTMT WMEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :85 SLDMIWDNMT WIQWEREIEN YTNLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 86 SLDMIWDNMT WIQWEREIEN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :87 SLERIWNNMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 88 SLDKIWDTMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 89 SLDMIWDNMT WIQWEREIEN YTDLIYSLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :90 TLDRIWNNMT WMEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :91 SLDMIWDKMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :92 SLEMIWDKMT WMEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :93 TLDRIWNNMT WMEWEREIDN YTGLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 94 TLDKIWNNMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :95 SLDGIWDNMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 96 TLGMIWNNMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: :97 TLAMIWNNMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 98 TLAMIWNNMT WIEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA

SEQ ID NO: 99 TLGMIWNNMT WMEWEREIDN YTDLIYNLLE ESQNQQEKNE QELLELDKWA 66ί 677 687 69 707 717

I . I I • · · I

SEQ ID NO 1 SLWNWF I TN WLWY VFAVLSIVNR VRQGYS PLSF SEQ ID NO 53 SLWNWFDITN WLWY IXIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 54 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 55 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 56 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 57 NLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 58 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 59 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 60 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI IFAVLSIVNR VRQGYSPLSF SEQ ID NO 61 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSL SEQ ID NO 62 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSL SEQ ID NO 63 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 64 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 65 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 66 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 67 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 68 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 69 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 70 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVSR VRQGYSPLSF SEQ ID NO 71 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNK VRQGYSPISF SEQ ID NO 72 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 73 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 74 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 75 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 76 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 77 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 78 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 79 SLWNWFDITN WLWYIRIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 80 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 81 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 82 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 83 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI IFAVLSIVNR VRQGYSPLSF SEQ ID NO 84 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 85 SLWSWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 86 SLWSWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSVVNR VRQGYSPLSF SEQ ID NO 87 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 88 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 89 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 90 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFTVLSVVNR VRQGYSPLSF SEQ ID NO 91 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 92 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 93 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFTVLSVVNR VRQGYSPLSF SEQ ID NO 94 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 95 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSVVNR VRQGYSPLSF SEQ ID NO 96 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 97 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPLSF SEQ ID NO 98 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFAVLSIVNR VRQGYSPISF SEQ ID NO 99 SLWNWFDITN WLWYIKIFIM IVGGLIGLRI VFTVLSVVNR VRQGYSPLSF 718 727 737 747 757 767

SEQ ID NO: :1 QTHLPTPRGP DRPEGIEEEG GERDRDRSIR LVNGSLALIW DDLRSLCLFS

SEQ ID NO: :53 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :54 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :55 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :56 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :57 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :58 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: :59 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: 60 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :61 QTRLPTPRGP DRPEGIEEEG GERDRDRSGV LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :62 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :63 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: 64 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :65 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: 66 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :67 QTRLPTPRGP DRPEGIEEEG GERDRDRSGV LVDGFLALFW DDLRSLCLFS

SEQ ID NO: 68 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: 69 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :70 QTRLPNPRGP DRPEGIEEEG GERNRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :71 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: :72 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: :73 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :74 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :75 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :76 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVGGFLALFW NDLRSLCLFS

SEQ ID NO: :77 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: :78 QTRLPNPRGP DRPEGIEEEG GERDRDRSRI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :79 QTRLPTPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: 80 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :81 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW ADLRSLCLFS

SEQ ID NO: 82 QTRLPNPRGP DRPEGIEEEG GERDRDRSGV LVDGFLALFW GDLRSLCLFS

SEQ ID NO: :83 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: 84 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: :85 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: 86 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW ADLRSLCLFS

SEQ ID NO: :87 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW DDLRSLCLFS

SEQ ID NO: 88 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW GDLRSLCLFS

SEQ ID NO: 89 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: :90 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW GDLRSLCLFS

SEQ ID NO: :91 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVGGFLALFW NDLRSLCLFS

SEQ ID NO: :92 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: :93 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: 94 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVGGFLALFW NDLRSLCLFS

SEQ ID NO: :95 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW GDLRSLCLFS

SEQ ID NO: 96 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVDGFLALFW ADLRSLCLFS

SEQ ID NO: :97 QTRLPNPRGP DRPEGIEEEG GERDRGRSGI LVDGFLALFW ADLRSLCLFS

SEQ ID NO: 98 QTRLPNPRGP DRPEGIEEEG GERDRGRSEI LVDGFLALFW NDLRSLCLFS

SEQ ID NO: 99 QTRLPNPRGP DRPEGIEEEG GERDRDRSGI LVGGFLALIW NDLRSLCLFS 768 77^' 787 797 807 817

I I I I I I I I

SEQ ID NO: :1 YHRLRDLLLI VTRIVELLGR RGWEALKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :53 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :54 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :55 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :56 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :57 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :58 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :59 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: 60 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :61 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :62 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :63 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: 64 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :65 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 66 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :67 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: 68 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: 69 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :70 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :71 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :72 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL QNSAVSLLNV SEQ ID NO: :73 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :74 YHRLRDLLLI VTRIVELLGR RGWEILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :75 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :76 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :77 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :78 YHRLRDLLLI VTRIVELLGR RGWEILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :79 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: 80 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :81 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: 82 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :83 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 84 YHRLRDLLLI VTRIVELLGR RGWEILKYWW NLLQYWSQEL KNSAISLLNV SEQ ID NO: :85 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 86 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :87 YHRLRDLLLI VTRIVELLGR RGWGILKYWW SLLQYWSQEL KNSAISLLNV SEQ ID NO: 88 YHRLRDLLLI VTRIVELLGR RGWEILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 89 YHRLRDLLLI VTRIVELLGR RGWEILKYWW NLLQYWSQEL KNSAVSLLNV SEQ ID NO: :90 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :91 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :92 YHRLRDLLLI VTRIVELLGR RGWGILKYWW SLLQYWSQEL KNSAVSLLNA SEQ ID NO: :93 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 94 YHRLRDLLLI VTRIVELLGR RGWGILKYWR SLLQYWSQEL KNSAVSLLNA SEQ ID NO: :95 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 96 YHRLRDLLLI VTRIVELLGR RGWEILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: :97 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 98 YHRLRDLLLI VTRIVELLGR RGWEILKYWW NLLQYWSQEL KNSAVSLLNA SEQ ID NO: 99 YHRLRDLLLI VTRIVELLGR RGWGILKYWW NLLQYWSQEL KNSAVSLLNA 818 827 837 847 857

SEQ ID NO: 1 TAIAVAEGTD RVIEWQGAC RAIRHIPRRI RQGLERILL*

SEQ ID NO: 53 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 54 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 55 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 56 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: :57 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 58 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGX

SEQ ID NO: 59 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 60 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: : 61 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: : 62 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: : 63 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 64 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 65 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 66 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 67 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 68 TAITVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 69 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 70 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: :71 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: ,12 TAIAVAEGTD RITELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: ,13 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: ,14 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: ,15 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 76 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: :77 TAIAVAEGTD RIIELIQRAY RAVLHIPRRI RQGFERALL*

SEQ ID NO: 78 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 79 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 80 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 81 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 82 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 83 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: 84 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 85 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 86 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 87 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 88 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 89 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 90 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 91 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 92 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 93 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 94 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL*

SEQ ID NO: : 95 TAIAVAEGTD RIIELIQRAY RAVLHIPRRI RQGFERALL-

SEQ ID NO: 96 TAIAVAEGTD RIIELIQRAY RAVLHIPRRI RQGFERALL-

SEQ ID NO: :97 TAIAVAEGTD RI IELVQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 98 TAIAVAEGTD RI IELVQRAY RAVLHVPRRI RQGFERALL-

SEQ ID NO: 99 TAIAVAEGTD RIIELIQRAY RAVLHVPRRI RQGFERALL- Alignment of HIV-2 Env B . GH .86. D205_AL . X61240 (SEQ ID NO:9) with HxB2 (SEQ ID NO:l)

10 20 27 37 47

SEQ ID NO:l MRVKEKYQHLWRWGWRWGTM—LLGMLMI -CSATEKLWVTVYYGVPVWKE 47 SEQ ID NO: 9 MAYFSS—RLPIALLLIGISGFVCK QYVTVFYGI PAWRN 37

57 67 77 87 97

SEQ ID NO:l ATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWK 97 SEQ ID NO: 9 ATVPLICATTN RDTWGTVQCLPDNGDYTEIRLN- ITEAFDAWD 79

107 117 127 137 147

SEQ ID NO:l NDMVEQMHEDI ISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRM 147 SEQ ID NO: 9 NTVTQQAVDDVWRLFETSIKPCVKLTPLCVAMNCSKTETNPGNASSTTTT 129

152 162 170

SEQ ID NO:l IME KGEIKNCSFNI STSIRGKV-Q- 170 SEQ ID NO: 9 KPTTTSRGLKTINETDPCIKNDSCTGLGEEEIMQCNFSMTGLRRDELKQY 179

180 188 198 208 218

SEQ ID NO:l KEYAFFYKLDI IPIDND— TSYKLTSCNTSVITQACPKVSFEPIPIHYC 218 SEQ ID NO: 9 KDTWYSE—DLECNNTRKY SRCYIRTCNT I IQESCDKHYWDSLRFRYC 227

228 238 247 257 267

SEQ ID NO:l APAGFAILKCNNKTFNGTGP-CTNVSTVQCTHGIRPVVSTQLLLNGSLAE 267 SEQ ID NO: 9 APPGFFLLRCNDTNYSGFMPNCSKVVASSCTRMMETQSSTWFGFNGTRAE 277

277 287 295 305 315

SEQ ID NO:l EEVVIRSVNFTDNAKTI IVQLNTSVE— INCTRPNNNTRKRIRIQRGPGR 315 SEQ ID NO: 9 NRTYIYWHEK-DN-RTI IS-LNTYYNLSIHCKRPGNKTVVPIRTVSGLLF 324

325 333 343 353

SEQ ID NO:l AFVTIGKIG —MRQAHCNI SRAKWNNTLKQIASKLREQFGNN 356 SEQ ID NO: 9 HS—QP-I-NKRPRQAWCWFKGNWTEAIKEVKR I IKHPRYKGGAKNI S 370

366 376 386 396 406

SEQ ID NO:l K I IFKQSSGGDPEIVTHSFNCGGEFFYCNS QLFNS WFNS WS EGSN 406 SEQ ID NO: 9 VKLVSEHGKGSDPETTYMWTNCRGEFLYCNMTWFLN WVENKTN 413

416 426 436 446 456

SEQ ID NO:l NTEGSDTITLPCRIKQI INMWQKVGKAMYAPPISGQIRCSSNI GLLLTR 456 SEQ ID NO: 9 TT RRNYAPCHIRQI INTWHKVGKNIYLPPREGELSCNSTVTSLIA— 458

466 475 485 495 504

SEQ ID NO:l DGGNSNNESEIFRPGGG-DMRDNWRSELYKYKVVKIEPLGVAPTKAKRR- 504 SEQ ID NO: 9 NINSDNSTTNISVSAEVSELYRLELGDYKLVEITPIGFAPTDV-RRY 504

512 522 532 542 552

SEQ ID NO:l —VVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQ 552 SEQ ID NO: 9 SSVKPRNKRGVMVLG-FLGFLAMAGSAMGATSLTLSAQSRTLLAGIVQQQ 553 562 572 582 592 602

NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKL 602 QQPVDVVKRQQELLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQ 603

612 622 632 642 652

ICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQ 652 VCHTTVPW PNETLTPNWNNMTWQQWEKQVHFLDANITALLEEAQIQ 649

662 672 682 692 702

QEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVL 702 QEKNMYELQKINSWDVFGNWFDLTSWIKYIHLGLYIVAGLVVLRIVVYIV 699

712 717 725 735 745

SIVNRVRQGY SPLSFQTHLPTP—RGPDRPEGIEEEGGERDRDRS 745

QMLARLRKGYRPVFSSPPSYTQQIPIRKDRGQPANEETEEGGGNDGDYRS 749

755 768 778 788

IRLVNGSLALIWDDLRSL CLFSYHRLRDLLLIVTRIVELLGRR 788

WPWQIEYIHFLLRQLRNLLIWLYNGCRTLLLKTFQILHQISTNLQPLRLP 799

798 808 818 828 838

GWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACR 838 VAYLQYGISWFQEALRAAARATGETLASAGETLWEAL RRAAR 841

848

AIRHIPRRIRQGLERILL* 857

AI IAIPRRIRQGLELTLL* 860

SEQ ΝΟ:1 = HxB2; SEQ NO : 2 = BG505 SOSIP; SEQ NO: 3 = BG505 SOSIP E64K ; SEQ NO : 4 = BG505 SOSIP H66R; SEQ NO: 5 = BG SOSIP A316W; SEQ NO : 6 = HIV-1 M: B_HXB2R; SEQ NO : 7 = HIV-1 M:B_HXB2R

40 50 60 70 80 90

I I I I I I I I I I I I ..

SEQ ID NO: 1 MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEWLVNVTEN

SEQ ID NO: 2 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEE SEQ ID NO: 3 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETKKHNVWATHACVPTDPNPQEIHLENVTEE SEQ ID NO: 4 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKRNVWATHACVPTDPNPQEIHLENVTEE SEQ ID NO: 5 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEE

10 SEQ ID NO: 6 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATCACVPTDPNPQEIHLENVTEE SEQ ID NO: 7 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHCCVPTDPNPQEIHLENVTEE

100 110 120 130 140 150 160 170 180

.. I I I I I I I I ... .1 I I I I I I I I I

15 SEQ ID NO:l FNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTD-LKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLD

SEQ ID NO: 2 FNMWKNNMVEQMH D11 SLWDQSLKPCVKLTPLCVTLQCTNVTNNI DDMRGELKNCSFNMTTELRDKKQKVYSLFYRLD

SEQ ID NO: 3 FNMWKNNMVEQMHTDI ISLWDQSLKPCVKLTPLCVTLQCTNVTNNIT DDMRGELKNCSFNMTTELRDKKQKVYSLFYRLD

SEQ ID NO: 4 FNMWKNNMVEQMHTD11 SLWDQSLKPCVKLTPLCVTLQCTNVTNNIT DDMRGELKNCSFNMTTELRDKKQKVYSLFYRLD

SEQ ID NO: 5 FNMWKNNMVEQMHTD11 SLWDQSLKPCVKLTPLCVTLQCTNVTNNIT DDMRGELKNCSFNMTTELRDKKQKVYSLFYRLD

20 SEQ ID NO: 6 FNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNIT DDMRGELKNCSFNMTTELRDKKQKVYSLFYRLD

SEQ ID NO: 7 FNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNIT DDMRGELKNCSFNMTTELRDKKQKVYSLFYRLD

190 200 210 220 230 240 250 260 270

I I I I I I I I I I I I I I I I I I .

SEQ ID NO: 1 IIPIDNDTT SYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEW

SEQ ID NO: 2 WQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVM

SEQ ID NO: 3 WQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVM

SEQ ID NO: 4 WQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVM SEQ ID NO: 5 WQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVM SEQ ID NO: 6 WQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVM SEQ ID NO: 7 WQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVM

10

280 290 300 310 320 330 340 350 360 ... I I I I I I I I I I ... .1 I I I I I I I I I

SEQ ID NO: 1 IRSVNFTDNAK I IVQLN SVEINCTRPNNNTRKRIRIQRGPGRAFV IGKI~GNMRQAHCNISRAKWNNTLKQIASKLREQFGNNK I IFKQSSGGDPE

SEQ ID NO: 2 IRSENI NNAKNILVQFN PVQINCTRPNNNTRKSIRI~~GPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNN I IRFANSSGGDLE

15 SEQ ID NO: 3 IRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRI—GPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRFANSSGGDLE

SEQ ID NO: 4 IRSENI NNAKNILVQFN PVQINCTRPNNNTRKSIRI~~GPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNN I IRFANSSGGDLE

SEQ ID NO: 5 IRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRI—GPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRFANSSGGDLE

SEQ ID NO: 6 IRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRI—GPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRFANSSGGDLE

SEQ ID NO: 7 IRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRI—GPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRFANSSGGDLE

20

380 390 400 410 420 430 440 450 460

I I I I I I I I I I I I I I I I I I .. . I ..

SEQ ID NO: 1 IVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQI INMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNN-ESEI SEQ ID NO: 2 VTTHSFNCGGEFFYCN SGLFNS WISN SVQ~GSNS GSNDSI LPCRIKQI INMWQRIGQAMYAPPIQGVIRCVSNI GLILTRDGGS NS ~ ET

SEQ ID NO: 3 VTTHSFNCGGEFFYCN SGLFNS WISN SVQ~GSNS GSNDSI LPCRIKQI INMWQRIGQAMYAPPIQGVIRCVSNI GLILTRDGGS NS ~ ET

SEQ ID NO: 4 VTTHSFNCGGEFFYCN SGLFNS WISN SVQ~GSNS GSNDSI LPCRIKQI INMWQRIGQAMYAPPIQGVIRCVSNI GLILTRDGGS NS ~ ET SEQ ID NO: 5 VTTHSFNCGGEFFYCN SGLFNS WISN SVQ~GSNS GSNDSI LPCRIKQI INMWQRIGQAMYAPPIQGVIRCVSNI GLILTRDGGS NS ~ ET SEQ ID NO: 6 VTTHSFNCGGEFFYCN SGLFNS WISN SVQ~GSNS GSNDSI LPCRIKQI INMWQRIGQAMYAPPIQGVIRCVSNI GLILTRDGGS NS ~ ET SEQ ID NO: 7 VTTHSFNCGGEFFYCN SGLFNS WISN SVQ~GSNS GSNDSI LPCRIKQI INMWQRIGQAMYAPPIQGVIRCVSNI GLILTRDGGS NS ~ ET

10

470 480 490 500 510 520 530 540 550 560 .. I I I I I I I I I . .1 I I I I I I I I I

SEQ ID NO: 1 FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQREKR—AVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQH

SEQ ID NO: 2 FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQH

15 SEQ ID NO: 3 FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQH

SEQ ID NO: 4 FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQH

SEQ ID NO: 5 FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQH

SEQ ID NO: 6 FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQC

SEQ ID NO: 7 FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQH

20

a a a a a a a

H H H H H H H

CO CO CO CO CO CO CO SEQ ID NO: 100 BG505 SOSIP.664

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 101 BG505 IP.664

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRVVGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNRNLSEIWDNMTWLQWDKEI SNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 102 BG505 IP.664 H72C-H564C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRVVGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQCLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNRNLSEIWDNMTWLQWDKEI SNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 103 BG505 IP.664 A73C-A558C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRVVGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRCPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNRNLSEIWDNMTWLQWDKEI SNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 104 BG505 IP.664 A73C-A561C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRVVGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 105 BG505 IP.664 A73C-A568C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRVVGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKCTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNRNLSEIWDNMTWLQWDKEI SNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 106 BG505 SOSIP.664 H66R

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKRNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 107 BG505 SOSIP.664 E64K

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETKKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 108 BG505 SOSIP.664 A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 109 BG505 SOSIP.664 H66R A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKRNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 110 BG505 SOSIP.664 E64K A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETKKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 111 BG505 SOSIP.664 H72C-H564C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQCLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 112 BG505 SOSIP.664 A73C-A561C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 113 BG505 SOSIP.664 H72C-H564C E64K A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETKKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQCLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEI SNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 114 BG505 SOSIP.664 A73C-A561C E64K A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETKKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 115 BG505 SOSIP.664 N554K

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSKLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 116 BG505 SOSIP.664 L555K

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNKLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 117 BG505 SOSIP.664 L556K

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLKRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 118 BG505 SOSIP.664 E49C-L555C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDACTTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNCLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 119 BG505 SOSIP.664 E49C-L556C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDACTTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLCRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 120 BG505 SOSIP.664 T50C-L555C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAECTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNCLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 121 BG505 SOSIP.664 T50C-L556C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAECTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLCRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 122 BG505 SOSIP.664 T51C-N554C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETCLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSCLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 123 BG505 SOSIP.664 H72C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 124 BG505 SOSIP.664 H72C-554C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSCLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 125 BG505 SOSIP.664 H72C-N554C

SEQ ID NO: 126 BG505 SOSIP.664 H72C-L555C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNCLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 127 BG505 SOSIP.664 H72C-L556C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLCRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 128 BG505 SOSIP.664 H72C-A558C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRCPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 129 BG505 SOSIP.664 H72C-P559C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRACEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 130 BG505 SOSIP.664 H72C-E560C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPCAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 131 BG505 SOSIP.664 H72C-A561C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 132 BG505 SOSIP.664 H72C-Q562C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEACQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 133 BG505 SOSIP.664 H72C-Q563C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQCHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 134 BG505 SOSIP.664 H72C-L565C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHCLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 135 BG505 SOSIP.664 H72C-L566C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLCKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 136 BG505 SOSIP.664 H72C-K567C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATCACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLCLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 137 BG505 SOSIP.664 A73C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 138 BG505 SOSIP.664 A73C-L555C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNCLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 139 BG505 SOSIP.664 A73C-L556C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLCRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 140 BG505 SOSIP.664 A73C-A558C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRCPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 141 BG505 SOSIP.664 A73C-P559C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRACEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 142 BG505 SOSIP.664 A73C-E560C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPCAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 143 BG505 SOSIP.664 A73C-A561C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 144 BG505 SOSIP.664 A73C-Q562C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEACQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 145 BG505 SOSIP.664 A73C-Q563C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQCHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 146 BG505 SOSIP.664 A73C-L565C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHCLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 147 BG505 SOSIP.664 A73C-L566C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLCKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 148 BG505 SOSIP.664 A73C-K567C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLCLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 149 BG505 SOSIP.664 A73C-L568C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKCTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 150 BG505 SOSIP.664 A73C-T569C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKTCVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 151 B41 SOSIP.664

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNI NT ITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRAFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQ ITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 152 B41 SOSIP.664 E64K

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNI NT ITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRAFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 153 B41 SOSIP.664 H66R

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVRNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNINNTNITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRAFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 154 B41 SOSIP.664 A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNINNTNITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD* SEQ ID NO: 155 B41 SOSIP.664 L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNI NT ITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRAFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQ ITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARNLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 156 B41 SOSIP.664 E64K A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNI NT ITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 157 B41 SOSIP.664 E64K A316W L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNINNTNITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARNLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 158 B41 SOSIP.664 H66R A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVRNV WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNINNTNITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD* SEQ ID NO: 159 B41 SOSIP.664 H66R A316W L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVR V WATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNI NT ITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNI SKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQ ITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARNLLSGIVQQQNNLLRAPEAQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 160 B41 SOSIP.664 L543N E64K 316W H72C-H564C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATCACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNI NT ITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNI SKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARNLLSGIVQQQNNLLRAPEAQQCMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 161 B41 SOSIP.664 L543N E64K 316W A73C-A561C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATCACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNINNTNITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNI SKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARNLLSGIVQQQNNLLRAPECQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

SEQ ID NO: 162 ZM197M SOSIP.664 E64K V535M

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARMEQLWVTVYYGVPVWKEAKATLFCASDAKAYEKKVRNV WATHACVPTDPNPQEI PLGNVTENFNMWKNDMADQMHEDI I SLWDQSLKPCVKLTPLCVTLNCSDATSNTTKN ATNTNTTSTDNRNATSNDTEMKGEIKNCTFNITTEVRDRKTKQRALFYKLDWPLEEEKNSSSKNSSYKEYRL ISCNTSTITQACPKVSFDPI PIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLA EEEI I IRSENLTDNTKTI IVHLNESVEINCTRPNNNTRKSVRIGPGQAFFATGEI IGDIRQAHCNLSKSNWTT TLKRIEKKLKEHFNNATIKFESSAGGDLEITTHSFNCRGEFFYCNTSGLFNSSLLNDTDGTSNSTSNATITLP CRIKQI INMWQEVGRAMYASPIAGI ITCKSNITGLLLTRDGGNKSAGIETFRPGGGNMKDNWRSELYKYKVVE IKPLGIAPTSCKRRWERRRRRRAAGIGAVILGFLGAAGSTMGAASMMLTVQARQLLSGIVQQQSNLLRAPEA QQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICCTAVPWNTSWSNKSKDEIWDNMTWMQWDRE IDNYTQVIYQLLEVSQNQQEKNENDLLALD* SEQ ID NO: 163 ZM197M SOSIP.664 E64K T316W V535M

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARMEQLWVTVYYGVPVWKEAKATLFCASDAKAYEKKVRNV WATHACVPTDPNPQEI PLGNVTENFNMWKNDMADQMHEDI I SLWDQSLKPCVKLTPLCVTLNCSDATSNTTKN ATNTNTTSTDNRNATSNDTEMKGEIKNCTFNITTEVRDRKTKQRALFYKLDWPLEEEKNSSSKNSSYKEYRL ISCNTSTITQACPKVSFDPI PIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLA EEEI I IRSENLTDNTKTI IVHLNESVEINCTRPNNNTRKSVRIGPGQWFFATGEI IGDIRQAHCNLSKSNWTT TLKRIEKKLKEHFNNATIKFESSAGGDLEITTHSFNCRGEFFYCNTSGLFNSSLLNDTDGTSNSTSNATITLP CRIKQI INMWQEVGRAMYASPIAGI ITCKSNITGLLLTRDGGNKSAGIETFRPGGGNMKDNWRSELYKYKVVE IKPLGIAPTSCKRRWERRRRRRAAGIGAVILGFLGAAGSTMGAASMMLTVQARQLLSGIVQQQSNLLRAPEA QQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICCTAVPWNTSWSNKSKDEIWDNMTWMQWDRE IDNYTQVIYQLLEVSQNQQEKNENDLLALD*

SEQ ID NO: 164 ZM197M SOSIP.664 H66R T316W V535M

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARMEQLWVTVYYGVPVWKEAKATLFCASDAKAYEKEVR V WATHACVPTDPNPQEI PLGNVTENFNMWKNDMADQMHEDI I SLWDQSLKPCVKLTPLCVTLNCSDATSNTTKN ATNTNTTSTDNRNATSNDTEMKGEIKNCTFNITTEVRDRKTKQRALFYKLDWPLEEEKNSSSKNSSYKEYRL ISCNTSTITQACPKVSFDPI PIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLA EEEI I IRSENLTDNTKTI IVHLNESVEINCTRPNNNTRKSVRIGPGQWFFATGEI IGDIRQAHCNLSKSNWTT TLKRIEKKLKEHFNNATIKFESSAGGDLEITTHSFNCRGEFFYCNTSGLFNSSLLNDTDGTSNSTSNATITLP CRIKQI INMWQEVGRAMYASPIAGI ITCKSNITGLLLTRDGGNKSAGIETFRPGGGNMKDNWRSELYKYKVVE IKPLGIAPTSCKRRWERRRRRRAAGIGAVILGFLGAAGSTMGAASMMLTVQARQLLSGIVQQQSNLLRAPEA QQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICCTAVPWNTSWSNKSKDEIWDNMTWMQWDRE IDNYTQVIYQLLEVSQNQQEKNENDLLALD*

SEQ ID NO: 165 ZM197M SOSIP.664 V535M H66R T316W H72C-H564C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARMEQLWVTVYYGVPVWKEAKATLFCASDAKAYEKEVRNV WATCACVPTDPNPQEI PLGNVTENFNMWKNDMADQMHEDI I SLWDQSLKPCVKLTPLCVTLNCSDATSNTTKN ATNTNTTSTDNRNATSNDTEMKGEIKNCTFNITTEVRDRKTKQRALFYKLDWPLEEEKNSSSKNSSYKEYRL ISCNTSTITQACPKVSFDPI PIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLA EEEI I IRSENLTDNTKTI IVHLNESVEINCTRPNNNTRKSVRIGPGQWFFATGEI IGDIRQAHCNLSKSNWTT TLKRIEKKLKEHFNNATIKFESSAGGDLEITTHSFNCRGEFFYCNTSGLFNSSLLNDTDGTSNSTSNATITLP CRIKQI INMWQEVGRAMYASPIAGI ITCKSNITGLLLTRDGGNKSAGIETFRPGGGNMKDNWRSELYKYKVVE IKPLGIAPTSCKRRWERRRRRRAAGIGAVILGFLGAAGSTMGAASVMLTVQARQLLSGIVQQQSNLLRAPEA QQCMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICCTAVPWNTSWSNKSKDEIWDNMTWMQWDRE IDNYTQVIYQLLEVSQNQQEKNENDLLALD*

SEQ ID NO: 166 ZM197M SOSIP.664 V535M H66R T316W A73C-A561C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARMEQLWVTVYYGVPVWKEAKATLFCASDAKAYEKEVRNV WATHCCVPTDPNPQEI PLGNVTENFNMWKNDMADQMHEDI I SLWDQSLKPCVKLTPLCVTLNCSDATSNTTKN ATNTNTTSTDNRNATSNDTEMKGEIKNCTFNITTEVRDRKTKQRALFYKLDWPLEEEKNSSSKNSSYKEYRL ISCNTSTITQACPKVSFDPI PIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLA EEEI I IRSENLTDNTKTI IVHLNESVEINCTRPNNNTRKSVRIGPGQWFFATGEI IGDIRQAHCNLSKSNWTT TLKRIEKKLKEHFNNATIKFESSAGGDLEITTHSFNCRGEFFYCNTSGLFNSSLLNDTDGTSNSTSNATITLP CRIKQI INMWQEVGRAMYASPIAGI ITCKSNITGLLLTRDGGNKSAGIETFRPGGGNMKDNWRSELYKYKVVE IKPLGIAPTSCKRRWERRRRRRAAGIGAVILGFLGAAGSTMGAASVMLTVQARQLLSGIVQQQSNLLRAPEC QQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICCTAVPWNTSWSNKSKDEIWDNMTWMQWDRE IDNYTQVIYQLLEVSQNQQEKNENDLLALD* SEQ ID NO: 167 AMC008 SOSIP.664

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSS ITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASITLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 168 AMC008 SOSIP.664 E64K

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASITLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 169 AMC008 SOSIP.664 H66R

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVRNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASITLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 170 AMC008 SOSIP.664 A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRWFYTTGEI IGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASITLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD* SEQ ID NO: 171 AMC008 SOSIP.664 I535M

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSS ITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 172 AMC008 SOSIP.664 L543Q

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 173 AMC008 SOSIP.664 L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASITLTVQARNLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 174 AMC008 SOSIP.664 E64K A316W

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRWFYTTGEI IGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASITLTVQARLLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD* SEQ ID NO: 175 AMC008 SOSIP.664 I535M L543Q

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSS ITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 176 AMC008 SOSIP.664 I535M L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 177 AMC008 SOSIP.664 A316W I535M L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRWFYTTGEI IGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 178 AMC008 SOSIP.664 E64K A316W I535M L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTKVHNV WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRWFYTTGEI IGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD* SEQ ID NO: 179 AMC008 SOSIP.664 H66R A316W I535M L543N

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVR V WATHACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSI IGPGRWFYTTGEI IGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSS ITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 180 AMC008 SOSIP.664 I535M L543N H66R A316W H72C-H564C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVR V WATCACVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRWFYTTGEI IGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQNNLLRAPEAQQCLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 181 AMC008 SOSIP.664 I535M L543N H66R A316W A73C-A561C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVRNV WATHCCVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRWFYTTGEI IGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSSNITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQNNLLRAPECQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD*

SEQ ID NO: 193 BG505 SOSIP.664 A73C-A561C E64K A316W E49C-L555C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDACTTLFCASDAKAYETKKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSIRIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNCLRAPECQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD* SEQ ID NO: 194 BG505 SOSIP.664 S306L

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKLIRIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI I MWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 195 BG505 SOSIP.664 R308L

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKSILIGPGQAFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 196 BG505 SOSIP.664 E64K A316W S306L R308L

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETKKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKLILIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD

SEQ ID NO: 197 BG505 SOSIP.664 A73C-A561C E64K A316W S306L R308L

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAENLWVTVYYGVPVWKDAETTLFCASDAKAYETKKHNV WATHCCVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI I SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDD MRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIP IHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQ FNTPVQINCTRPNNNTRKLILIGPGQWFYATGDI IGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTI IRF ANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDS ITLPCRIKQI INMWQRIGQAMY APPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRWGR RRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPECQQHLLKLTVWGIKQLQAR VLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALD SEQ ID NO: 198 AMC008 SOSIP.664 I535M L543N H66R A316W A73C-A561C T49C- R557C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARTEKLWVTVYYGVPVWKEACTTLFCASDAKAYDTEVRNV WATHCCVPTDPSPQEVILENVTENFNMWTNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCTNELKNTTKT NNSSWGGEMKNCSFNVTTSIRDKVQKEYALFYKLDIVPI DDDNNTSNYRLINCNTSVITQACPKITFEPIPIQ FCTPAGFAILKCNNKKFNGKGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEVI IRSDNFTDNAKTI IVQLN ESWINCTRPNNNTRKSINIGPGRWFYTTGEIIGDIRQAHCNLSRTQWNNTLKQIAIKLREQFENKTIVFNQS SGGDPEIVMHSFNCGGEFFYCNTTKLFNSTWNDTDIRGNNTEGNDTITI PCRIKQIVNMWQEVGKAMYAPPIR GQIRCSS ITGLLLTRDGGSESNTTEI FRPGGGDMRDNWRSELYKYKWRIEPLGVAPTKCKRRVVQRRRRRR AVGTIGAMFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQNNLLCAPECQQHLLQLTVWGIKQLQARVLAV ERYLRDQQLLGIWGCSGKLICCTSVPWNTSWSNKSLDKIWNNMTWMEWEREIDNYTSLIYTLLEESQNQQEKN EQELLELD

SEQ ID NO: 199 B41 SOSIP.664 L543N E64K 316W A73C-A561C T49C-L555C

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARAAKKWVTVYYGVPVWKEACTTLFCASDAKAYDTKVHNV WATCACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDI I SLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNN STNATI SDWEKMETGEMKNCSFNVTTS IRDKIKKEYALFYKLDWPLENKNNI NT ITNYRLINCNTSVITQ ACPKVSFEPI PIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSEN ITDNAKTI IVQLNEAVEINCTRPNNNTRKSIHIGPGRWFYATGDI IGNIRQAHCNISKARWNETLGQIVAKLE EQFPNKTI IFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQ ITLQCRIKQI INMW QGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTA CKRRWQRRRRRRAVGLGAFILGFLGAAGSTMGAASMALTVQARNLLSGIVQQQNNCLRAPECQQHMLQLTVW GIKQLQARVLAVERYLRDQQLLGIWGCSGKI ICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYT LLEVSQIQQEKNEQELLELD*

Claims

An isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus, the Env polypeptide comprising:

- a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2; wherein said gpl20 or gpl25 polypeptide portion comprises a first cysteine residue at an amino acid position equivalent to amino acid position 49, 50, 51, 71, 72 or 73,

and wherein said gp41 or gp36 ectodomain polypeptide portion comprises a second cysteine residue at an amino acid position equivalent to an amino acid position selected from the group consisting of: 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569 and 570, wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said first and said second cysteine residues form a disulfide bond between said gpl20 and said gp41 polypeptide portions, or between said gpl25 and said gp36 polypeptide portions.

The isolated, recombinant or synthetic polypeptide according to claim 1 , wherein said second cysteine residue is at an amino acid position equivalent to an amino acid position selected from the group consisting of: 554, 555, 556, 557, 558, 561, 564, 565, 566, 568, and 569 of SEQ ID NO: l .

The isolated, recombinant or synthetic polypeptide according to claim 1 or 2, wherein said first and second cysteinse are at the amino acid positions equivalent to a pair of amino acid positions selected from the group consisting of:

a. position 49 and a position selected from the group consisting of positions 555, 556, 557 and 560, and

b. position 50 and a position selected from the group consisting of positions 555 and 556, 557 and

c. position 51 and position 554, and

d. position 72 and a position selected from the group consisting of positions 554, 555, 556,

557, 558, 559, 560, 561, 562, 563, 564, 565, 566, and 567, and

e. position 73 and a position selected from the group consisting of positions 555, 556, 557,

558, 559 ,560, 561, 562, 563, 565, 566, 567, 568, and 569 wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1.

4. The isolated, recombinant or synthetic polypeptide according to any one of claims 1-3, wherein said first cysteine is at a position selected from the group consisting of positions 49, 50 and 51 of SEQ ID NO: 1 , and wherein said second cysteine is at a position selected from the group consisting of positions 554, 555, 556, 557 and 560 of SEQ ID NO: 1.

5. The isolated, recombinant or synthetic polypeptide according to any one of claims 1-3, wherein said first and second cysteines are at the amino acid positions equivalent to a pair of amino acid positions selected from the group consisting of:

- positions 72 and 564, and

- positions 73 and 558, and

- positions 73 and 561, and

- positions 73 and 568,

wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1.

6. An isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus, the Env polypeptide comprising:

- a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one of amino acid residue selected from the group consisting of: a K at a position equivalent to position 64, a R at a position equivalent to position 66, a F or W at a position equivalent to position 302, a F or W at a position equivalent to position 304, a L at a position equivalent to position 306, a F or W at a position equivalent to position 307, a L at a position equivalent to position 308, a Y or K or I or W or F or V at a position equivalent to position 315, a Y or K or I or F or W or V at a position equivalent to position 316, a K at a position equivalent to position 555, a K at a position equivalent to position 556, and an A at a position equivalent to position 137, wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1.

7. The isolated, recombinant or synthetic polypeptide according to claim 6, further comprising an intermolecular disulfide bond between said first and second cysteine residue as defined in any of claims 1-5.

8. The isolated, recombinant or synthetic polypeptide according to any of claims 1-7, wherein said gp41 ectodomain polypeptide portion comprises the amino acid sequence of the amino acids of positions 512-664 of SEQ ID NO: l or an amino acid sequence having sequence identity of at least 70% thereto.

9. The isolated, recombinant or synthetic polypeptide according to any of claims 1-8, wherein said gpl20 polypeptide portion comprises the amino acid sequence of the amino acids of positions 31 -511 of SEQ ID NO: 1 or an amino acid sequence having sequence identity of at least 70% thereto.

10. The isolated, recombinant or synthetic polypeptide according to any of claims 1-9, wherein said Env polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and at least the amino acid sequences corresponding to the gpl40 portions of any of the Env polypeptide sequences of SEQ ID NOs: 10-99.

11. The isolated, recombinant or synthetic polypeptide according to any of claims 1-10, further comprising a disulfide bond between cysteine residues at positions equivalent to positions 501 and 605 of SEQ ID NO 1.

12. The isolated, recombinant or synthetic polypeptide according to any of claims 1-11, comprising further at least one amino acid residue corresponding to an amino acid residue selected from the group consisting of: 25W, 51C, 54C, 60E, 64K, 66N, 66R, 66A, 71C, 112A, 112S, 112R, 114C, 120C, 121W, 121F, 121C, 122C, 125W, 127F, 127W, 128C, 129F, 129C, 134C, 136C, 136N, 139W,140I, 151C, 152C, 153C, 153F, 153W, 154C, 154F, 154W, 155C, 156C, 161C, 162F, 162W, 163F, 163W, 164F, 164W, 165F, 165W, 165Y, 166Y, 166F, 166W, 166C, 167C, 167F, 167E, 167N, 172F, 172W, 175C, 175F, 175W, 176W, 177C, 177W, 178C, 178C, 179C, 179F,

179W, 180F, 180W, 181F, 181W, 182F, 182W, 191F, 191W, 193C, 193F, 193W, 194F, 194W, 194W, 198F, 198W, 199C, 200C, 201C, 202C, 202F, 202W, 203C, 204F, 204W, 210A, 210S, 210R, 21 IK, 220C, 221C, 244C, 293F, 293S, 302F, 302W, 304F, 304W, 306L ,307F, 307W, 308L, 313C, 315Y, 315K, 3151, 315W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, 316V, 317C, 318C, 319W, 320C, , 420C, 421C, 421F,421W, 422F, 422W, 423F, 423W, 430F, 430W,

432F, 432W, 432P, 433P, 433C, 436F, 436W, 437C, 520W, 523C, 535M543N, 543Q, 554K, 555K, 556K, 559C, 560C, 561C, 562C, 567K, 570C, 571C, 574C, 578C, 582C, wherein said amino acid residues are numbered according to SEQ ID NO: l .

13. The isolated, recombinant or synthetic polypeptide according to claim 12, wherein said at least one amino acid residue corresponds to an amino acid residue selected from the group consisting of 302F, 302W, 304F, 304W, 306L ,307F, 307W, 308L, 313C, 315Y, 315K, 3151, 315W, 315F, 315 V, 316Y, 316K, 3161, 316F, 316 W and 316V, said group most preferably consisting of 316F, 316W, 315 W and 315F, wherein said amino acid residues are numbered according to SEQ ID NO: l .

14. The isolated, recombinant or synthetic polypeptide according to claim 12, wherein said at least one amino acid residue corresponds to an amino acid residue selected from the group consisting of: 60E, 64K, 66N, 66R and 66A, wherein said amino acid residues are numbered according to SEQ ID NO: l .

15. The isolated, recombinant or synthetic polypeptide according to any of claims 1-14, having at least one amino acid residue according to claim 13 and at least one amino acid residue according to claim 14.

16. The isolated, recombinant or synthetic polypeptide according to any one of claims 1-15,

comprising a combination of amino acid residues corresponding to a combination of amino acid residues selected from the group consisting of:

- 64K and 316W;

- 125W and 316W; - 535M and 543Q;

- 535M and 543N; - 64K, 316W nd 543N;

- 535M, 543N, 316W and 64K;

- 535M, 543N, 316W and 66R; - 535M, 543N, 66R and 316W;

- 64K and 535M; - 64K, 316W and 535M; - 66R, 316W and 535M;

- 535M and 543Q; - 535M and 543N;

- 316W, 535M and 543N;

- 64K, 316W, 535M and 543N;

- 66R, 316W, 535M and 543N;

- 559P, 316W, 64K, 73C, 561C, 49C and 555C,; - 559P, 535M, 543N, 316W and 64K;

- 559P, 535M, 543Q, 316W and 64K;

- 559P, 535M, 543N, 66R, 316W, 73C and 561C - 64K, 316W, 306L and 308L;

64K, 316W, 72C and 564C;

- 64K, 316W, 73C and 561C;

- 64K, 316W, 73C, 561C, 201C and 433C;

- 64K, 316W, 73C, 561C, 306L and 308L;

- 64K, 316W, 73C, 561C, 49C, and 555C ;

- 66R, 316W, 72C and 564C; - 66R, 316W, 73C and 561C;

-535M, 543N, 66R, 316W, 72C and 564C;

-535M, 543N, 66R, 316W, 73C, 561C, 201C and 433C; and

-535M, 543N, 66R, 316W, 73C, 561C, 49C and 557C, wherein said residues are numbered according to SEQ ID NO: l .

17. The isolated, recombinant or synthetic polypeptide according to any one of claims 1-16, having an amino acid sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID

NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 111, SEQ ID NO: 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO: 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO: 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO: 131, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO: 136, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO: 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO: 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 160, SEQ ID NO: 161, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 180, SEQ ID NO 181, SEQ ID NO: 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO : 196, SEQ ID NO: 197, SEQ ID NO: 198 and SEQ ID NO : 199 and amino acid sequences having sequence identity of at least 70% thereto.

18. The isolated, recombinant or synthetic polypeptide according to any one of the above claims, wherein said Env polypeptide is of HIV-1.

19. A trimer comprising the isolated, recombinant or synthetic polypeptide according to any of claims 1 -18.

20. A virus like particle (VPL) or pseudoparticle comprising the isolated, recombinant or synthetic polypeptide according to any one of claims 1-18 or the trimer according to claim 19.

21. A nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein, wherein said at least 25 contiguous amino acids from said monomeric ferritin subunit protein are joined to a HIV Env protein, such that the nanoparticle comprises HIV Env trimers on its surface.

22. The nanoparticle of claim 21, wherein the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin, an insect ferritin, a fungal ferritin and a mammalian ferritin.

23. The nanoparticle of claim 21 or 22, wherein the monomeric ferritin subunit protein is a

monomeric subunit of a Helicobacter pylori ferritin protein.

24. The nanoparticle according to any of claims 21-23, wherein the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids of an amino acid sequence of the isolated, recombinant or synthetic polypeptide according to any of claims 1-18.

25. The nanoparticle according to any of claims 21-24, wherein the monomeric ferritin subunit protein comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, and 181.

26. The nanoparticle of any of claims 21-25, wherein the fusion protein comprises a linker sequence, preferably having the amino acid sequence GSG.

27. A pharmaceutical composition comprising:

- the isolated, recombinant or synthetic polypeptide according to any one of claims 1-18, or the trimer according to claim 19, or the virus like particle according to claim 20, or the nanoparticle according to any of claims 21-26; and

- a pharmaceutically acceptable carrier, excipient, or diluent, and

- optionally, an adjuvant.

28. The pharmaceutical composition according to claim 27, comprising at least 2 different Env

polypeptides.

29. The pharmaceutical composition according to claim 27 or 28, further comprising an anti- retroviral agent.

30. The isolated, recombinant or synthetic polypeptide according to any one of claims 1-18, the trimer according to claim 19, the virus like particle according to claim 20, the nanoparticle according to any of claims 21-26, or the pharmaceutical composition according to claim 27, 28, or 29 for use in a medical treatment.

31. The isolated, recombinant or synthetic polypeptide according to any one of claims 1-18, the trimer according to claim 19, the virus like particle according to claim 20, the nanoparticle according to any of claims 21-26, or the pharmaceutical composition according to claim 27, 28 or 29, for use in the treatment of an HIV infection or for use in the prevention of an HIV infection.

32. The isolated, recombinant or synthetic polypeptide according to any one of claims 1-18, the trimer according to claim 19, the virus like particle according to claim 20, the nanoparticle according to any of claims 21-26, or the pharmaceutical composition according to claim 27, 28 or 29 for use in the treatment of an HIV-1 infection or for use in the prevention of an HIV-1 infection.

33. A nucleic acid encoding the isolated, recombinant or synthetic polypeptide according to any one of claims 1-18 or the nanoparticle according to any of claims 21-26.

34. A vector comprising the nucleic acid according to claim 33.

35. An isolated or recombinant eukaryotic or prokaryotic host cell comprising the vector of claim 34.

36. Use of a polypeptide having the amino acid sequence according to any of SEQ ID NO: 10-99 as immunogen.

37. Use of a polypeptide having the amino acid sequence according to any of SEQ ID NO: 10-99 or a nucleic acid encoding said polypeptide as an Env polypeptide for introducing at least one mutation which improves its immunogenicity.