WO2010047829A1 - Mutant hepatitis c virus e2 polypeptides for hcv treatment - Google Patents

Mutant hepatitis c virus e2 polypeptides for hcv treatment Download PDF

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WO2010047829A1
WO2010047829A1 PCT/US2009/005785 US2009005785W WO2010047829A1 WO 2010047829 A1 WO2010047829 A1 WO 2010047829A1 US 2009005785 W US2009005785 W US 2009005785W WO 2010047829 A1 WO2010047829 A1 WO 2010047829A1
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polypeptide
mutant
antibody
virus
hcv
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PCT/US2009/005785
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French (fr)
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Mansun Law
Dennis R. Burton
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • mutant hepatitis C virus (HCV) E2 polypeptides and nucleic acid molecules encoding the polypeptides are mutant hepatitis C virus (HCV) E2 polypeptides and nucleic acid molecules encoding the polypeptides.
  • the mutant E2 polypeptides and encoding nucleic acid molecules can be used for diagnosis and therapy of hepatitis C virus (HCV) infection and can be employed as vaccines for the prevention and treatment of HCV infection.
  • the mutant E2 polypeptides also can be employed for the production of antibodies against HCV.
  • HCV infection predisposes the patient to chronic liver cirrhosis, cancer and liver failure. About 85 % of individuals initially infected with HCV become chronically infected. Once established, chronic HCV infection causes an inflammation of the liver, and this can progress to scarring and eventually, liver cirrhosis. Some patients with cirrhosis will go on to develop liver failure or liver cancer. In the United States and Western Europe, the complications of chronic hepatitis and cirrhosis are the most common reasons for liver transplantation. In addition, liver disease caused by HCV is the leading cause of death in patients co-infected with human immunodeficiency virus. Given the large number of infected people worldwide, HCV infection can be a burden on health care systems worldwide.
  • mutant E2 polypeptides Provided herein are mutant E2 polypeptides, compositions containing mutant
  • the polypeptides can be employed for the prophylaxis and therapy of hepatitis C virus infection.
  • the polypeptides can be used as immunogens to elicit antibodies that can protect against infection by a hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • the mutant HCV E2 polypeptides provided herein display conserved neutralizing AR3 epitopes recognized by conformation-dependent cross-neutralizing anti-HCV antibodies.
  • nucleic acid molecules encoding mutant HCV E2 polypeptides and expression vectors containing the nucleic acid molecules for their production.
  • cells that contain nucleic acid molecules encoding mutant HCV E2 polypeptides and cells that contain expression vectors containing the nucleic acid molecules.
  • preparations and pharmaceutical compositions containing a mutant HCV E2 polypeptide are also provided herein.
  • methods of eliciting an immune response in a mammal comprising administering a mutant HCV E2 polypeptide provided herein, methods for determining whether a mammal has been infected with an HCV, and methods for identifying an anti-HCV agent.
  • a mutant HCV E2 polypeptide contains the amino acid sequence which includes, from the amino to the carboxy termini: (1) a first segment that corresponds to amino acid residues 412 to 459 of a select HCV, (2) a second segment that corresponds to amino acid residues 486 to 569 of the select HCV, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the select HCV.
  • the segments of the mutant HCV E2 polypeptide can be linked directly or via a linker, typically a polypeptide linker.
  • mutant HCV E2 polypeptides provided herein contain deletions of amino acid residues corresponding to amino acids 384 to 411 and amino acids 718 to 746 of the fiill-length E2 polypeptide of a select hepatitis C virus and does not include a contiguous sequence of amino acids that corresponds to amino acid residues 411 to 662 of the E2 polypeptide of the select hepatitis C virus.
  • mutant E2 polypeptides provided herein are polypeptides that can specifically or selectively bind to an antibody that is immunospecific for a conformational epitope on the E2 polypeptide that contains at least amino acids 411 to 462.
  • the conformational epitope contains amino acids corresponding to amino acids 412 to 424, 436 to 447 and 523 to 540 of the select hepatitis C virus.
  • the mutant E2 polypeptides provided herein contain (1) a first segment that corresponds to amino acid residues 412 to 459 of the E2 polypeptide of a select hepatitis C virus, (2) a second segment that corresponds to amino acid residues 486 to 569 of the E2 polypeptide of the select hepatitis C virus, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the E2 polypeptide of the select hepatitis C virus, where the segments are linked directly or via a polypeptide linker, with the proviso that the mutant polypeptide does not include a contiguous sequence of amino acids corresponding to all of amino acid residues 411 to 662 of the E2 polypeptide of the select hepatitis C virus; the mutant polypeptide has deletions of amino acid residues corresponding to amino acids 384 to 411 and amino acids 718 to 746 relative to the full-length E2 polypeptide of the select hepatitis C virus.
  • the mutant E2 polypeptides provided herein do not contain the contiguous amino acid residues that correspond to amino acid residues 460 to 485 and/or 570 to 580 of the E2 polypeptide of the select hepatitis C virus.
  • the segments of the mutant E2 polypeptides provided herein can be linked directly or via a linker, such as a polypeptide linker provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope.
  • the linker located between the first and second segments contains 10, 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, or more amino acid residues provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope.
  • the linker located between the second and third segments contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more amino acid residues provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope.
  • the linker located between the first and second segments contains about 26 amino acid residues. In some examples, the linker located between the second and third segments contains about 12 amino acid residues. In some examples, the first and second segments are linked via a linker that comprises amino acids corresponding to amino acids 460 to 485 of the full-length E2 polypeptide of a select hepatitis C virus. In some examples, the second and third segments are linked via a linker that comprises amino acids corresponding to amino acids 570 to 580 of the full-length E2 polypeptide of a select hepatitis C virus.
  • the segments of the mutant E2 polypeptide correspond to amino acid segments in E2 polypeptides in select hepatitis C virus such as subtypes Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p or 6t.
  • the select hepatitis C virus is a subtype 1 a, Ib, or 1 c virus.
  • the select hepatitis C virus is H77, HCV-L2, or HCV-G9.
  • the first segment of the mutant E2 polypeptide provide herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %,
  • the second segment of the mutant E2 polypeptide provide herein is 65 %
  • the third segment of the mutant E2 polypeptide provided herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 581 to 645 of hepatitis C virus H77.
  • the first amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 888-912 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
  • the second amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS : 913-937 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
  • the third amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 938-962 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
  • the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 727-730 and 740-742 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
  • the mutant E2 polypeptide further includes a polypeptide tag, such as amino or carboxy terminal tag.
  • the tag is an N- terminal ubiquitin signal, a poly-histidine sequence, a FLAG (DYKDDDDK) sequence(SEQ ID NO:607), an HA sequence, a myc sequence, a V5 sequence, a chitin binding protein sequence, a maltose binding protein sequence or a glutathione- S-transferase sequence.
  • nucleic acid molecules that encode a mutant E2 polypeptide provided herein.
  • expression vectors that contain a nucleic acid molecule sequence that encodes a mutant E2 polypeptide provided herein.
  • the nucleic acid molecule encoding a polypeptide provided herein is operably linked to an expression control sequence or regulatory sequence.
  • the expression control sequence or regulatory sequence is a promoter.
  • the promoter is a viral promoter, a bacterial promoter or a mammalian promoter.
  • the promoter is a SV40 promoter, a Rous Sarcoma Virus promoter, or a cytomegalovirus immediate early promoter.
  • cells that contain a mutant E2 polypeptide provided herein are also provided herein. Also provided herein are cells that contain a nucleic acid molecule encoding a mutant E2 polypeptide provided herein. Also provided herein are cells that contain an expression vector that contains a nucleic acid molecule sequence encoding a mutant E2 polypeptide provided herein.
  • the cell is a bacterial cell, a mammalian cell, such as, for example, a Chinese hamster ovary cell.
  • the subject is a mammal, such as, but not limited to, mouse, sheep, goat, horse, rabbit, hamster, rat or human.
  • the subject is a human.
  • the method also involves obtaining a sample from the mammal, such as a blood, lymph, or urine sample.
  • the method includes administering to the subject a second dose of the polypeptide at a selected time after the first administration.
  • the methods provided herein further can involve isolating an antibody or antibody-producing cell from the mammal.
  • the antibody is a cross-neutralizing antibody.
  • the antibody is a conformation- dependent antibody.
  • the method further includes fusing the antibody-producing cells from the mammal with a myeloma cell to obtain an antibody-producing hybridoma.
  • the polypeptide is in a pharmaceutical composition that includes a pharmaceutically acceptable carrier. In some examples, the polypeptide is in an amount effective to prevent or treat HCV infection in the mammal.
  • the antibody can be a full length antibody (e.g. an IgG) or an antibody fragment (e.g. a Fab or F(ab')2).
  • the antibody is a monoclonal antibody, a single chain variable fragment (scFv), or other antigen binding fragment.
  • the antibody is a murine antibody.
  • the antibody is a conformation dependent antibody.
  • the antibody is a cross- neutralizing antibody.
  • the method involves administering to the mammal an expression vector that includes a nucleic acid molecule sequence encoding a polypeptide provided herein.
  • the nucleic acid molecule can include a sequence encoding a polypeptide of SEQ ID NO: 866, 867, 868, 869 or 870.
  • the nucleic acid molecule sequence can include the sequence of SEQ ID NO: 874, 875, 876, 877, 878, 879, 880 or 881.
  • the nucleic acid molecule is operably linked to an expression control sequence.
  • the expression control sequence can be a viral, phage, bacterial, or mammalian promoter.
  • the promoter can be a SV40 promoter, a Rous Sarcoma Virus promoter, or a cytomegalovirus immediate early promoter.
  • pharmaceutical compositions that include (1) a mutant E2 polypeptide provided herein, (2) a nucleic acid molecule that encodes a mutant E2 polypeptide provided herein, (3) a vector, such as an expression vector, that includes a nucleic acid molecule sequence encoding a mutant E2 polypeptide provided herein, or (4) an antibody that binds to a mutant E2 polypeptide provided herein, and a pharmaceutically acceptable carrier.
  • immunostimulatory or immunogenic compositions and vaccines that include a mutant E2 polypeptide provided herein or a nucleic acid molecule that encodes a mutant E2 polypeptide provided herein.
  • uses of a mutant E2 polypeptide provided herein or a nucleic acid molecule that encodes a mutant E2 polypeptide for the preparation of a pharmaceutical composition for the treatment or prevention of HCV infection in a subject are examples of a mutant E2 polypeptide provided herein or a nucleic acid molecule that encodes a mutant E2 polypeptide for the preparation of a pharmaceutical composition for the treatment or prevention of HCV infection in a subject.
  • purified preparations of the polypeptides provided herein in which at least 80 % of the polypeptides are in a conformation capable of binding to a conformation-dependent cross-neutralizing antibody.
  • purified preparations of the antibody provided herein in which the antibody is at least 5 % of the antibodies in the preparation are provided herein.
  • the sample can be a cell lysate, for example, a cell lysate from a bacterial, yeast, insect, or mammalian cell.
  • the method also involves separating the polypeptide from the antibody to obtain a preparation that has at least 50 % polypeptides.
  • the polypeptide is separated from the antibody by elution with 0.2 M glycine at pH 2.2; 2M sodium thiocyanate at pH 7.4; or 0.2 M glycine at pH 11.5.
  • the method further involves purifying the polypeptide using size- exclusion chromatography.
  • preparations obtained by a method provided herein in which at least 50 %, at least 75 %, at least 85 %, at least 90 %, or at least 95 % of the polypeptides in the preparation are in a conformation capable of binding to a conformation dependent antibody.
  • the conformation-dependent antibody binds specifically with a conformational epitope that includes: (1) amino acids 396-424 having the sequence TAGLVGLLTPGAKQNIQLINTNGSWHINS (SEQ ID NO: 694); (2) amino acids 436-447 having the sequence GWLAGLFYQHKF (SEQ ID NO: 695), and (3) amino acids 523-540 having the sequence GAPTYSWGANDTDVFVLN (SEQ ID NO: 696).
  • the conformation-dependent antibody binds specifically with a conformational epitope that includes: (1) amino acids 412-424 having the sequence QLINTNGSWHINS (SEQ ID NO: 688); (2) amino acids 436-447 having the sequence GWLAGLFYQHBCF (SEQ ID NO: 695), and (3) amino acids 523-540 having the sequence GAPTYSWGANDTDVFVLN (SEQ ID NO: 696).
  • the conformation-dependent antibody binds specifically with a conformational epitope on the HCV E2 polypeptide.
  • determining whether a mammal has been infected with a HCV involves contacting a sample, such as for example, a blood sample, from the mammal with a polypeptide provided herein and determining whether the polypeptide binds specifically with an antibody from the sample (e.g., a blood sample) of the mammal to form a polypeptide-antibody complex, wherein the presence of the complex indicates that the mammal has been infected with an HCV and the absence of the complex indicates that the mammal has not been infected with the virus.
  • a sample such as for example, a blood sample
  • identifying an anti-HCV agent that involves contacting a candidate agent with the E2 polypeptide provided herein, identifying the candidate agent as an anti-HCV agent if the candidate agent binds to the E2 polypeptide and prevents its binding with a cell receptor such as CD81 or prevents its ability to inhibit viral replication.
  • the purified polypeptides are useful as immunogens for raising broadly neutralizing antibodies against HCV, as reagents for diagnosis of HCV infection and for screening of new anti-HCV antiviral agents.
  • the polypeptides and corresponding coding nucleic acid molecules can be used as polypeptide- and nucleic acid-based vaccines to elicit a protective immune response against HCV.
  • methods of immunizing a mammal against HCV that involves administering a polypeptide- or nucleic acid-based vaccine.
  • FIGS IA-C illustrate properties of anti-HCV E2 Fabs isolated as described herein.
  • FIG. IA illustrates the specificity of anti-E2 Fabs. The binding of Fabs to GST-E1E2 complex and E2 is compared. The GST-E 1E2 fusion protein was captured by a goat anti-GST antibody while soluble E2 and ovalbumin were coated directly onto ELISA plates. Fabs were added to the antigens and subsequently detected with phosphatase-conjugated goat anti-human F(ab)'2 IgG. Recombinant Fabs were produced in cleared lysate of E. coli transformed with the corresponding phagemids.
  • FIG. IA illustrates the specificity of anti-E2 Fabs. The binding of Fabs to GST-E1E2 complex and E2 is compared. The GST-E 1E2 fusion protein was captured by a goat anti-GST antibody while soluble E2 and ovalbumin were coated directly onto ELISA plates
  • IB illustrates competition between MAb AR3A and anti-E2 Fabs.
  • Vaccinia-expressed El E2 was captured onto ELISA wells by lectin and preincubated with saturating concentration of soluble Fabs before the addition of MAb AR3A.
  • Binding of MAb AR3A was detected with a goat anti-human IgG Fc antibody and the % reduction of binding compared to that in the absence of a Fab is shown. Lightly-shaded bars indicate that Fabs bind E2 better than El E2; while bars of medium shading indicate that Fabs bind E1E2 better than E2.
  • FIG. 1C illustrates the inhibition of anti-E2 Fab binding to El E2 by mouse MAb H53.
  • FIG. 2 shows neutralization of HCVpp by human Fabs. Infectivity in Relative
  • RLU Light Units
  • MMV murine leukemia virus
  • GT Ia H77
  • GT Ib H77
  • GT Ib OH8
  • GT Ib GT Ib
  • FIG. 2E J6 (GT 2a)
  • ARl-Fabs B2, Dl & E
  • AR2-Fabs F & G
  • AR3- Fabs Cl, J2, J3 & L4.
  • Control anti-HIV gpl20 Fab bl2; Empty, background infectivity from pseudotype virus generated without Env gene.
  • Dotted lines indicate HCVpp infectivity in the absence of any antibody. Error bars represent SEM calculated from three experiments performed in the same manner.
  • FIG. 3 is a schematic diagram of E2 regions important for binding of AR3- specific antibodies.
  • E2 (residues 384-746) is a transmembrane glycoprotein, and a truncated form of E2 (residues 384-661) can be expressed as a soluble protein that retains its ability to bind cell lines expressing HCV receptors and CD81 -LEL (Michalak, J.P. et al. J. Gen. Virol. 78, 2299-2306 (1997)).
  • the regions that were investigated by antibody competition and alanine mutagenesis are indicated by dotted and solid boxes, respectively.
  • the AR3 discontinuous epitopes include: (1) amino acids 396-424 having the sequence TAGLVGLLTPGAKQNIQLINTNGSWHINS (SEQ ID NO: 694); (2) amino acids 436-447 having the sequence GWLAGLFYQHKF (SEQ ID NO: 695), and (3) amino acids 523-540 having the sequence GAPTY S WGANDTD VF VLN (SEQ ID NO: 696). Important residues in these regions include S424, G523, P525, G53O, D535, V538 and N540. The locations of the N-linked glycans are indicted by branched forks. The hypervariable regions (see Troesch, M. et al. Virology 352, 357-367 (2006) and the transmembrane regions are indicated by the designation HVRs and TM.
  • FIG. 4 illustrates the levels of human MAb in human liver-chimeric mice 24 hours post-administration.
  • Intravenous administration of human serum is the most reliable way to assure delivery of human serum but a stressful procedure: 5 of 18 treated mice did not recover after the procedure.
  • Human IgG in mouse sera were quantified by a quantitative sandwich ELISA using conjugated and unconjugated goat anti-human F(ab)' 2 antibody.
  • FIG. 5 demonstrates passive antibody protection against an HCV population.
  • FIG. 6 is a sequence comparison of a viral quasispecies population in the HCV genotype la-infected human serum.
  • Partial E2 amino acid sequences (residues 384-622) of a total of 40 clones represented by KP S9 (SEQ ID NO: 701), KP R14 (SEQ ID NO: 702), KP S6 (SEQ ID NO: 703), KP S 18 (SEQ ID NO: 704), KP S 16 (SEQ ID NO: 705), KP R8 (SEQ ID NO: 706), KP S20 (SEQ ID NO: 707), KP S4 (SEQ ID NO: 708), KP R3 (SEQ ID NO: 709), KP S3 (SEQ ID NO: 710), KP S 12 (SEQ ID NO: 711), KP S15 (SEQ ID NO: 712), KP S5 (SEQ ID NO: 713), KP R7 (SEQ ID NO: 714), KP Rl 1 (SEQ ID NO: 715), KP Rl (SEQ ID NO: 716), KP R12 (SEQ ID NO: 717), KP
  • the top sequence, clone KP S9 represents the consensus and dominant sequence in this infectious serum.
  • the periods indicate regions of amino acid sequence identity.
  • the frequency of each clone is bracketed.
  • Hypervariable regions (HVRs) are within the dashed-line boxes. Regions important for binding of AR3-antibodies are within the solid-line boxes.
  • the corresponding sequences of isolates H77 (SEQ ID NO: 723) and UKNlbl2.16 (SEQ ID NO: 724), sharing 87% and 75% amino acid identity with KP S9, respectively, are shown for comparison.
  • FIG. 7 is a schematic illustration of a panel of recombinant E2 fragments.
  • Full length E2 (residues 384-746) is shown at the top and the relative locations of N- gl yeans and cysteines are marked by light and darker vertical lines, respectively.
  • the hypervariable region 1 (HVRl) at the N-terminus and transmembrane region at the C- terminus of E2 are shaded.
  • the positions of N- or C-terminal truncation in the mutants are indicated, and the Flag tags are indicated by boxes at the C-termini. Fragments are named according to the primer sets used in gene amplification.
  • disulfide bridges are predicted to form between C1-C16 (i.e. residues C429-C644), C2-C4 (C452-C486), C8-C9 (C552-C564), C13-C14 (C597-C607), and C7-C11 (C508-581) or C7-C12 (C581-585).
  • FIG. 8A-H illustrate the binding properties of E2 fragments.
  • 293T cells were transfected with DNA plasmids encoding the E2 fragments depicted in FIG. 7 and the expression of the corresponding proteins was assayed by sandwich ELISA.
  • ELISA wells were pre-coated with MAbs specific to the 3 different E2 antigenic regions (ARl, AR2 and AR3), or CD81-LEL, to capture the recombinant proteins in serially diluted cell supernatants. The reagents used in the capture are indicated on the left of the bar charts.
  • Bound E2 fragments were detected using the mouse anti-Flag tag M2 MAb (Sigma). Data shown are means of duplicate measurements.
  • E2flr2a produced in the presence of kifunensine was purified using a MAb AR3A- affinity column and was eluted with low pH (0.2 M glycine pH 2.2), 2 M NaSCN (pH 7.4) or high pH (0.2 M glycine pH 11.5) buffer.
  • the different purified E2flr2a monomers were titrated from 4 ⁇ g/mL ( ⁇ 145 nM, 5-fold serial dilution) to investigate their antigenicities.
  • the purified proteins were captured onto microwells precoated with Galanthus nivalis lectins (5 ⁇ g/mL) and the captured proteins detected with the indicated human anti-E2 monoclonal antibodies (MAbs).
  • MAbs human anti-E2 monoclonal antibodies
  • CD81-LEL microwells coated with maltose binding protein (MBP)-fused CD81-large extracellular loop (LEL) (10 ⁇ g/mL) were used to capture the purified proteins and bound proteins were detected with the mouse anti-FLAG tag MAb M2.
  • MBP maltose binding protein
  • LEL CD81-large extracellular loop
  • Bound human or mouse MAbs were detected with peroxidase-conjugated anti-human or anti-mouse secondary antibodies and TMB substrate. The results show that E2flr2a monomers eluted by buffers with different pH are similar antigenically.
  • FIG. 10A-E are graphs illustrating the antigenic properties of E2 ⁇ TM.
  • E2 ⁇ TM produced in the presence of kifunensine was purified using a MAb AR3A- affinity column and was eluted with 2 M NaSCN buffer (pH 7.4). The effect of pH on the antigenicity of the protein was investigated. Purified E2 ⁇ TM monomers were exposed briefly to low or high pH by adding 10-fold excess volume of 0.2M glycine pH 2.2 or pH 11.5, respectively. After 10 minutes, the solutions were neutralized by adding equal volume of 2M Tris-HCl pH 7.4 and treated and untreated E2 ⁇ TM monomers were titrated from 5.5 ⁇ g/mL ( ⁇ 145 nM, 5-fold serial dilution).
  • the purified proteins were captured onto microwells precoated with Galanthus nivalis lectins (5 ⁇ g/mL) and the captured proteins detected with the indicated human anti-E2 monoclonal antibodies (MAbs).
  • MAbs human anti-E2 monoclonal antibodies
  • CD81-LEL microwells coated with maltose binding protein (MBP)-fused CD81 -large extracellular loop (LEL) (10 ⁇ g/mL) were used to capture the purified proteins and bound proteins were detected with the mouse anti-FLAG tag MAb M2.
  • Bound human or mouse MAbs were detected with peroxidase-conjugated anti-human or anti-mouse secondary antibodies and TMB substrate. The results show that pH does not have a significant effect on the antigenicity of E2 ⁇ TM. DETAILED DESCRIPTION
  • hepatitis C virus includes different viral genotypes, subtypes, quasispecies and isolates. It includes any noncytopathic RNA virus that has a single and positive-stranded RNA genome belonging to the Hepacivirus genus of the Flaviviridae family.
  • the term includes different isolates of HCV such as, without limitation, those having polyprotein sequences and accession numbers shown above, as well as any others in the NCBI database. Examples of different genotypes encompassed by this term include, without limitation, genotype 1, 2, 3, 4, 5 and 6, as described in Simmonds et al. (Hepatology 42:962-973). Reference to HCV also includes those of any additional genotypes that are established.
  • Examples of different subtypes include, without limitation, Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p and 6t .
  • the term also includes cell culture HCVs (HCVcc) and pseudotype HCVs (HCVpp), as well as HCV quasispecies. Various HCVs are described by Simmonds P.
  • HCV nucleotide sequences are known in the art. For example, see the Viral Bioinfomatics Research Center (hcvdb.org) and the Hepatitis C Virus database (hcv.lanl.gov/).
  • An "E2" polypeptide is the HCV viral envelope protein that forms a heterodimer with the El glycoprotein through non-covalent interactions.
  • HCV El and E2 envelope glycoproteins are exposed on the viral surface where they function in viral attachment and fusion to target cells.
  • the E2 glycoprotein is residues 384 to 746.
  • the term "mutant" as used in reference to an HCV E2 polypeptide provided herein means that the polypeptide is modified by one or more substitutions or deletions relative to a naturally occurring HCV E2 polypeptide.
  • the mutant polypeptide provided herein is free of sequences in the hypervariable region of the HCV E2 polypeptide, in particular, sequences that correspond to the segment defined by amino acid residues 384 to 395.
  • mutant E2 polypeptides provided herein that contain one or more deletions relative to a naturally occurring E2 are able to fold into a conformation that preserves a conformational epitope, such as for example, a conformational epitope recognized by an AR3A, AR3B, AR3C, or AR3D antibody.
  • Exemplary mutant E2 polypeptides provided herein differ from the corresponding naturally-occurring E2 amino acid sequence in that the mutant E2 polypeptide provided herein does not include one or more segments defined by (1) amino acid residues 384 to 411; (2) amino acid residues 460 to 485; (3) amino acid residues 570-580, (4) amino acid residues 646-661, (5) amino acid residues 662-717 or 718-746, or any combination thereof, relative to the naturally-occurring E2 polypeptide.
  • the mutant E2 polypeptides provided herein also can have one or more amino acid modifications that decrease the immunogenicity of particular immunodominant epitopes, such as for example epitopes recognized by the ARIA and ARlB antibodies.
  • Such modifications include, but are not limited to deletion or substitution of amino acids at positions 416, 417, 483, 484, 485, 538, 540, 544, 545, 547, 549 or any combinations thereof relative to the E2 polypeptide sequence of HCV (e.g. HCV strain H77).
  • the recitation that a sequence of amino acids "corresponds to" particular amino acids from a selected isolate of HCV means the corresponding amino acids in another isolate. Since E2 and other proteins may include slight amino acid variations from isolate-to-isolate, the exact amino acid residues or positions may vary by one or two residues.
  • One of skill in the art readily can align, using standard programs, or by eye, since the amount of variation is minimal, polypeptides from different isolates to identify corresponding segments or residues.
  • a "modification" is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively.
  • Methods for modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies or direct synthesis.
  • deletion when referring to a nucleic acid molecule or polypeptide sequence, refers to the deletion of one or more nucleotides or amino acids compared to a sequence, such as a target polynucleotide or polypeptide or a native or wild-type sequence.
  • insertion when referring to a nucleic acid molecule or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids, within a target, native, wild-type or other related sequence.
  • a nucleic acid molecule that contains one or more insertions compared to a wild-type sequence contains one or more additional nucleotides within the linear length of the sequence.
  • additions to nucleic acid and amino acid molecules describe addition of nucleotides or amino acids onto either termini compared to another nucleic acid or amino acid molecule.
  • substitution refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid molecule or polypeptide with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule.
  • substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule.
  • Substitution mutations compared to a particular polypeptide can be expressed in terms of the number of the amino acid residue along the length of the polypeptide sequence.
  • a modified polypeptide having a modification in the amino acid at the 19 l position of the amino acid sequence that is a substitution of Isoleucine (He; I) for cysteine (Cys; C) can be expressed as Il 9C, Ilel9C, or simply C 19, to indicate that the amino acid at the modified 19 th position is a cysteine.
  • the molecule having the substitution has a modification at He 19 of the unmodified polypeptide.
  • polypeptide refers to a polymer of three or more amino acids, regardless of post-translational modifications such as methylation, glycosylation or phosphorylation.
  • a "peptide” refers to a polypeptide that is from 2 to about or 40 amino acids in length.
  • an “amino acid” is an organic compound containing an amino group and a carboxylic acid group.
  • a polypeptide contains two or more amino acids.
  • amino acids contained in the antibodies provided include the twenty naturally-occurring amino acids (Table 1), non-natural amino acids, and amino acid analogs (e.g., amino acids wherein the ⁇ -carbon has a side chain).
  • the amino acids, which occur in the various amino acid sequences of polypeptides appearing herein are identified according to their well-known, three- letter or one-letter abbreviations (see Table 1).
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are generally in the "L” isomeric form. Residues in the "D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide.
  • amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl- terminus.
  • amino acid residue is defined to include the amino acids listed in the Table of Correspondence (Table 1), modified, non-natural and unusual amino acids.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH 2 or to a carboxyl-terminal group such as COOH.
  • Suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224).
  • naturally occurring amino acids refer to the 20 L-amino acids that occur in polypeptides.
  • non-natural amino acid refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid.
  • Non- naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids.
  • non-natural amino acids are known to those of skill in the art, and include, but are not limited to, 2- Aminoadipic acid (Aad), 3-Aminoadipic acid (Baad), ⁇ -alanine/ ⁇ -Amino-propionic acid (BaIa), 2-Aminobutyric acid (Abu), 4-Aminobutyric acid/piperidinic acid (4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe), 2-Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm), 2,4- Diaminobutyric acid (Dbu), Desmosine (Des), 2,2'-Diaminopimelic acid (Dpm), 2,3- Diaminopropionic acid (Dpr), N-Ethylglycine (EtGIy), N-Ethylasparagine (EtA
  • a "native polypeptide” or a “native nucleic acid” molecule is a polypeptide or nucleic acid molecule, respectively, that can be found in nature.
  • a native polypeptide or nucleic acid molecule can be the wild-type form of a polypeptide or nucleic acid molecule.
  • a native polypeptide or nucleic acid molecule can be the predominant form of the polypeptide, or any allelic or other natural variant thereof.
  • the variant polypeptides and nucleic acid molecules provided herein can have modifications compared to native polypeptides and nucleic acid molecules.
  • the wild-type form of a polypeptide or nucleic acid molecule is a form encoded by a gene or by a coding sequence encoded by the gene. Typically, a wild-type form of a gene, or molecule encoded thereby, does not contain mutations or other modifications that alter function or structure.
  • the term wild-type also encompasses forms with allelic variation as occurs among and between species and virus genotype and subtype variations.
  • a predominant form of a polypeptide or nucleic acid molecule refers to a form of the molecule that is the major form produced from a gene. A "predominant form" varies from source to source.
  • polypeptides for example, by alternative splicing and/or by alternative protein processing.
  • a different polypeptide can be a "predominant form.”
  • numeric terms identifying amino acid residues or positions in a polypeptide i.e. the protein or polypeptide "coordinates," for example, the term “residues 396 to 424," “residue 416,” or “amino acid 416,” are based on the absolute amino acid numbering system for HCV described by Kuiken et al. in Hepatology 44: 1355-1361 (2006), which is incorporated herein by reference in its entirety.
  • the polyprotein sequence of HCV strain WIl is used as a reference in the numbering system, and the first amino acid of the core protein is amino acid residue number 1.
  • Other HCV polyprotein sequences are compared with the H77 polyprotein sequences by sequence alignment. Insertions in other non-H77 sequences are identified using a residue number/alphabet designation relative to the H77 reference. For example, three inserted amino acids in a non-H77 polyprotein sequence inserted between amino acid residues 396 and 397 of the reference H77 sequence would be identified as follows: residue 396a, 396b and 396c.
  • Insertions longer than the length of the alphabet would be identified as follows: ...396x, 396y, 396z, 396aa, 396ab, 396ac, ... 396ax, 396ay, 396az, 396ba, 396bb ... .
  • Deletions in a non-H77 sequence relative to the H77 reference sequence can be indicated by identifying the residue deleted. For example, a missing residue, i.e. a "deletion”, in a non-H77 sequence relative to the H- 77 reference sequence identified in a sequence alignment such as a deletion of amino acid residue 396 is indicated by the term "del 396".
  • a polypeptide coordinate or coordinates such as “amino acid 396,” “residue 396,” or “amino acids 396 to 424,” refer to analogous residues or segments in HCV polyproteins from different isolates, strains, subtypes or genotypes. Analogous residues or segments can be identified by sequence alignment as described below. A similar system is used for identifying HCV nucleotide sequence.
  • amino acid sequences of two or more HCV E2 polypeptides can be compared by alignment using methods known in the art including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M.
  • Two HCV polyprotein sequences can be compared by sequence alignment in a manner to produce the highest degree of sequence similarity or identity. Upon such alignment, sequence identity is determined on a position-by-position basis, e.g., the sequences are "identical" at a particular position if at that position, the amino acid residues are identical. Exemplary methods to determine sequence identity between two sequences are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs.
  • Such programs include, but are not limited to, the GCG program package (Devereux, et ah, Nucleic Acids Research, 12:387 (1984)), BLASTP, BLASTN and FASTA (Altschul et al., J. Molec. Biol., 215:403 (1990)).
  • the BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul et ah, NCVI NLM NIH
  • HCV sequence analysis tools are known and available in the art, such as for example, but not limited to, online tools available from the Hepatitis C Virus (HCV) Database Project provided by the Los Alamos National laboratory (hcv.lanl.gov) and the European HepCVax Cluster provided by the Leiden University Medical Center, The Netherlands (euhcvdb.ibcp.fr).
  • HCV Hepatitis C Virus
  • amino acid 396 or “amino acids 396 to 424" refers to analogous residues in different HCVs including, for example, HCVs of different isolates, strains, species, quasispecies, subtypes or genotypes.
  • antibody refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantly, produced, including any fragment thereof containing at least a portion of the variable region of the immunoglobulin molecule that retains the binding specificity ability of the full-length immunoglobulin.
  • an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site).
  • Antibodies include antibody fragments, such as anti-RSV antibody fragments.
  • the term antibody thus, includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above.
  • Fab fragments such as, but not limited to, Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fab
  • Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b).
  • immunoglobulin type e.g., IgG, IgM, IgD, IgE, IgA and IgY
  • any class e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass e.g., IgG2a and IgG2b.
  • an “antibody fragment” refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody that binds antigen (e.g. one or more CDRs and/or one or more antibody combining sites) and thus retains the binding specificity, and at least a portion of the specific binding ability of the full-length antibody; antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically, e.g. recombinantly produced derivatives. An antibody fragment is included among antibodies.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd' fragments other fragments, including modified fragments (see, for example, Methods in Molecular Biology, VoI 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1 ; p 3-25, Kipriyanov).
  • the fragment can include multiple chains linked together, such as by disulfide bridges and/or by peptide linkers.
  • An antibody fragment generally contains at least or about 50 amino acids and typically at least or about 200 amino acids.
  • an Ig domain is a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond.
  • Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (C L ), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (C H ).
  • VL variable region domain
  • C L constant region domain
  • C H variable region domain
  • Each V L , C L , V H, and CH domain is an example of an immunoglobulin domain.
  • variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies.
  • Each light chain and each heavy chain has one variable region domain, V L and V H , respectively.
  • the variable domains provide antigen specificity, and thus are responsible for antigen recognition.
  • Each variable region contains CDRs that are part of the antigen-binding site domain and framework regions (FRs).
  • antigen-binding domain As used herein, "antigen-binding domain,” “antigen-binding site,” “antigen combining site” and “antibody combining site” are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with cognate antigen.
  • a native conventional full-length antibody molecule has two conventional antigen-binding sites, each containing portions of a heavy chain variable region and portions of a light chain variable region.
  • a conventional antigen-binding site contains the loops that connect the anti-parallel beta strands within the variable region domains.
  • the antigen combining sites can contain other portions of the variable region domains.
  • Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions also are called complementarity-determining regions (CDRs).
  • framework regions are the domains within the antibody variable region domains that are located within the beta sheets; the FRs are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
  • a "constant region” domain is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved than that of the variable region domain.
  • each light chain has a single light chain constant region (C L ) domain and each heavy chain contains one or more heavy chain constant region (C H ) domains, which include, C H I, C H 2, C H 3 and C H 4.
  • C L constant region
  • C H heavy chain constant region
  • Full-length IgA, IgD and IgG isotypes contain CHU CH2, CH3 and a hinge region, while IgE and IgM contain C H I, C H 2, C H 3 and C H 4.
  • C H I and C L domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms.
  • Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g. through interactions with various cells, biomolecules and tissues.
  • the term "binds specifically” or “specifically binds,” with reference to an antibody/antigen interaction means that the antibody binds with a particular antigen without substantially binding to other unrelated antigens.
  • the antibody has at least 50 % or greater affinity, such as about 75 % or greater affinity, such as, for example, about 90 % or greater affinity, to a particular polypeptide than to other unrelated polypeptides.
  • an antibody that specifically binds (or that immunospecifically binds) to a virus antigen or virus is one that binds to a virus antigen (or to the antigen in the virus or to the virus) with an affinity constant (K 3 ) of about or IxIO 7 M '1 or Ix 10 8 M ' 'or greater (or a dissociation constant (K d ) of Ix 10 "7 M or IxIC 8 M or less).
  • Affinity and dissociation constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11 :54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336
  • neutralize means that the antibody can prevent or reduce HCV infection or replication in a cell culture or in a mammal, as well as alleviate one or more symptoms associated with HCV infection in a mammal.
  • reduce means a decrease in any amount such as a 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 % or more than 65 %.
  • HCV infection or replication can be detected by examining HCV RNA levels, virus particles count or clinical symptoms associated with HCV infection using methods known to those of skill in the art.
  • Whether an antibody will prevent or reduce HCV infection or replication or alleviate associated symptoms can be determined using methods known in the art, as well as the methods described herein, including determining the level of HCV RNA in a sample from a mammal that has been infected with HCV or detecting reduction of signals from a reporter gene encoded by the virus such as, for example, the relative light unit (RLU) for luciferase or the mean fluorescence intensity (MFI) of green fluorescent protein (GFP).
  • RLU relative light unit
  • MFI mean fluorescence intensity
  • an antibody will bind selectively to HCV and neutralize it can be determined using methods known in the art, as well as the methods described herein, including determining the level of HCV RNA or detecting reduction of signals from a reporter gene encoded by the virus such as, for example, the relative light unit (RLU) for luciferase or the mean fluorescence intensity (MFI) of green fluorescent protein (GFP).
  • RLU relative light unit
  • MFI mean fluorescence intensity
  • GFP green fluorescent protein
  • epitopic determinants typically comprise chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds, and is made up of one or more segments of amino acids.
  • An epitope can be a linear or conformational epitope, and can be continuous or discontinuous. Typically, linear epitopes are continuous, i.e. made up of one continuous stretch of amino acids. Conformational epitopes can be discontinuous i.e. made up of two or more discontinuous segments of amino acids that come together to form an epitope when the antigen is folded. Methods for determining whether antibodies bind to the same epitope are known in the art. Epitopes can be defined or mapped by standard methods well known in art. For example, epitopes can be mapped using assays, such as ELISA assays, utilizing peptide libraries or site-directed mutagenesis of the antigen (such as alanine-scanning of the antigen).
  • binds to the same epitope with reference to two or more antibodies means that the antibodies compete for binding to an antigen and bind to the same, overlapping or encompassing continuous or discontinuous segments of amino acids.
  • Those of skill in the art understand that the phrase "binds to the same epitope” does not necessarily mean that the antibodies bind to exactly the same amino acids.
  • the precise amino acids to which the antibodies bind can differ.
  • a first antibody can bind to a segment of amino acids that is completely encompassed by the segment of amino acids bound by a second antibody.
  • a first antibody binds one or more segments of amino acids that significantly overlap the one or more segments bound by the second antibody.
  • such antibodies are considered to "bind to the same epitope.”
  • Antibody competition assays can be used to determine whether an antibody "binds to the same epitope" as another antibody. Such assays are well known in the art and are described herein (see, e.g. Examples 1-4). Typically, competition of 70 % or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more, of an antibody known to interact with the epitope by a second antibody under conditions in which the second antibody is in excess and the first saturates all sites, is indicative that the antibodies "bind to the same epitope.” To assess the level of competition between two antibodies, for example, radioimmunoassays or assays using other labels for the antibodies, such as biotin (see, e.g., Example 1) can be used.
  • an HCV antigen such as the E1E2 complex
  • a saturating amount of a first anti-HCV antibody or antigen-binding fragment thereof conjugated to a labeled compound e.g., 3 H, 125 I or biotin
  • a labeled compound e.g. 3 H, 125 I or biotin
  • the amount of labeled antibody that is bound to the antigen in the presence of the unlabeled blocking antibody is then assessed and compared to binding in the absence of the unlabeled blocking antibody. Competition is determined by the percentage change in binding signals in the presence of the unlabeled blocking antibody compared to the absence of the blocking antibody.
  • first and second antibody of 70 % or more such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more
  • first antibody inhibits binding of the second antibody (or vice versa) to the antigen by 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more (compared to binding of the antigen by the second antibody in the absence of the first antibody).
  • inhibition of binding of a first antibody to an antigen by a second antibody of 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more indicates that the two antibodies bind to the same epitope.
  • the term "bind selectively” or “selectively binds,” in reference to a polypeptide or an antibody provided herein, means that the polypeptide or antibody binds with a selected epitope without substantially binding to another epitope.
  • an antibody or fragment thereof that selectively binds to a selected epitope specifically binds to the epitope, such as with an affinity constant (K a ) of about or IxIO 7 M “1 or 1 x 10 8 M-Or greater, as defined below.
  • telomere binding fragment As used herein, “specifically binds” or “immunospecifically binds,” with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of the antibody and the antigen.
  • the antigen can be an isolated antigen or presented in a virus.
  • an antibody that specifically binds to a virus antigen or virus is one that binds to the virus antigen (or to the antigen in the virus or to the virus) with an affinity constant (K a ) of about or IxIO 7 M “1 or Ix 10 8 M '1 or greater (or a dissociation constant (K ⁇ 0 of 1 x 10 '7 M or 1 x 10 "8 M or less).
  • affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst.
  • ITC isothermal titration calorimetry
  • other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of anti-HCV antibodies).
  • Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).
  • an antibody that specifically binds to a virus antigen can bind to other peptides, polypeptides, or proteins or viruses with equal or lower binding affinity.
  • an antibody or antigen-binding fragment thereof provided herein that binds specifically binds to a HCV E2 protein does not cross-react with other antigens or cross reacts with substantially (at least 10-100 fold) lower affinity for such antigens.
  • an antibody or antigen-binding fragment thereof provided herein that binds specifically to a HCV E1E2 complex does not cross-react with other antigens or cross reacts with substantially (at least 10-100 fold) lower affinity for such antigens.
  • An antibody or antigen-binding fragment thereof provided herein that binds selectively to E2 typically also binds to the El E2 complex.
  • Antibodies or antigen- binding fragments that selectively bind to a particular virus antigen can be identified, for example, by immunoassays, such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISAs), surface plasmon resonance, or other techniques known to those of skill in the art.
  • An antibody or antigen-binding fragment thereof that specifically binds to an epitope on a HCV E2 or El E2 typically is one that binds to the epitope (presented in the protein or virus) with a higher binding affinity than to any cross-reactive epitope as determined using experimental techniques, such as, but not limited to, immunoassays, surface plasmon resonance, or other techniques known to those of skill in the art.
  • a recombinantly produced protein such as HCV E1E2
  • the affinity for the antibody or antigen-binding fragments for virus or the antigen as presented in the virus can be determined.
  • the target such as the isolated protein or the virus, will be identified.
  • the term "conformation-dependent,” in reference to an antibody, means that the antibody recognizes and binds specifically with discontinuous epitopes composed of amino acid residues that are located at some distance from each other, i.e. the residues are discontinuous in the polypeptide sequence.
  • the discontinuous epitopes come together through proper folding of the polypeptide to form a binding site, i.e. a conformational epitope that is recognized by a conformation-dependent antibody.
  • cross-neutralizing means the ability to neutralize at least two HCV strains, isolates, species, quasispecies, subtypes or genotypes.
  • neutralize as used herein in reference to an antibody, means that the antibody can prevent or reduce HCV infection or replication in a cell culture or in a mammal, as well as alleviate one or more symptoms associated with HCV infection in a mammal.
  • reduce means a decrease in any amount such as a 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 % or more than 65 %.
  • HCV infection or replication can be detected by examining HCV RNA levels, virus particles count or clinical symptoms associated with HCV infection. Whether an antibody will prevent or reduce HCV infection or replication or alleviate associated symptoms can be determined using methods known in the art, as well as the methods described herein, including determining the level of HCV RNA in a sample from a mammal that has been infected with HCV or detecting reduction of signals from a reporter gene encoded by the virus such as, for example, the relative light unit (RLU) for luciferase or the mean fluorescence intensity (MFI) of green fluorescent protein (GFP).
  • RLU relative light unit
  • MFI mean fluorescence intensity
  • GFP green fluorescent protein
  • linker or “spacer” peptide refers to short sequences of amino acids, such as 2, 5 or 10 to 20, 30, 40, 50, 60, 70 or 80, that join two polypeptide sequences (or nucleic acid encoding such an amino acid sequence).
  • Peptide linker refers to the short sequence of amino acids joining the two polypeptide sequences. Linkers are well-known and any known linkers can be used in the provided methods. Exemplary of polypeptide linkers are (Gly-Ser) n amino acid sequences, with some GIu or Lys residues dispersed throughout to increase solubility. Other exemplary linkers are described herein; any of these and other known linkers can be used with the provided compositions and methods.
  • Linkers with reference to the mutant E2 polypeptides also include all or part of the native sequence joining the segments in the native E2 polypeptide, as long as the resulting polypeptide does not correspond to amino acids about 412-462 of the E2 polypeptide of the particular HCV isolate.
  • a "tag” or an “epitope tag” refers to a sequence of amino acids, typically added to the N- or C- terminus of a polypeptide, such as the polypeptides provided herein.
  • tags fused to a polypeptide can facilitate polypeptide purification and/or detection.
  • a tag or tag polypeptide refers to a polypeptide that has enough residues to provide an epitope recognized by an antibody or can serve for detection or purification, yet is short enough such that it does not interfere with activity of the chimeric polypeptide to which it is linked.
  • the tag polypeptide typically is sufficiently unique so an antibody that specifically binds thereto does not substantially cross-react with epitopes in the polypeptide to which it is linked. Suitable tag polypeptides generally have at least 5 or 6 amino acid residues and usually between about 8-50 amino acid residues, typically between 9-30 residues.
  • the tags can be linked to one or more chimeric polypeptides in a multimer and permit detection of the multimer or its recovery from a sample or mixture. Such tags are well known and can be readily synthesized and designed.
  • Exemplary tag polypeptides include those used for affinity purification and include, histidine (His) tags, the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5, (Field et al.
  • nucleic acid refers to a polymer of deoxyribose nucleic acids (DNA), as well as ribose nucleic acids (RNA).
  • the term includes linear molecules, as well as covalently closed circular molecules. It includes single stranded molecules, as well as double stranded molecules.
  • Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid).
  • RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded nucleic acids.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine.
  • RNA the uracil base is undine.
  • Nucleic acids can contain nucleotide analogs, including, for example, mass modified nucleotides, which allow for mass differentiation of nucleic acid molecules; nucleotides containing a detectable label such as a fluorescent, radioactive, luminescent or chemiluminescent label, which allow for detection of a nucleic acid molecule; or nucleotides containing a reactive group such as biotin or a thiol group, which facilitates immobilization of a nucleic acid molecule to a solid support.
  • a nucleic acid also can contain one or more backbone bonds that are selectively cleavable, for example, chemically, enzymatically or photolytically cleavable.
  • a nucleic acid can include one or more deoxyribonucleotides, followed by one or more ribonucleotides, which can be followed by one or more deoxyribonucleotides, such a sequence being cleavable at the ribonucleotide sequence by base hydrolysis.
  • a nucleic acid also can contain one or more bonds that are relatively resistant to cleavage, for example, a chimeric oligonucleotide primer, which can include nucleotides linked by peptide nucleic acid bonds and at least one nucleotide at the 3' end, which is linked by a phosphodiester bond or other suitable bond, and is capable of being extended by a polymerase.
  • Peptide nucleic acid sequences can be prepared using well-known methods (see, for example, Weiler et al. (1997) Nucleic Acids Res. 25:2792-2799).
  • polynucleotide and “nucleic acid molecule” refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), joined together, typically by phosphodiester linkages.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Polynucleotides also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid).
  • Polynucleotides include single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) as well as analogs or derivatives of either RNA or DNA.
  • RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded polynucleotides.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine.
  • RNA the uracil base is undine.
  • Polynucleotides can contain nucleotide analogs, including, for example, mass modified nucleotides, which allow for mass differentiation of polynucleotides; nucleotides containing a detectable label such as a fluorescent, radioactive, luminescent or chemiluminescent label, which allow for detection of a polynucleotide; or nucleotides containing a reactive group such as biotin or a thiol group, which facilitates immobilization of a polynucleotide to a solid support.
  • a polynucleotide also can contain one or more backbone bonds that are selectively cleavable, for example, chemically, enzymatically or photolytically cleavable.
  • a polynucleotide can include one or more deoxyribonucleotides, followed by one or more ribonucleotides, which can be followed by one or more deoxyribonucleotides, such a sequence being cleavable at the ribonucleotide sequence by base hydrolysis.
  • a polynucleotide also can contain one or more bonds that are relatively resistant to cleavage, for example, a chimeric oligonucleotide primer, which can include nucleotides linked by peptide nucleic acid bonds and at least one nucleotide at the 3' end, which is linked by a phosphodiester bond or other suitable bond, and is capable of being extended by a polymerase.
  • Peptide nucleic acid sequences can be prepared using well-known methods (see, for example, Weiler et al. (1997) Nucleic Acids Res. 25:2792-2799).
  • Exemplary of the nucleic acid molecules (polynucleotides) provided herein are oligonucleotides, including synthetic oligonucleotides, oligonucleotide duplexes, primers, including fill- in primers, and oligonucleotide duplex cassettes.
  • DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
  • a "DNA segment” is a portion of a larger DNA molecule having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5' to 3' direction, encodes the sequence of amino acids of the specified polypeptide.
  • a positive strand polynucleotide refers to the "sense strand" of a polynucleotide duplex, which is complementary to the negative strand or the "antisense” strand.
  • the sense strand is the strand that is identical to the mRNA strand that is translated into a polypeptide, while the antisense strand is complementary to that strand.
  • Positive and negative strands of a duplex are complementary to one another.
  • isolated as used herein with reference to a nucleic acid molecule, means that the nucleic acid molecule is free of unrelated nucleic acid sequences, i.e.
  • nucleic acid sequences encoding other genes or non-E2 polypeptide sequences, or those involved in the expression of such other genes, that flank it's 5' and 3' ends in the naturally-occurring genome of the organism from which the nucleic acid provided herein is derived. Accordingly, an "isolated nucleic acid” provided herein has a structure that is different from that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes.
  • isolated nucleic acid molecule includes, for example, (1) a DNA molecule that has the sequence of part of a naturally occurring genomic DNA molecule, but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (2) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally-occurring vector or genomic DNA; (3) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (4) a recombinant nucleotide sequence that is part of a hybrid gene, i.e.
  • PCR polymerase chain reaction
  • nucleic acids present in mixtures of (1) DNA molecules, (2) transfected cells, and (3) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
  • a regulatory region or an "expression control sequence" of a nucleic acid molecule means a cis-acting nucleotide sequence that influences expression, positively or negatively, of an operatively linked gene.
  • Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration gene expression can be decreased.
  • Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.
  • Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or as part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.
  • Regulatory regions or expression control sequences also include, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and stop codons, leader sequences and fusion partner sequences, internal ribosorne binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.
  • IRIS internal ribosorne binding site
  • a "host cell” is a cell that is used to receive, maintain, reproduce and amplify a vector.
  • a host cell also can be used to express the polypeptide encoded by the vector.
  • the nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.
  • the host cell is a genetic package, which can be induced to express the variant polypeptide on its surface.
  • the host cell is infected with the genetic package.
  • the host cells can be phage-display compatible host cells, which can be transformed with phage or phagemid vectors and accommodate the packaging of phage expressing fusion proteins containing the variant polypeptides.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation.
  • Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide.
  • the vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression/display of the polypeptide encoded by the nucleic acid.
  • the vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome.
  • vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
  • a vector also includes "virus vectors” or “viral vectors.”
  • viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.
  • an "expression vector” is a nucleic acid molecule capable of transporting and/or allowing for the expression of another nucleic acid to which it has been linked.
  • Expression vectors contain appropriate expression control sequences that direct expression of a nucleic acid that is operably linked to the expression control sequence to produce a transcript.
  • the product of that expression is referred to as a messenger ribose nucleic acid (mRNA) transcript.
  • mRNA messenger ribose nucleic acid
  • the expression vector also can include other sequences such as enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modified polyadenylation and transcriptional termination sequences, e.g. bovine growth hormone or rabbit beta-globulin polyadenylation sequences, to improve expression of the nucleic acid encoding the E2 polypeptide.
  • TPL adenovirus tripartite leader
  • expression refers to the process by which polypeptides are produced by transcription and translation of polynucleotides.
  • the level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA 5 Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.
  • the term "purified" with reference to a polypeptide or antibody preparation means that the polypeptide or antibody in the preparation is substantially free of naturally-associated components, i.e. components that accompany it in its natural state.
  • the term “purified” also encompasses a biological sample such as a blood sample that has been subject to at least one separation step, for example, centrifugation to separate cellular components from non- cellular components.
  • a polypeptide provided herein can constitute at least about 25 % by weight of a sample containing the polypeptide provided herein, and usually constitutes at least about 50%, at least about 75 %, at least about 85 %, at least about 90 % of a sample, particularly at least about 95 % of the sample or 99 % or more.
  • prevent refers to use in a prophylactic manner that includes, for example, preventing a new infection or viral replication or reducing the probability of infection, as well as preventing the onset of symptoms and/or their underlying cause.
  • the terms “treat,” “treating” and “treatment,” include reducing viral replication, reducing the severity and/or frequency of symptoms, eliminating the symptoms and/or underlying cause or improving or remediating damage associated with the infection.
  • reduce means a decrease in any amount, for example, a decrease of 5 %, 10 %, 20 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more than 90 %.
  • a "pharmaceutically effective agent” includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.
  • a "therapeutic effect” means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition.
  • a “therapeutically effective amount” or a “therapeutically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.
  • therapeutic efficacy refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, or composition containing a compound has been administered.
  • a prophylactically effective amount or a “prophylactically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses.
  • a prophylactically effective amount can be administered in one or more administrations.
  • amelioration of the symptoms of a particular disease or disorder by a treatment refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.
  • diagnosisically effective amount refers to the quantity of an agent, compound, material, or composition containing a detectable compound that is at least sufficient for detection of the compound following administration to a subject.
  • a diagnostically effective amount of an anti- RSV antibody or antigen-binding fragment thereof such as a detectably-labeled antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment thereof that can be detected by a secondary agent, administered to a subject for detection is the quantity of the antibody or antigen-binding fragment thereof which is sufficient to enable detection of the site having the HCV antigen for which the antibody or antigen-binding fragment thereof is specific.
  • a detectably labeled antibody or antigen-binding fragment thereof is given in a dose which is diagnostically effective.
  • a label or detectable moiety is a detectable marker (e.g., a fluorescent molecule, chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product) that can be attached or linked directly or indirectly to a molecule or associated therewith and can be detected in vivo and/or in vitro.
  • a detectable marker e.g., a fluorescent molecule, chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product
  • a detectable marker e.g., a fluorescent molecule, chemiluminescent molecule
  • the detection method can be any method known in the art, including known in vivo and/or in vitro methods of detection (e.g., imaging by visual inspection, magnetic resonance (MR) spectroscopy, ultrasound signal, X-ray, gamma ray spectroscopy (e.g., positron emission tomography (PET) scanning, single-photon emission computed tomography (SPECT)), fluorescence spectroscopy or absorption).
  • Indirect detection refers to measurement of a physical phenomenon, such as energy or particle emission or absorption, of an atom, molecule or composition that binds directly or indirectly to the detectable moiety.
  • the term "subject" refers to an animal, including a mammal, such as a human being.
  • a patient refers to a human subject.
  • animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and other animals.
  • rodents such as mice and rats
  • fowl such as chickens
  • ruminants such as goats, cows, deer, sheep
  • ovine such as pigs and other animals.
  • Non-human animals exclude humans as the contemplated animal.
  • the enzymes provided herein are from any source, animal, plant, prokaryotic and fungal. Most enzymes are of animal origin, including mammalian origin.
  • pharmaceutically acceptable it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof, for example, a buffered aqueous, oil or organic medium containing optional stabilizing agents and adjuvants for stimulation of immune binding.
  • unit dose form refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.
  • a single dosage formulation refers to a formulation for direct administration.
  • an “article of manufacture” is a product that is made and sold.
  • Fluids refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
  • composition refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.
  • a "combination" refers to any association between or among two or more items.
  • the combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof.
  • the elements of a combination are generally functionally associated or related.
  • combination therapy refers to administration of two or more different therapeutics, such as two or more different anti-RSV antibodies and/or anti- RSV antibodies and antigen-binding fragments thereof.
  • the different therapeutic agents can be provided and administered separately, sequentially, intermittently, or can be provided in a single composition.
  • kits are packaged combinations that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof, for a purpose including, but not limited to, activation, administration, diagnosis, and assessment of a biological activity or property.
  • the singular forms "a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
  • reference to a polypeptide, comprising "an immunoglobulin domain” includes polypeptides with one or a plurality of immunoglobulin domains.
  • the term “or” is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
  • ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 amino acids” means “about 5 amino acids” and also “5 amino acids.”
  • “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • an optionally variant portion means that the portion is variant or non- variant.
  • the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem.
  • mutant E2 polypeptides Provided herein are mutant E2 polypeptides, compositions containing them, as well as methods for their production, purification and use.
  • the polypeptides provided herein can be used as immunogens to elicit antibodies that can protect against infection by a hepatitis C virus (HCV).
  • HCV E2 polypeptides provided herein display the conserved neutralizing AR3 epitopes recognized by conformation- dependent cross-neutralizing anti-HCV antibodies.
  • mutant E2 polypeptides displaying conserved neutralizing epitopes, nucleic acids encoding these polypeptides and expression vectors for their production.
  • cells comprising such nucleic acids or expression vectors, a preparation or pharmaceutical composition comprising a mutant HCV E2 polypeptide, as well as (1) a method of eliciting an immune response in a mammal comprising administering a mutant HCV E2 polypeptide, (2) a method for determining whether a mammal has been infected with an HCV, and (3) a method for identifying an anti-HCV agent.
  • a preparation or pharmaceutical composition comprising a mutant HCV E2 polypeptide
  • Hepatitis C virus is a noncytopathic, positive-stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family that causes acute and chronic hepatitis and hepatocellular carcinoma (Hoofhagle, J. H. (2002) Hepatology 36, S21-29).
  • the hepatocyte is the primary target cell, although various lymphoid populations, especially B cells and dendritic cells also can be infected at lower levels (Kanto et al, Immunol. 162, 5584-5591 (1999); Auffermann-Gretzinger et al, Blood 97, 3171-3176 (2001); Hiasa et al. (1998) Biochem. Biophys. Res. Commun.
  • HCV infection A striking feature of HCV infection is its tendency towards chronicity with at least 70 % of acute infections progressing to persistence (Hoofhagle, J. H. (2002) Hepatology 36, S21-29). HCV chronicity is often associated with significant liver disease, including chronic active hepatitis, cirrhosis and hepatocellular carcinoma (Alter, H. J. & Seeff, L. B. (2000) Semin. Liver Dis. 20, 17-35). With over 170 million people currently infected (id.), HCV represents a growing public health concern.
  • HCV viruses can be categorized into several genotypes and subtypes.
  • Exemplary HCV genotypes include, but are not limited to, genotype 1, 2, 3, 4, 5 and 6.
  • Exemplary of HCV subtypes include, but are not limited to, Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p and 6t .
  • the single stranded HCV RNA genome has a single open reading frame (ORF) encoding a large polyprotein.
  • the polyprotein has about 3010-3033 amino acids (Q.-L. Choo, etal. Proc. Natl. Acad. ScL USA 88, 2451-2455 (1991); N. Kato et al., Proc. Natl. Acad. Sci. USA 87, 9524-9528 (1990); A. Takamizawa et al., J. Virol. 65, 1105-1113 (1991)).
  • Nucleic acid and amino acid sequences for different isolates of HCV can be found in the art, for example, in the National Center for Biotechnology Information (NCBI) database (see ncbi.nlm.nih.gov).
  • HCV subtype 1 a is strain H77, which can be found in the NCBI database as accession number AF009606. Its polyprotein sequence (AAB66324) is as follows:
  • HCV subtype Ib is strain HCV-L2, which can be found in the NCBI database as accession number UOl 214 (gi 437107). Its polyprotein sequence (AAA75355 ) is as follows:
  • HCV polyprotein sequences are known in the art and can be used to generate the mutant HCV E2 polypeptide provided herein (see for example, hcvdb.org/viruses.asp; .ncbi.nlm.nih.gov; and hcv.lanl.gov and HCV sequence databases referenced in Kuiken et al. (2005) Bioinformatics 21 (3):379-84 and Yusim et al. (2005) Applied Bioinformatics 4(4)). Additional examples include a Taiwan isolate of hepatitis C virus available in the NCBI database at accession number P29846 (gi: 266821).
  • HCV polyprotein sequences include, but are not limited to, NCBI accession number AF009606, AY734971, AJ238799, AY545953, AY734974, AB047639, AFl 77036, AY734977, AY734982, AY734984, AY734987, EF427672, and AY736194.
  • NCBI accession number AF009606, AY734971, AJ238799, AY545953, AY734974, AB047639, AFl 77036, AY734977, AY734982, AY734984, AY734987, EF427672, and AY736194 C. Mutant E2 Polypeptides
  • mutant HCV E2 polypeptides differs from a naturally-occurring E2 polypeptide of HCV in that the mutant E2 polypeptide provided herein has amino acid deletions relative to the naturally-occurring E2 polypeptide.
  • Exemplary regions of an E2 polypeptide that can be removed include, for example, the region defined by amino acid residues corresponding to 384-411, 460 to 485, 570-580, 646-647, 648-661, 662-717 or 718- 746, (the amino acid positions corresponding to the amino acid positions in an HCV polyprotein) or any combination thereof can be deleted from a naturally-occurring E2 polypeptide of HCV to generate a mutant E2 polypeptide provided herein.
  • a mutant E2 polypeptide provided herein thus has an amino acid sequence that comprises, from the amino to the carboxy termini: (1) a first segment that corresponds to amino acid residues 412 to 459 of a select hepatitis C virus polyprotein, (2) a second segment that corresponds to amino acid residues 486 to 569 of the select hepatitis C virus polyprotein, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the select hepatitis C virus polyprotein.
  • one or more immunodominant epitopes in the naturally-occurring E2 polypeptide are eliminated or its immunogenicity to particular epitopes attenuated, while the immunogenicity of conserved or cross-neutralizing epitopes are augmented.
  • immunodominant epitopes such as, for example, the hypervariable region 1 (amino acid residues 384 to 412) or the epitopes recognized by the ARIA and ARlB antibodies that include the residues T416, T416, N417, R483, P484, Y485, V538, N540, P544, P545, G547 and W549.
  • mutant E2 polypeptide provided herein also can have one or more amino acid substitutions or deletions at positions 416, 417, 483, 484, 485, 538, 540, 544, 545, 547, 549 or any combinations thereof relative to the E2 polypeptide sequence of HCV (e.g. corresponding to the HCV strain H77).
  • the segments of the mutant E2 polypeptide can be linked directly or indirectly, in any order, via a linker provided that the polypeptide properly folds to present the conserved HCV E2 conformational epitope.
  • linkers are known in the art and include, for example, polypeptide linkers.
  • the polypeptide linker can link the first and second segments, and the polypeptide linker is at least 10 amino acids, e.g.
  • the polypeptide linker can link the second and third segments, and the polypeptide linker is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more than 28 amino acid residues in length provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope.
  • the first and second segments are linked via a polypeptide linker which is about 26 amino acid residues
  • the second and third segments are linked via a polypeptide linker which is about 12 amino acid residues in length.
  • any HCV E2 polypeptide can be modified to produce the mutant E2 polypeptides provided herein.
  • the amino acid segments can be derived for an HCV E2 polypeptide of any genotype (e.g. 1, 2, 3, 4, 5, or 6) or subtype (e.g. Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p or 6t).
  • Exemplary naturally-occurring HCV E2 sequences that can be modified include, but are not limited to, HCV E2 polypeptides shown in Table 2 below.
  • amino acid sequence segments from select hepatitis C viruses that can be employed to produce the mutant E2 polypeptides provided herein include, but are not limited, to the segments that correspond to the polypeptide segments shown in Table 3.
  • the first segment of the mutant E2 polypeptide provide herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino . acids 412 to 459 of a hepatitis C virus E2 polypeptide, such as H77.
  • the second segment of the mutant E2 polypeptide is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 486 to 569 of a hepatitis C virus E2 polypeptide, such as H77.
  • the third segment of the mutant E2 polypeptide provided herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 581 to 645 of a hepatitis C virus E2 polypeptide, such as H77.
  • the first amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 888-912 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to any one of SEQ ID NOS: 888-912.
  • the second amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 913-937 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to any one of SEQ ID NOS: 913- 937.
  • the third amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 938-962 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to any one of SEQ ID NOS: 938-962.
  • the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 727 '-730 and 740-742 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %,
  • Exemplary mutant E2 polypeptides provided herein differ from the corresponding naturally-occurring E2 amino acid sequence in that the mutant E2 polypeptide provided herein does not include one or more segments defined by (1 ) amino acid residues 384 to 411 of the hypervariable region 1 of the naturally- occurring E2 polypeptide; (2) amino acid residues 460 to 485; (3) amino acid residues 570-580, (4) amino acid residues 647-661, (5) amino acid residues 662-717 or (6) 718-746, or any combination thereof.
  • the mutant E2 polypeptide provided herein also can differ from the corresponding naturally-occurring E2 amino acid sequence in that the mutant E2 polypeptide can have at least one amino acid substitution at position 416, 417, 483, 484, 485, 538, 540, 544, 545, 547, 549 or any combinations thereof.
  • a mutant E2 polypeptide provided herein can have at least two amino acid substitutions at these positions, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions.
  • the amino acid that can be substituted at these positions can be one that has a different chemical or physical property from the naturally-occurring residue.
  • the proline residues at position 484, 544 or 545 can be substituted with an amino acid residue that enable the polypeptide to be more flexible such as for example an alanine, valine or other non-cyclic residues.
  • the glycine residue at position 547 can be substituted with an amino acid residue that has a bulkier side chain such as, for example, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine or glutamine, while the tryptophan residue at position 549 can be substituted with an amino acid residue that has a less bulky side chain, for example, glycine, alanine, valine, serine, cysteine, or threonine.
  • the threonine residue at position 416 can be substituted with a residue that does not have a hydroxyl or sulfur-containing side chain.
  • the acidic asparagine residue at position 417 or 540 can be substituted with, for example, a basic amino acid residue such as histidine, lysine or arginine, while the basic arginine residue at position 483, for example, can be substituted with, for example, an acidic residue such as aspartic acid, glutamic acid, asparagine or glutamine.
  • the aromatic amino acid tyrosine at position 485 can be substituted with, for example, a non-aromatic residue, while the valine at position 538 can be substituted with a residue having a bulkier side chain, a basic or acidic residue, or one with an aromatic, hydroxyl or sulfur-containing side chain.
  • An exemplary substitution or combination of substitutions is one that decreases the immunogenicity or function of epitopes recognized by the ARl antibodies such as ARIA and ARlB.
  • the mutant E2 polypeptide provided herein also can have one or more other substitutions, insertions or deletions relative to a naturally-occurring E2 polypeptide as long as the mutant E2 polypeptide sequence includes the discontinuous epitopes described herein that come together to form a conformational epitope recognized by a conformation-dependent cross-neutralizing antibody such as the AR3A, AR3B, AR3C or AR3D antibody.
  • the mutant E2 polypeptide provided herein contain one or more discontinuous epitopes of an E2 polypeptide.
  • the polypeptides provided herein include the following amino acid regions: (1) amino acid residues 412 to 424; (2) amino acid residues 436 to 447; and (3) amino acid residues 523 to 540 relative to the naturally- occurring E2 polypeptide (e.g. HCV strain H77 polypeptide and other HCV strains, isolates, species, quasispecies, subtypes or genotypes). Sequences of the discontinuous epitopes can be determined based on sequence alignment of the HCV E2 or HCV polyprotein sequence with the sequence of strain H77 using the methods described above.
  • mutant E2 polypeptides provided herein include the mutant E2 polypeptides shown in Table 5 below.
  • a polypeptide provided herein also can include non-E2 sequences at the N or C terminus.
  • Non-E2 sequences can be, for example, a tag such as an N-terminal ubiquitin signal, a poly-histidine sequence (SEQ ID NO:685), a FLAG sequence (SEQ ID NO:607), an HA sequence (SEQ ID NO:608), a myc sequence (SEQ ID NO:609), a V5 sequence (SEQ ID NO:610), a chitin binding protein sequence, a maltose binding protein sequence (SEQ ID NO:687) or a glutathione-S-transferase sequence.
  • D. Nucleic Acids Encoding Mutant E2 Polypeptides Provided herein are isolated nucleic acids encoding modified E2 polypeptides.
  • Nucleic acids encoding mutant E2 polypeptides provided herein can be generated from nucleic acids encoding the naturally-occurring HCV polyprotein using methods known to those skilled in the art. For example, nucleic acids encoding mutant E2 polypeptides containing various amino acid substitutions can be produced by site- specific mutagenesis and polymerase chain reaction (PCR) amplification from the nucleic acids encoding the naturally-occurring HCV polyprotein. Nucleic acids encoding mutant E2 polypeptides, i.e.
  • polypeptides that do not include amino acid residues 384 to 410 of the hypervariable region of the naturally occurring E2 protein can be produced by PCR using primers that do not encompass the nucleotides coding for amino acid residues 384 to 410.
  • Nucleic acid sequences encoding the naturally- occurring HCV polyproteins are disclosed at the NCBI website (ncbi.nlm.nih.gov).
  • Selected accession numbers for nucleic acids encoding the naturally-occurring HCV polyproteins are as follows: AF009606; D10749; U01214; AY051292; AY746460; AY232731; D50409; DQ155561; AB031663; DQ437509; D49374; D63821; Yl 1604; DQ516083; EF589160; AF064490; AY859526; NC009827; EF420130; DQ314805 ; DQ835764; D63822; D84264; DQ835763; and DQ278894.
  • Nucleic acids encoding a polypeptide provided herein can be used for recombinant expression of the E2 polypeptide provided herein. Nucleic acids encoding a polypeptide provided herein also can be used in a nucleic acid-based vaccine to elicit an immune response against an HCV.
  • Nucleic acid encoding a polypeptide provided herein can be operably-linked to an expression control sequence in an expression vector, which can be introduced into a host cell for expression of the encoded polypeptide or administered to a mammal to elicit an immune response against the polypeptide.
  • nucleic acid sequences encoding mutant E2 polypeptides provided herein are shown in Table 6 below.
  • Nucleic acids encoding E2 polypeptides provided herein can be incorporated into viral, bacterial, insect, yeast or mammalian expression vectors.
  • nucleic acids encoding E2 polypeptides can be operably-linked to expression control sequences such as viral, bacterial, insect, yeast or mammalian promoters and enhancers.
  • expression control sequences such as enhancers and promoters include viral promoters such as SV 40 promoter, Rous Sarcoma Virus (RSV) promoter, and cytomegalovirus (CMV) immediate early promoter.
  • viral vectors include retrovirus-based vectors, e.g. lentiviruses, adenoviruses and adeno-associated viruses.
  • nucleic acid encoding an E2 polypeptide provided herein also can be linked to nucleic acid sequences that code for unrelated amino acid sequences such as N-terminal ubiquitin signals to improve antigen targeting, a poly-histidine sequence, a FLAG (DYKDDDDK, SEQ ID NO:607) sequence, an HA sequence, a myc sequence, a V5 sequence, a chitin binding protein sequence, a maltose binding protein sequence or a glutathione-S-transferase sequence.
  • unrelated amino acid sequences such as N-terminal ubiquitin signals to improve antigen targeting, a poly-histidine sequence, a FLAG (DYKDDDDK, SEQ ID NO:607) sequence, an HA sequence, a myc sequence, a V5 sequence, a chitin binding protein sequence, a maltose binding protein sequence or a glutathione-S-transferase sequence.
  • Expression vectors containing nucleic acids encoding E2 polypeptides can be introduced into bacterial, insect, yeast or mammalian host cells (e.g. CHO, Balb/3T3, HeLa 3 MT2, mouse NSO (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293 (e.g. 293T), 293S, 2B8, and HKB cells) for expression using conventional methods including, without limitation, transformation, transduction and transfection.
  • mammalian host cells e.g. CHO, Balb/3T3, HeLa 3 MT2, mouse NSO (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293 (e.g. 293T), 2
  • Expression vectors containing nucleic acids encoding E2 polypeptides, in saline for example can be introduced into a mammal, e.g. mammalian tissues, using standard methods including, for example, injection using a standard hypodermic needle, by a gene gun delivery, jet injection or liposome-mediated delivery. Injection can be intramuscular or intradermal. Electroporation, myotoxins such as bupivacaine or hypertonic solutions of saline or sucrose also can aid in delivery.
  • E2 polypeptides provided herein When expressed in bacterial, yeast, insect or mammalian host cells, E2 polypeptides provided herein can be purified using a method provided herein. Specifically, E2 polypeptides provided herein are purified by affinity chromatography using a cross-neutralizing antibody such as, for example, AR3A, AR3B, AR3C or AR3D in combination with size exclusion chromatography. More specifically, an E2 polypeptide provided herein can be separated from unrelated proteins by affinity chromatography using a conformation-dependent antibody provided herein such as AR3A.
  • a cross-neutralizing antibody such as, for example, AR3A, AR3B, AR3C or AR3D
  • the E2 polypeptide can be eluted at acidic, neutral or basic pH using: (1) 0.2M glycine pH 2.2, (2) 2M sodium thiocyanate (pH adjusted to pH 7.4 with 5OmM Tris-HCl); or (3) 0.2M glycine pH 11.5, and then further purified by size-exclusion chromatography.
  • the method provided herein for purifying E2 polypeptide allows for the purification of E2 polypeptides that properly fold to form the conformational epitope described herein.
  • the methods of purification using a conformation dependent antibody such as, but not limited to AR3A, provide for the purification of E2 polypeptide monomers that properly fold to form the E2 conformational epitope.
  • E2 polypeptides incorporated in a viral vector, for example, can be used as a nucleic acid-based vaccine to elicit an immune response against HCV.
  • the antibody is a cross-neutralizing antibody, i.e. one that neutralizes at least two HCV strains, isolates, species, quasispecies, subtypes or genotypes.
  • An antibody provided herein can be a polyclonal or monoclonal antibody.
  • Polyclonal antibodies can be obtained by immunizing a mammal with a mutant polypeptide provided herein, and then isolating antibodies from the blood of the mammal using standard techniques including, for example, enzyme linked immunosorbent assay (ELISA) to determine antibody titer and protein A chromatography to obtain the antibody-containing IgG fraction.
  • ELISA enzyme linked immunosorbent assay
  • a monoclonal antibody is a population of molecules having a common antigen binding site that binds specifically with a particular antigenic epitope.
  • a monoclonal antibody can be obtained by selecting an antibody-producing cell from a mammal that has been immunized with a mutant polypeptide provided herein and fusing the antibody-producing cell, e.g. a B cell, with a myeloma to generate an antibody- producing hybridoma.
  • a monoclonal antibody provided herein also can be obtained by screening a recombinant combinatorial library such as an antibody phage display library using, for example, a mutant polypeptide provided herein.
  • An immunologically-active fragment of an antibody is the biologically active fragment of an immunoglobulin molecule, for example, the F(ab) or F(ab') 2 fragment generated by cleavage of the antibody with an enzyme such as pepsin.
  • An antibody provided herein also can be a murine, chimeric, humanized or fully human antibody.
  • a murine antibody is an antibody derived entirely from a murine source, for example, an antibody derived from a murine hybridoma generated from the fusion of a mouse myeloma cell and a mouse B-lymphocyte cell.
  • a chimeric antibody is an antibody that has variable regions derived from a non-human source, e.g. murine or primate, and constant regions derived from a human source.
  • a humanized antibody has antigen-binding regions, e.g. complementarity-determining regions, derived from a mouse source, and the remaining variable regions and constant regions derived from a human source.
  • a fully human antibody is an antibody from human cells or derived from transgenic mice carrying human antibody genes.
  • a polyclonal antibody provided herein can be prepared by immunizing a suitable mammal with a mutant polypeptide provided herein.
  • the mammal can be, for example, a rabbit, goat, sheep, rabbit, hamster, cow, or mouse.
  • antibody molecules can be isolated from the mammal, e.g. from the blood or other fluid of the mammal, and further purified using standard techniques that include, without limitation, precipitation using ammonium sulfate, gel filtration chromatography, ion exchange chromatography or affinity chromatography using protein A.
  • an antibody-producing cell of the mammal can be isolated and used to prepare a hybridoma cell that secretes a monoclonal antibody provided herein.
  • Techniques for preparing monoclonal antibody-secreting hybridoma cells are known in the art. See, for example, Kohler and Milstein, Nature 256:495-97 (1975) and Kozbor etai, Immunol Today 4: 72 (1983).
  • a monoclonal antibody provided herein also can be prepared using other methods known in the art, such as, for example, expression from a recombinant DNA molecule, or screening of a recombinant combinatorial immunoglobulin library using a mutant polypeptide provided herein.
  • a chimeric antibody can be produced by expression from a nucleic acid that encodes a non-human variable region and a human constant region of .an antibody molecule. See, for example, Morrison et al., Proc. Nat. Acad. ScL U.S.A. 86: 6851 (1984).
  • a humanized antibody can be produced by expression from a nucleic acid that encodes non-human antigen-binding regions (complementarity-determining regions) and a human variable region (without antigen-binding regions) and human constant regions.
  • a mutant HCV E2 polypeptide or cross-neutralizing antibody provided herein can be used to detect the presence of HCV in a sample obtained from a subject, such as a mammal.
  • a diagnostic use is based on the detection of antibodies generated by a subject (e.g. a mammal) that has been infected with HCV.
  • Diagnostic use also can be based on detection of HCV antigens. Detection of an antibody-antigen complex indicates that the mammal has been exposed to or infected with HCV.
  • a mutant polypeptide provided herein can be used to detect the presence of anti-HCV antibodies in a sample from the mammal.
  • a cross-neutralizing antibody provided herein can be used to detect HCV particles or antigens in the sample.
  • the sample from the mammal can be a biological fluid such as blood or a cell or tissue sample.
  • the mutant E2 polypeptides or antibodies provided herein can be labeled with a detectable label.
  • the polypeptide or cross-neutralizing antibody provided herein can be labeled with a detectable molecule, which can be an enzyme such as, but not limited to, alkaline phosphatase, acetylcholinesterase, ⁇ - galactosidase or horseradish peroxidase; a prosthetic group such as, but not limited to, streptavidin, biotin, or avidin; a fluorescent group such as dansyl chloride, dichlorotriazinylamine, dichlorotriazinylamine fluorescein, fluorescein, fluorescein isothiocyanate, phycoerythrin, rhodamine, umbelliferone; a luminescent group such as luminal; a bioluminescent group such as a
  • a polypeptide provided herein can be used to generate cross-neutralizing antibodies against HCV.
  • a polypeptide provided herein can be used to elicit an immune response in a subject, such as a mammal.
  • Antibodies that bind specifically with the mutant E2 polypeptide provided herein can be isolated using known methods as described above.
  • a mutant polypeptide provided herein is particularly useful to focus the immune response to the conserved AR3 neutralizing epitopes as the immunogenicity of the hypervariable regions and the ARl residues are dampened by deletion of a large portion of the hypervariable region and substitution of important selected ARl residues.
  • kits for eliciting an immune response in a subject comprising administering to the subject a mutant E2 polypeptide provided herein and then isolating antibodies or antibody producing cells from the subject using methods known to those of skilled in the art.
  • the subject can be a rabbit, rat, mouse, sheep, cow, monkey, horse, goat or a pig.
  • the method is particularly useful to generate antibodies against conserved HCV epitopes.
  • the method can be used to develop passive vaccines containing one or more anti-HCV antibodies provided herein.
  • a polypeptide provided herein also can be used to screen for anti-HCV agents, such as those that block viral entry into target cells.
  • an E2 polypeptide provided herein can be used to screen for agents that bind to an E2 polypeptide provided herein and prevent binding of the E2 polypeptide with a cell receptor.
  • a polypeptide or cross-neutralizing antibody provided herein can be used to prevent or treat a new or recurring HCV infection, or prevent or reduce HCV replication, as well as treat the associated disease condition or clinical symptoms.
  • the mutant HCV polypeptide provided herein can be used to provide immune protection against HCV.
  • the immune protection against HCV provided by the immunogenic polypeptide can be any immune response, cellular or humoral, that either inhibits or helps to prevent HCV infection.
  • the immunogenic polypeptide of the present invention can bind to CD81, induce antibodies associated with resolving HCV infection, induce production of cytokines, induce antibodies that can neutralize HCV binding to host cells, or prime an immune system against secondary HCV infection or exposure.
  • the immune protection provided by the immunogenic polypeptide of the present invention can be protective against more than one of the HCV genotypes.
  • HCV infection or replication is indicated by the amount of HCV particles or the amount of HCV RNA in a sample from the subject determined using methods known in the art and also those described herein.
  • HCV infection is also indicated by clinical symptoms described further below.
  • the E2 polypeptide provided herein, corresponding nucleic acid or cross- neutralizing antibody provided herein can be used to prevent or reduce transmission, to prevent or treat disease progression, and to prevent or reduce HCV replication or reduce viral load.
  • Treatment includes the alleviation or diminishment of at least one symptom typically associated with the infection.
  • the treatment cures, e.g., substantially inhibits viral infection and/or eliminates the symptoms associated with the infection.
  • Symptoms of HCV exposure or infection include, without limitation, inflammation of the liver, decreased appetite, fatigue, abdominal pain, jaundice, flu- like symptoms, itching, muscle pain, joint pain, intermittent low-grade fevers, sleep disturbances, nausea, dyspepsia, cognitive changes, depression headaches and mood changes.
  • Subjects that can benefit from the polypeptide, nucleic acid or antibody provided herein can be identified using the diagnostic and screening techniques discussed above.
  • HCV infection can be diagnosed by detecting antibodies to the virus using the mutant E2 polypeptide provided herein, detecting the HCV itself using a cross-neutralizing antibody provided herein, detecting liver inflammation by biopsy, liver cirrhosis, portal hypertension, thyroiditis, cryoglobulinemia and glomerulonephritis.
  • diagnosis of exposure or infection or identification of one who is at risk of exposure to HCV can be based on medical history, abnormal liver enzymes or liver function tests during routine blood testing.
  • infection can be diagnosed using polymerase chain reaction (PCR) for detecting viral nucleic acids, enzyme linked immunosorbent assay (ELISA) for detecting viral antigens or anti-viral antibodies, and immunofluorescence for detecting viral antigens.
  • PCR polymerase chain reaction
  • ELISA enzyme linked immunosorbent assay
  • a polypeptide or antibody provided herein can be combined with an appropriate sample from the patient to produce a complex.
  • the complex in turn can be detected with a marker reagent for binding with such a complex.
  • Typical marker reagents include secondary antibodies selective for the complex, secondary antibodies selective for certain epitopes of the polypeptide or antibody or a label attached to the polypeptide or antibody itself.
  • radioimmunoassay RIA
  • radioallergosorbent test RAST
  • radioimmunosorbent test RIST
  • immunoradiometric assay IRMA
  • Fair assay fluorescence immunoassay
  • FOA fluorescence immunoassay
  • ELISA enzyme linked immunosorbent assay
  • Labels including radioactive labels, chemical labels, fluorescent labels, luciferase and the like also can be directly attached to the polypeptide according to the techniques described in U.S. Patent No. (BN patent cite), the disclosure of which is incorporated herein by reference.
  • a subject that can benefit from a polypeptide, nucleic acid or cross-neutralizing antibody provided herein includes one who is likely to be or has been exposed to HCV.
  • exemplary subjects include, without limitation, someone present in an area where HCV is prevalent or commonly transmitted, e.g., Africa, Southeast Asia, China, South Asia, Australia, India, the United States, Russia, as well as Central and South American countries.
  • a subject who is likely to be or has been exposed to HCV also includes a recipient of donated body tissues or fluids including, for example, a recipient of blood or one or more of its components such as plasma, platelets, or stem cells and an organ or cell transplant recipient such as a liver transplantee.
  • a subject who is likely to be or has been exposed to HCV also can include medical, clinical or dental personnel handling body tissues and fluids.
  • a subject e.g., a mammal
  • who has been exposed to HCV includes, without limitation, someone who has had contact with the body tissue or fluid, e.g. blood, of an infected person or otherwise have come in contact with HCV.
  • a subject e.g., a mammal
  • who can benefit from a polypeptide or cross-neutralizing antibody provided herein includes one who is susceptible to HCV infection or one who has recurring HCV infection.
  • kits for preventing a new or recurring HCV infection and its associated symptoms and/or complications such as by preventing or reducing HCV replication in a subject (e.g. a mammal) infected with HCV.
  • a polypeptide, nucleic acid or cross-neutralizing antibody provided herein can be used prophylactically to prevent a susceptible individual from being infected with HCV or to prevent recurring HCV infection, for example, in an individual who has received a liver transplant.
  • a polypeptide or cross-neutralizing antibody provided herein can be used to prevent or treat infection of a cell, e.g. a mammalian cell, such as a human cell.
  • a polypeptide, nucleic acid or cross-neutralizing antibody provided herein can be used to prevent or treat acute or chronic HCV infection, or prevent or reduce HCV replication, in a subject, e.g. a mammal such as a human.
  • an E2 polypeptide or a nucleic acid encoding an E2 polypeptide provided herein can be used as an active vaccine, a nucleic acid or DNA-based vaccine, or be incorporated into vaccine carriers, to elicit a protective immune response in a subject.
  • Exemplary vaccines include vaccines that are effective for the prevention of HCV infection by one or more HCV genotypes.
  • Methods of preventing or treating HCV infection include contacting a cell with an effective amount of an antibody provided herein; mixing biological fluids, cells or tissues to be administered or transplanted into a subject with a polypeptide, nucleic acid or antibody provided herein prior to the administration or transplant; or administering to a subject such as a human a therapeutically effective amount of a polypeptide, nucleic acid or antibody provided herein.
  • in vitro methods of preventing HCV infection or transmission by contacting biological samples such as fluids, cells or tissues containing the virus with an effective amount of the polypeptide, nucleic acid or antibody provided herein, as well as in vivo methods of treating or preventing HCV infection by administering the polypeptide, nucleic acid or antibody to the subject.
  • a polypeptide, nucleic acid or antibody provided herein can be administered in a variety of ways.
  • Routes of administration include, without limitation, oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, vaginal, dermal, transdermal (topical), transmucosal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.
  • the means of administration can be by injection, using a pump or any other appropriate mechanism.
  • a polypeptide, nucleic acid or antibody provided herein can be administered in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the polypeptide, nucleic acid or antibody provided herein can be essentially continuous over a pre-selected period of time or can be in a series of spaced doses.
  • methods of eliciting an immune response in a subject e.g.
  • a mammal that involves administering a modified polypeptide, nucleic acid or antibody provided herein at a select time and then administering a second, third, fourth or additional doses at select times after the first administration. Both local and systemic administrations are contemplated.
  • the dosage to be administered to a subject can be any amount appropriate to reduce or prevent viral infection or to treat at least one symptom associated with the viral infection.
  • a subject e.g. a mammal
  • dosages can be any amount appropriate to reduce or prevent viral infection or to treat at least one symptom associated with the viral infection.
  • Some factors that determine appropriate dosages are well known to those of ordinary skill in the art and can be addressed with routine experimentation. For example, determination of the physicochemical, toxicological and pharmacokinetic properties can be made using standard chemical and biological assays and through the use of mathematical modeling techniques known in the chemical, pharmacological and toxicological arts. The therapeutic utility and dosing regimen can be extrapolated from the results of such techniques and through the use of appropriate pharmacokinetic and/or pharmacodynamic models.
  • the dosage will also depend on the polypeptide or antibody chosen and whether prevention or treatment is to be achieved, and if the polypeptide or antibody is chemically mutant. Such factors can be readily determined by the clinician employing viral infection models such as in vitro HCV infection system described herein, or other animal models or test systems that are available in the art. The precise amount to be administered to a subject (e.g. a mammal) such as a human will be the responsibility of the attendant physician. The amount useful to establish treatment of HCV can be determined by diagnostic and therapeutic techniques well known to those of ordinary skill in the art.
  • the dosage can be determined by titrating a sample of the patient's blood sera with the polypeptide or antibody to determine the end point beyond which no further immunocomplex is formed. Such titrations can be accomplished by the diagnostic techniques discussed below. Available dosages include administration of from about 1 to about 1 million effective units of antibody per day, wherein a unit is that amount of polypeptide, which will provide at least 1 microgram of antigen-polypeptide complex. In some examples, about 10 to about 100,000 units of antibody per day can be administered.
  • one or more mutant polypeptides or antibody provided herein can be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500, 750 or 1000 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages can provide beneficial results.
  • the one or more polypeptide or antibody provided herein can be administered as soon as possible, e.g. within 24 hours if possible, after exposure to HCV. To prevent recurrent HCV infection, e.g.
  • a mutant polypeptide or antibody provided herein can be administered prior to and after transplantation.
  • the polypeptide or antibody provided herein can be administered during the anhepatic phase, as well as during the post-operative phase.
  • the polypeptide, nucleic acid or antibody provided herein can be administered daily, biweekly or monthly after the transplant.
  • the polypeptide, nucleic acid or antibody provided herein can be administered daily for the first week after transplant, weekly for two, three or more weeks after the transplant and then monthly.
  • the absolute weight of a polypeptide or antibody included in a unit dose can vary widely.
  • the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.
  • the daily dose of a polypeptide, nucleic acid or antibody provided herein can vary as well. Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.
  • a polypeptide, nucleic acid or antibody provided herein can be used alone or in combination with a second medicament.
  • the second medicament can be another polypeptide or antibody provided herein, a known antiviral agent such as, for example, an interferon-based therapeutic or another type of antiviral medicament such as ribavirin.
  • a polypeptide, nucleic acid or antibody provided herein also can be used in combination with one or more agents to enhance the immune response in a subject.
  • the polypeptide, nucleic acid or antibody provided herein can be administered in combination with other therapeutic agents including, without limitation, immunoregulatory agents, immunoglobulin, cytokines, lymphokines, and chemokines, e.g., IL-2, modified IL-2 (e.g. Cl 25S), GM-CSF, IL-12, gamma- interferon, IP-IO, MIPl ⁇ , or RANTES.
  • the second medicament also can be an anticancer, antibacterial, or another antiviral agent.
  • the antiviral agent can act at any step in the life cycle of the virus from initial attachment and entry to egress.
  • the second antiviral agent can interfere with attachment, fusion, entry, trafficking, translation, viral polyprotein processing, viral genome replication, viral particle assembly, egress or budding.
  • the antiviral agent can be an attachment inhibitor, entry inhibitor, a fusion inhibitor, a trafficking inhibitor, a replication inhibitor, a translation inhibitor, a protein processing inhibitor, an egress inhibitor, in essence an inhibitor of any viral function.
  • the effective amount of the second medicament will follow the recommendations of the manufacturer of the second medicament, as well as the judgment of the attending physician, and will be guided by the protocols and administrative factors for amounts and dosing as indicated in the PHYSICIAN'S DESK REFERENCE.
  • the polypeptide, nucleic acid or antibody provided herein can be administered in combination with an adjuvant.
  • the polypeptide, nucleic acid or antibody provided herein can be administered with any suitable adjuvant for stimulating immune response, e.g., providing immune protection.
  • it can be a particulate or a non-particulate adjuvant.
  • a particulate adjuvant usually includes, without limitation, aluminum salts, calcium salts, water-in-oil emulsions, oil-in water emulsion, immune stimulating complexes (ISCOMS) and ISCOM matrices (U.S. Pat. No.
  • a non-particulate adjuvant usually includes, without limitation, muramyl dipeptide (MDP) and derivatives, e.g., treonyl MDP or murametide, non-ionic block copolymers, saponins, e.g., Quil A and QS21, lipid A or its derivative 4' monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), various cytokines including gamma-interferon and interleukins 2 or 4, carbohydrate polymers, derivatized polysaccharides, e.g., di ethyl aminoethyl dextran, and bacterial toxins, e.g., cholera toxin or E. coli labile toxin.
  • the polypeptide e.g., treonyl MDP or murametide, non-ionic block copolymers
  • saponins e.g., Quil A and QS21
  • MPL
  • nucleic acid or antibody provided herein for inhibition and treatment of HCV infection, methods available in the art and those described herein can be used.
  • the effectiveness of the method of treatment can be assessed by monitoring the patient for signs or symptoms of the viral infection as discussed above, as well as determining the presence and/or amount of viral particle or viral RNA present in the blood, e.g. the viral load, using methods known in the art.
  • Viral infection or replication in the presence or absence of a polypeptide or antibody provided herein can be evaluated, for example, by determining intracellular viral RNA levels or the number of viral foci by immunoassays using antibodies against viral proteins as described herein.
  • a polypeptide or antibody is effective for treatment and inhibition of HCV if it can inhibit or reduce viral infection or replication by any amount, for example, by 2 fold or more than 2 fold.
  • a polypeptide or antibody provided herein can inhibit or reduce HCV infection by 2-5 folds, 5-10 folds, or more than 10 folds.
  • a polypeptide, nucleic acid or antibody provided herein also can be used to increase the safety of blood and blood products, to increase the safety of clinical laboratory samples and to increase the safety of biological tissues as well as articles, devices, or instruments intended for preventative or therapeutic use.
  • a polypeptide, nucleic acid or antibody provided herein can be added to blood or blood products such as plasma, platelets, and blood or marrow cells prior to use.
  • a polypeptide, nucleic acid or antibody provided herein can be combined with cells or tissues prior to use or administration. It can be coated on articles, devices or instruments such as, for example, valves, bags and stents.
  • purified preparations containing a mutant polypeptide provided herein or a preparation containing a cross-neutralizing antibody provided herein are provided herein.
  • a purified preparation of a mutant polypeptide provided herein at least 50 % of the mutant polypeptides in the preparation are folded in a conformation such that the discontinuous epitopes (i.e. amino acid segments corresponding to amino acids 412 to 424, amino acids 436 to 447 and amino acids 523 to 540 of HCV strain H77) come together to form a conformational epitope that can bind with a conformation-dependent antibody such as a cross-neutralizing antibody, for example, AR3A, AR3B, AR3C or AR3D.
  • a conformation-dependent antibody such as a cross-neutralizing antibody
  • polypeptide preparation at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 % or 95 % of the mutant polypeptides are folded as described above.
  • mutant polypeptides are folded as described.
  • a larger proportion of the antibodies are cross-neutralizing antibodies.
  • an antibody preparation can be a biological sample such as blood or plasma obtained from a subject (e.g. a mammal) that has been immunized with a mutant polypeptide provided herein.
  • the blood sample contains a larger proportion of cross- neutralizing antibodies than a blood sample obtained from a similar animal that has been immunized with a naturally-occurring E2 polypeptide.
  • Such a cross-neutralizing antibody preparation can be a partially purified or purified polypeptide preparation, i.e. a preparation resulting from one or more protein purification steps known in the art as well as those discussed herein.
  • Such cross- neutralizing antibody preparation contains at least about 2 %, 5%, 10 %, 20 %, 30 %, 40 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, or 80 % cross- neutralizing anti-HCV antibodies.
  • such cross-neutralizing antibody preparation can contain about 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, 50 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %, 58 %, 59 %, 60 %, 62 %, 63 %, 64 %, 65 %, 66 %, 67 %, 68
  • mutant polypeptides and cross-neutralizing antibodies provided herein are described above. Preparations of these can be obtained using protein purification procedures known to those skilled in the art. See, for example, CURRENT PROTOCOLS IN PROTEIN SCIENCE, Coligan et al, eds., John Wiley & Sons, Inc., 1997.
  • compositions comprising a mutant polypeptide, nucleic acid or antibody provided herein.
  • a mutant polypeptide or antibody provided herein is obtained, e.g. by expression in a host cell or using polymerase chain reaction, purified as necessary or desired and then lyophilized and stabilized.
  • the polypeptide, nucleic acid or antibody can then be adjusted to the appropriate concentration and then combined with other agent(s) or pharmaceutically acceptable carrier(s).
  • a pharmaceutical formulation containing therapeutic amounts of one or more polypeptides, nucleic acids or antibodies provided herein can be prepared by procedures known in the art using well-known and readily available ingredients.
  • one or more polypeptides, nucleic acids or antibodies can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like.
  • excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives.
  • Binding agents also can be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone.
  • Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution also can be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds also can be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols also can be included. Preservatives also can be added. The compositions provided herein also can contain thickening agents such as cellulose and/or cellulose derivatives.
  • polypeptides, nucleic acids or antibodies can be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum.
  • the active polypeptide also can be presented as a bolus, electuary or paste.
  • the formulations can, where appropriate, be conveniently presented in discrete unit dosage forms and can be prepared by any of the methods well known to the pharmaceutical arts including the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
  • One or more polypeptides, nucleic acids or antibodies provided herein also can be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes.
  • a pharmaceutical formulation containing one or more therapeutic polypeptides, nucleic acids or antibodies provided herein also can take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.
  • polypeptides, nucleic acids or antibodies can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelf life of the dosage form.
  • the polypeptides, nucleic acids or antibodies and other ingredients can form suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • polypeptides, nucleic acids or antibodies and other ingredients can be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water
  • formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol,” polyglycols and polyethylene glycols, C1-C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol,” isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.
  • organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol,” polygly
  • antioxidants chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings.
  • Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and ⁇ -tocopherol and its derivatives can be added.
  • the one or more polypeptides, nucleic acids or antibodies are formulated as a microbicide, which is administered topically or to mucosal surfaces such as the vagina, the rectum, eyes, nose and the mouth.
  • the therapeutic agents can be formulated as is known in the art for direct application to a target area.
  • Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap.
  • an agent provided herein can be formulated as a vaginal cream or a microbicide to be applied topically.
  • Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols.
  • the one or more polypeptides, nucleic acids or antibodies provided herein can be delivered via patches or bandages for dermal administration.
  • the polypeptides, nucleic acids or antibodies can be formulated to be part of an adhesive polymer, such as polyacrylate or acryl ate/vinyl acetate copolymer.
  • the backing layer can be any appropriate thickness that will provide the desired protective and support functions.
  • a suitable thickness will generally be from about 10 to about 200 microns.
  • Ointments and creams can, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions can be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the active agents also can be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051 ,842.
  • the percent by weight of one or more polypeptides, nucleic acids or antibodies provided herein present in a topical formulation will depend on various factors, but generally will be from 0.01 % to 95 % of the total weight of the formulation, and typically 0.1-85 % by weight.
  • Drops such as eye drops or nose drops, can be formulated with one or more of the polypeptides, nucleic acids or antibodies in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents.
  • Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
  • the one or more polypeptides, nucleic acids or antibodies further can be formulated for topical administration in the mouth or throat.
  • the active ingredients can be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition provided herein in a suitable liquid carrier.
  • the pharmaceutical formulations provided herein can include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art.
  • pharmaceutically acceptable carriers such as physiologically buffered saline solutions and water.
  • diluents such as phosphate buffered saline solutions pH 7.0-8.0.
  • polypeptides, nucleic acids or antibodies provided herein also can be administered to the respiratory tract.
  • aerosol pharmaceutical formulations and dosage torms tor use in the methods provided herein comprise an amount of at least one of the polypeptides, nucleic acids or antibodies provided herein effective to treat or prevent the clinical symptoms of the viral infection. Any statistically significant attenuation of one or more symptoms of the infection that has been treated pursuant to the method provided herein is considered to be a treatment of such infection within the scope provided herein.
  • the composition can take the form of a dry powder, for example, a powder mix of one or more polypeptides, nucleic acids or antibodies and a suitable powder base such as lactose or starch.
  • the powder composition can be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder can be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung. Clarke. S. W. and Davia, D. eds., pp. 197-224, Butterworths, London, England, 1984).
  • MDI pressurized metered dose inhaler
  • the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung. Clarke. S. W. and Davia, D. e
  • the one or more polypeptides, nucleic acids or antibodies provided herein also can be administered in an aqueous solution when administered in an aerosol or inhaled form.
  • other aerosol pharmaceutical formulations can comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/mL and about 100 mg/mL of one or more of the polypeptides, nucleic acids or antibodies provided herein specific for the indication or disease to be treated.
  • Dry aerosol in the form of finely divided solid polypeptide, nucleic acid or antibody particles that are not dissolved or suspended in a liquid are also useful in the practice provided herein.
  • Polypeptides, nucleic acids or antibodies provided herein can be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 ⁇ m, alternatively between 2 and 3 ⁇ m.
  • Finely divided particles can be prepared by pulverization and screen filtration using techniques well known in the art.
  • the particles can be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units.
  • the effective amount can be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.
  • the one or more polypeptides, nucleic acids or antibodies provided herein are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs can comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Nebulizers include, but are not limited to, those described in U.S. Patent Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co., (Valencia, CA). For intra-nasal administration, the therapeutic agent also can be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).
  • An exemplary formulation involves lyophilized polypeptides, nucleic acids or antibodies and separate pharmaceutical carrier. Immediately prior to administration, the formulation is constituted by combining the lyophilized polypeptides, nucleic acids or antibodies and pharmaceutical carrier. Administration by a parenteral or oral regimen will deliver the polypeptides, nucleic acids or antibodies to the desired site of action.
  • Pharmaceutical formulations of the polypeptides, nucleic acids or antibodies provided herein can be prepared as liquids, gels and suspensions. Exemplary formulations are suitable for injection, insertion or inhalation. Injection can be accomplished by needle, cannula, catheter and the like. Insertion can be accomplished by lavage, trochar, spiking, surgical placement and the like.
  • Inhalation can be accomplished by aerosol, spray or mist formulation.
  • the polypeptides, nucleic acids or antibodies provided herein also can be administered topically such as to the epidermis, the buccal cavity and instillation into the ear, eye and nose.
  • the polypeptides, nucleic acids or antibodies can be present in the pharmaceutical formulation at concentrations ranging from about 1 percent to about 50 percent, such as about 1 percent to about 20 percent, such as, for example, about 2 percent to about 10 percent by weight relative to the total weight of the formulation.
  • a polypeptide, nucleic acid or antibody provided herein also can be used in combination with one or more known therapeutic agents, for example, a pain reliever; an antiviral agent such as an anti-HBV, other anti-HCV (HCV inhibitor, HCV protease inhibitor) or an anti-herpetic agent; an antibacterial agent; an anti-cancer agent; an anti-inflammatory agent; an antihistamine; a bronchodilator; an immunomodulatory agent; and appropriate combinations thereof, whether for the conditions described or some other condition.
  • a pain reliever an antiviral agent such as an anti-HBV, other anti-HCV (HCV inhibitor, HCV protease inhibitor) or an anti-herpetic agent
  • an antibacterial agent such as an anti-cancer agent
  • an anti-inflammatory agent such as an antihistamine; a bronchodilator; an immunomodulatory agent; and appropriate combinations thereof, whether for the conditions described or some other condition.
  • articles of manufacture that include a pharmaceutical composition containing one or more polypeptides, nucleic acids or antibodies provided herein for controlling microbial infections.
  • Such articles can be a useful device such as a vaginal ring, a condom, a bandage or a similar device.
  • the device holds a therapeutically effective amount of a pharmaceutical composition for controlling viral infections.
  • the device can be packaged in a kit along with instructions for using the pharmaceutical composition for control of the infection.
  • the pharmaceutical composition includes at least one polypeptide, nucleic acid or antibody provided herein, in a therapeutically effective amount such that viral infection is controlled.
  • An article of manufacture also can be a vessel or filtration unit that can be used for collection, processing or storage of a biological sample containing a polypeptide or antibody provided herein.
  • the vessel can be evacuated.
  • Vessels include, without limitation, a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter.
  • the filtration unit can be part of another device, for example, a catheter for collection of biological fluids.
  • the one or more polypeptides or antibodies provided herein also can be adsorbed onto or covalently attached to the article of manufacture, for example, a vessel or filtration unit.
  • the material in the article when material in the article is decanted therefrom or passed through, the material will not retain substantial amounts of the polypeptides or antibodies.
  • Adsorption or covalent attachment of the one or more polypeptides or antibodies to the article kills viruses or prevents their transmission, thereby helping to control viral infection.
  • the one or more polypeptides or antibodies provided herein can be in filtration units integrated into biological collection catheters and vials, or added to collection vessels to remove or inactivate viral particles that can be present in the biological samples collected, thereby preventing transmission of the disease.
  • compositions comprising one or more polypeptides, nucleic acids or antibodies provided herein and one or more clinically useful agents such as a biological stabilizer.
  • Biological stabilizer includes, without limitation, an anticoagulant, a preservative and a protease inhibitor.
  • Anticoagulants include, without limitation, oxalate, ethylene diamine tetraacetic acid, citrate and heparin.
  • Preservatives include, without limitation, boric acid, sodium formate and sodium borate.
  • Protease inhibitors include inhibitors of dipeptidyl peptidase IV.
  • compositions comprising an agent provided herein and a biological stabilizer can be included in a collection vessel such as a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter or any other container or vessel used for the collection, processing or storage of biological samples.
  • a collection vessel such as a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter or any other container or vessel used for the collection, processing or storage of biological samples.
  • compositions comprising one or more polypeptides, nucleic acids or antibodies provided herein and a biological sample such as blood, semen or other body fluids that is to be analyzed in a laboratory or introduced into a recipient subject (e.g. a mammal).
  • a biological sample such as blood, semen or other body fluids that is to be analyzed in a laboratory or introduced into a recipient subject (e.g. a mammal).
  • one or more polypeptides, nucleic acids or antibodies provided herein can be mixed with blood prior to laboratory processing and/or transfusions.
  • the one or more polypeptides, nucleic acids or antibodies are present in at least about 0.15 mg/mL of the sample, e.g.
  • the one or more polypeptides, nucleic acids or antibodies provided herein can be included in physiological media used to store and transport biological tissues, including transplantation tissues.
  • physiological media used to store and transport biological tissues including transplantation tissues.
  • liver, heart, kidney and other tissues can be bathed in media containing the present agents to inhibit viral transmission to transplant recipients.
  • the one or more polypeptides, nucleic acids or antibodies are present in at least about 1.5 mg/kg of the sample, e.g.
  • Huh-7 (Zhong, J. et al., Proc. Natl. Acad. ScL U.S.A. 102, 9294-9299 (2005)) and 293T cells were grown in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% fetal calf serum (FCS) (Invitrogen).
  • D-MEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • bone marrow mononuclear cell RNA from a 35-year-old female patient with Sjogren's syndrome and chronic HCV infection was used as source material for an IgGl Fab phage display library (Maruyama, T. etal, Am. J. Pathol. 165, 53-61 (2004)).
  • the donor was diagnosed with HCV in 1991 and developed mixed cryoglobulinemia, symptoms of Sjogren's syndrome and tested positive for antinuclear antibody in 1994.
  • the donor was treated with interferon- ⁇ with initial decrease in viral load but the treatment was stopped due to severe drop in platelet count (idiopathic thrombocytopenic purpura).
  • Bone marrow samples were collected for the evaluation of neutropenia as an outpatient clinical procedure at Scripps Clinic. After meeting the needs of clinical pathology, a fraction of the biopsy was used to construct the antibody library. The human subjects protocol was approved by the Human Subjects Committee for General Clinical Research Center of Scripps Clinic and informed consent was obtained from the donor. Due to subsequent relapse of HCV, the donor underwent a liver transplant in 2000 and has been maintained on anti-rejection medications since. The viral genotype in this donor was not determined at the time of tissue donation but was found to be genotype 1 a seven years later.
  • the Fab heavy chains were expressed as a fusion protein with the phage gene HI surface protein for display.
  • the library was amplified in XL-I Blue cells (Stratagene) using 0.3% SeaPrep agarose (BioWhittaker) in SuperBroth (SB) Medium by a semisolid phase amplification method.
  • the phagemid library was transformed into E. coli (XL-I Blue) (Stratagene) by electroporation and the phage was propagated overnight with VCS-Ml 3 helper phage (Stratagene).
  • Recombinant E2 glycoprotein (genotype Ia, amino acids 388- 644; Lesniewski, R. et al., J. Med. Virol. 45, 415-422 (1995)) was coated directly onto a microtiter plate overnight at 4 0 C (Costar). The wells were washed and then blocked with 4% non-fat dry milk in phosphate-buffered saline (PBS).
  • the phage library was added to the wells and incubated for 1-2 hours at 37°C and unbound phage washed away with PBS. Bound phage were eluted and used to infect freshly grown E. coli (XLl -Blue) (Stratagene) for titration on LB agar plates with carbenicillin. The phage libraries were panned for four consecutive rounds with increasing washing stringency.
  • Library panning by an epitope masking strategy was repeated using recombinant E1E2 fused to glutathione S transferase (GST-E1E2; Chan-Fook, C. et al., Virology TTb, 60-66 (2000)) pre-incubated with Fabs obtained above.
  • GST-E1E2 was first captured with goat anti-GST antibody (Amersham Biosciences) and the wells were washed and blocked with 4% non-fat dry milk in PBS.
  • Fabs obtained from the panning using E2 antigen above were added to the captured antigens to mask corresponding specific epitopes.
  • the epitope-masked GST-El E2 was used to pan the phage library as described above. It is important to note that, highly isolate-specific antibodies, e.g. those against HVRl, were not selected due to the use of heterologous antigens in panning. Screening of Fab displayed phage. Single individual colonies were isolated from titration plates after the 2nd, the
  • Vector plgGl is a derivative of pDR12 in which heavy and light chain cloning sites were altered to XhollBstEll and SacllXbal sites to facilitate direct cloning of the antibody gene fragments.
  • the heavy and light chain genes of Fab Cl were amplified by PCR then inserted sequentially into the SacVXbal and Hindlll/EcoRl sites of the vector (Burton, D.R.
  • plgGl the heavy and light chain gene fragments were excised from the phagemids and inserted sequentially into the XhollBstEll and SacVXbal sites of the vector.
  • the recombinant plasmids were transfected into Chinese hamster ovarian (CHO) cells. Stable cell clones were established by selection with L-methionine sulfoxide (MSX) and by limiting dilution. Cell clones expressing high IgG levels were amplified and the IgGs were purified using a protein A-agarose column (Pharmacia).
  • GST-El E2 (8 ⁇ g/mL) captured by pre-coated goat anti-GST-antibody (10 ⁇ g/mL), or with ovalbumin (4 ⁇ g/mL). Specific binding was detected by alkaline phosphatase (AP)-conjugated goat anti-human IgG F(ab') 2 antibody (Pierce) (1 :500) in 1% BSA/PBS and disodium /j-nitrophenyl phosphate (Sigma), (ii) To study the relationship of different ARs to the mouse MAb epitope H53 (Cocquerel et al., J. Virol.
  • Non-fat milk 4%, BioRad
  • PBS lectin-captured antigens
  • the ELISA plates were washed after a 1 hour incubation and binding of human Fabs was detected by peroxidase (HRP)-conjugated goat anti-human IgG F(ab') 2 antibody (1 :2000) (Pierce) and TMB substrate (Pierce).
  • HRP peroxidase
  • TMB substrate TMB substrate
  • vaccinia-expressed E1E2 was either captured directly onto ELISA wells pre- coated with lectin (folded protein), or unfolded with 0.1% SDS, 50 mM DTT and incubated at 100 0 C for 5 minutes before capture onto ELISA wells (unfolded protein). Binding of the MAbs to folded and unfolded proteins was detected using the peroxidase system.
  • Mouse MAb A4 Dubuisson, J. et al., J. Virol.
  • CD81-LEL Two forms of recombinant CD81-LEL, either in fusion with glutathione S-transferase (GST) (Owsianka, A.M. et al, J. Virol. 80, 8695- 8704 (2006)) or maltose binding protein (MBP) (Chan-Fook, C.
  • GST glutathione S-transferase
  • MBP maltose binding protein
  • the binding of human MAbs was detected by HRP-conjugated goat anti- human IgG F(ab') 2 antibody as above. Non-infected/non-transfected cell lysate were used as negative controls to determine background for each MAb.
  • MAbs recognizing linear epitopes bind to both folded and unfolded proteins but the biotinylated human MAbs bind conformational epitopes on folded E2. Consequently, competition is performed with the MAbs to linear epitopes as blocking MAbs to eliminate potential non-specific signals caused by misfolded proteins in the system. After incubation for 1 h, the ELISA plates were washed and binding of biotinylated MAbs was detected with HRP-conjugated streptavidin (1 :2000, Sigma- Aldrich) in PBS with 1% BSA and TMB substrate (Pierce).
  • HCVpp human immunodeficiency virus pp neutralization
  • HCVpp was generated by co-transfection of 293T cells with pNL4-3.1ucR-E- (Connor et al, Virology 206, 935-944 (1995); He, J. et al., J. Virol. 69, 6705-671 1 (1995)) and the corresponding expression plasmids encoding the El E2 genes at 4:1 ratio by polyethylenimine (Boussif, O. et al, Proc. Natl. Acad. Sci. U.S.A.
  • RNA in the HCV GT 1 a-infected human serum KP 140 ⁇ l was purified using a QIAamp Viral RNA Mini Kit (Qiagen).
  • First strand cDNA was generated using either a reverse primer specific to HCVIa (HCVlaOuterR, GGGATGCTGCATTGAGTA, (SEQ ID NO: 697); Lavillette, D. et al., Hepatology 41, 265-274 (2005)) or random hexamer using the Superscript III reverse transcriptase (Invitrogen).
  • the GTIa E1E2 genes were amplified by a nested PCR as described previously (Lavillette, D.
  • E1E2 proteins were confirmed by the presence of folded E2 proteins in cell lysates, prepared from 293T cells transfected with the corresponding DNA plasmids, by ELISA using MAb AR3A. Antibody protection studies. Human liver-chimeric mice were prepared as described previously. Mercer,
  • mice Only mice with serum levels of hAAT greater than 60 ⁇ g/mL at 6 weeks and 100 ⁇ g/mL at 8 weeks, an indication for successful transplantation, were used in the protection study ( ⁇ 50% of transplanted mice).
  • Mice with low level of human liver chimerism were used in preliminary experiments to measure the toxicity and kinetics of MAbs in Alb-uPA/SCID mice, and the level of human IgG present in mice injected with a genotype 1 a HCV-infected human serum KP. This serum, serially diluted from 1 : 50 to 1 :4050, did not neutralize HCVpp-H77 (data not shown).
  • HCV RNA in mouse serum was quantified by a real-time TaqMan PCR assay.
  • the two primers in the real-time PCR system were designed to produce a 194 bp PCR fragment corresponding to the 5 ' non-coding region with maximum specificity to all HCV genotypes.
  • AGGTTTAGGATTCGTGCTCAT (SEQ ID NO: 699) were designed with the aid of software Primer Express (PE biosystems) and were purchased from PE Applied Biosystems.
  • PE biosystems guanidinium thiocyanate
  • silico method Bax, R. et al., J. Clin. Microbiol. 28, 495-503 (1990) was used. Briefly, 30 ⁇ L of serum was mixed with 500 ⁇ l GuSCN lysis buffer and 20 ⁇ L size- fractionated silica particles for 15 minutes. The silica particles were pelleted and washed twice with 500 ⁇ L washing buffer, twice with 70% ethanol and once with acetone.
  • Superscript Il First-Strand Synthesis Kit (Invitrogen) was used to synthesize first- strand cDNA for PCR.
  • a 50 ⁇ L mixture contained 9 ⁇ L of template HCV cDNA, 1 x TaqMan
  • a serial dilution of HCV cDNA including 1.5 X lO 6 , 1.5 x 10 s , 1.5 x 10 4 , 1.5 x 10 3 , 1.5 x 10 2 , 1.5 x 10 1 , 1.5 x 10° IU, was used to generate a standard curve for calculation of HCV RNA copy number.
  • the dynamic range of HCV RNA detection for the two step RT-PCR procedure is 6.0 x 10 2 IU/ml to 3.0 x 10 8 IU/mL.
  • Each assay run incorporates in duplicate a negative control and an HCV RNA positive control.
  • the positive control is the OptiQual HCV RNA 1 Control purchased from AcroMetrix which has been calibrated to the WHO first International Standard for HCV RNA.
  • Statistical analysis is the OptiQual HCV RNA 1 Control purchased from AcroMetrix which has been calibrated to the WHO first International Standard for HCV RNA.
  • GraphPad Prism 4 software was used for statistical analysis of the antibody protection experiment. Animals seropositive for HCV RNA by the quantitative PCR assay at or after day 7 post-infection were scored as "infected" subjects and animals seronegative up to week 6 were scored as "censored” subjects. The scores were used to construct the Kaplan-Meier survival (infection in this case) curves to calculate statistical significance between the neutralizing antibody-treated and isotype antibody control groups by a two-tailed log rank test within the experimental period. Motulsky, H. Survival curves, in GraphPad Prism4 Statistics Guide: Statistical analyses for laboratory and clinical researchers 107-117 (GraphPad Software, San Diego, 2005).
  • a total of 115 clones that exhibit specific binding to HCV E2 glycoprotein were isolated from an antibody antigen-binding fragment (Fab) phage display library generated from a donor chronically infected with HCV (see Example 1).
  • DNA sequence analysis identified 36 distinct Fabs with 13 unique heavy chain sequences. The sequences of the 36 distinct Fabs belonging to 13 groups based on the heavy chain sequences are also shown in Table 7 below. Fabs with the same designation and * or ** have the same heavy chain but distinct light chains, e.g. Hl, Hl* and Hl** have the same heavy chain, but 3 different light chains.
  • the numbers in parenthesis denote the percentage of clones recognizing each AR in the phage-display panning. It is important to note that highly isolate-specific antibodies, e.g. those against HVRl, would unlikely be selected in this study due to the use of heterologous antigens in the panning. Fab K was excluded in this table due to its poor signal in FIG. 1.
  • ⁇ 1-E2 produced by transfected 293T cells dApparent affinity is defined as the antibody concentration required to achieve half-maximal binding in an ELISA Data shown are the means of at least two independent experiments All mAbs bind natively folded, but not reduced and denatured, E2 GTIa indicates genotype Ia, GT2a indicates genotype 2a and dashes indicate that no significant inhibition or binding was observed with the highest mAb concentration tested
  • mAbs at 50, 25, 10, 5 or 1 ⁇ g/mL were tested for virus neutralization, and the lowest antibody concentrations that reduced >50% of virus infectivity are shown Dashes indicate no or ⁇ 50% virus neutralization with 50 ⁇ g/mL mAb. Data shown are the means of at least two experiments.
  • b Neutralization of HC Vpp was determined by the reduction in luciferase activity in Huh-7 cells infected with HCVpp displaying Env from different HCV isolates.
  • the panel of HCVpps shown includes HCV Env proteins that produce a signal at least tenfold higher than the background signal induced by the control pseudotype virus generated without HCV Env cDNA
  • HCV Env proteins including CH35 (genotype Ib), UKN3A1.28c (genotype 3a), UKN6.5.8 (genotype 6) and 13 different
  • KP Env clones did not produce a consistent signal tenfold higher than background and were excluded from this analysis
  • Numbers indicate percentage of residual binding signals of biotinylated human mAbs in the presence of blocking mAbs. Origin: h, human; m, mouse; r, rat.
  • the panel of variants includes substitutions at conserved residues in the putative CD81 -binding regions of E2. Substitutions important for CD81 binding are shaded and include L413A, W420A, H421A, I422A, N423A, S424A, G523A, T526A, Y527A, W529A, G530A, D535A, V538A, N540A and F550A. (Owsianka, A.M. et al. J. Virol 80, 8695-8704 (2006)).
  • the antibody competition study shows that mAbs AP33 and 3/11 (*) recognize epitopes partially dependent on proper protein folding (Tarr, A.W. et al, Hepatology 43, 592-601 (2006)).
  • the results confirm the broad designation of the antigenic regions and suggest that the discontinuous epitopes in AR3 are formed by at least three segments between amino acids 396-424, 436—447 and 523-540; the first and third segments also contribute to the CD81 -binding domain of E2 (Owsianka, A.M. et al, J. Virol.
  • a key question is whether broadly neutralizing AR3-specific antibodies can protect against infection by heterologous HCV quasispecies.
  • the human liver-chimeric Alb-uPA/SCID mouse model was used (Kneteman, N.M. et al, Hepatology 43, 1346-1353 (2006); Lindenbach, B.D. et al, Proc. Natl. Acad. Sd. USA 103, 3805-3809 (2006)).
  • this animal model is not suitable for studying virus pathogenesis, owing to its lack of a functional adaptive immune system, the question of whether antibodies can protect against HCV challenge is appropriate.
  • Previous passive antibody studies in animal models have reported relatively high antibody concentrations are needed for protection.
  • the kinetics and tolerability were first established in the animal model for the antibodies AR3 A, AR3B and a human isotype control IgGl to HIV-I , b6.
  • Transplanted Alb-uPA/SCID mice with a low level of human liver chimerism were injected intraperitoneally with 100, 150 or 200 mg/kg MAb, and blood samples were collected by tail bleed.
  • Human antibody in the murine sera was measured by a quantitative sandwich ELISA using conjugated and unconjugated goat anti-human F(ab)' 2 antibody. The antibodies did not show adverse effects in control mice. No specific weight loss or signs of illness associated with the administration of the MAbs were noted in the mice during the experiment.
  • mice One mouse (N457) was euthanized due to unrelated morbidity at Day 7. A dose of 200 mg/kg given through intraperitoneal injection was required to achieve mean serum titers approximately 100 x higher than in vitro neutralization titers. Such titers have previously been found to be necessary to achieve sterilizing immunity in other viral disease models.
  • the neutralizing activity in mouse sera collected ten days after injection was determined by HCVpp-H77 neutralization assay.
  • Mouse sera containing anti-HCV MAbs AR3A and AR3B neutralized 50% of HCVpp infectivity (IC 5 o) in the range of 1:200 to 1:1000.
  • the IC 50 titers of mouse sera containing anti-HCV MAbs AR3A and AR3B were in the range of 0.4-1.1 (mean 0.8 ⁇ s.d. 0.3) and 0.5-3 (mean 1.2 ⁇ s.d. 0.9) ⁇ g/mL, respectively.
  • Isotype control MAbs b6 & DEN3 did not neutralize HCVpp.
  • the observed half-lives of mAbs AR3 A, AR3B and b6 were 6.0 ⁇ 2.2 d, 9.0 ⁇ 1.3 d and 7.3 ⁇ 1.8 d (mean ⁇ s.d.), respectively, and their specific neutralizing activities (that is, neutralizing activity relative to serum mAb concentration) were stable for at least 10 days in the mice.
  • the mAbs were administered intraperitoneally in passive transfer experiments to mice with high levels of human liver chimerism (see Example 1), and the mean serum titers of mAbs AR3A, AR3B and the control mAb b6, at 24 hours after injection were -2.5 ⁇ 0.3 mg/mL, 3.1 ⁇ 0.5 mg/mL and 2.6 ⁇ 0.3 mg/mL, respectively (FIG. 4).
  • the partial amino acid sequences (residues 384-622) of forty HCVs found in the viral quasispecies population in the HCV genotype la-infected human serum are shown below.
  • HCV E2 glycoprotein is a major target for virus neutralizing antibodies and an important component in a HCV vaccine.
  • E2 has encoded several features to evade antibodies. First, E2 encodes regions that are highly mutable. Rapid changes in viral sequence facilitate virus escape. Second, E2 is highly glycosylated and the associated glycans help shield the neutralizing epitopes from antibodies. Despite these escape features, we have identified the antigenic region 3 (AR3) on E2 as a relatively conserved target for antibody neutralization in vitro and antibody protection in vivo. The amino acid residues important for the binding of AR3-specific antibodies is described above. The following show how these residues organize together to form the AR3 conformational epitopes.
  • AR3 antigenic region 3
  • E2 that displays AR3 properly while silencing some of the variable sequences that are usually immunogenic but are not targets of broadly neutralizing antibodies
  • a panel of E2 truncation mutants were constructed.
  • the minimal E2 fragment that displays the CD81 -binding sites and the broadly neutralizing epitopes correctly the binding of these E2 mutants with CDE81- LEL or various mAb were studied.
  • E2 mutants were constructed by deletion of highly variable regions, specific N-glycosylation signals, or every other cysteine residues from C- or N- terminus of wildtype (WT) E2.
  • WT wildtype
  • the panel of E2 mutants in fusion with the Flag tags at their C-termini are illustrated in FIG. 7, and their sequences are shown in Table 16.
  • Table 16 Hepatitis C virus E2 Glycoprotein Mutants
  • the cDNA encoding these mutants were generated by polymerase chain reaction (PCR) or by splicing by overlap extension polymerase chain reaction (SOE- PCR) as described in Horton et al., Biotechniques 8:528-535 (1990).
  • PCR polymerase chain reaction
  • SOE- PCR overlap extension polymerase chain reaction
  • the plasmid pCV-H77c (Genbank accession# AFOl 1751) encoding wildtype E2 gene of the isolate H77 was used as a template.
  • the primers used in the reactions are enlisted below.
  • E2wtF 744 AATAACGCGTGAAACCCACGTCACCGG
  • E2flF 745 AATAACGCGTCAACTGATCAACACCAACG
  • E2f2F 746 AATAACGCGTTTGGCCAGCTGCCGACGC
  • E2f4F 748 AATAACGCGTGTATATTGCTTCACTCCCAG
  • E2f5F 749 AATAACGCGTACTGGATTCACCAAAGTGTG
  • E2wtR 750 TATTCTCGAGCTCCCACTTAATGGCCCAG
  • E2r2 752 TATTCTCGAGCCGCGTCCAGTTGCAGGC
  • E2r2a 753 TATTCTCGAGGTTGCAGGCCGCTTCCAGC
  • E2r3 754 TATTCTCGAGGTAGTCGACCATGCACCTG
  • E2r5 756 TATTCTCGAGCACCCCTCCGATGACACAAG
  • E2r7 758 TATTCTCGAGCGGGCCACACACGCTCTTTG delHVR2F 759 TGCGGCTCTAGCGGATGCTGGCACTACCCTCCAAG delHVR2R 760 CAGCATCCGCTAGAGCCGCAGCTGGCCAACCTCTC delHVR3F 761 TGTGGAAGCTCTGGCTGCCCCACTGATTGCTTCC delHVR3R 762 GCAGCCAGAGCTTCCACAAGGGGGCGCTCCGCAC
  • PCR conditions 94°C, 3 min; 25 cycles of (94°C, 30 s; 55°C, 30 s; 70 0 C, 90 s); & 70 0 C, 10 min; PCR system: Platinum Pfx DNA polymerase (Invitrogen); PCR instrument: GeneAmp PCR System 9700 (Applied Biosystems).
  • the PCR products generated in Table 18 were resolved by agarose gel electroporesis and the DNA bands of correct size were excised and purified.
  • the products were either used as templates in a second PCR, or were digested with MIu I and Xho I restriction enzymes.
  • the digested products were gel-purified and inserted between the BssH II and Xho I sites of the plasmid pCMV-Tag4A-tpaJR-FLgpl20 (Pantophlet et al., J Virol 77:642-658 (2003); Law et ai, J Virol 81 :4272-4285 (2007)).
  • the inserted products replaced the HIV genes in the plasmid and are in frame with a 5 '-signal peptide and a 3'-FLAG tag to facilitate protein secretion and for detection.
  • the nucleotide sequences of the E2 mutants were verified by DNA sequencing.
  • E2 mutants were expressed by transient transfection of 293T cells.
  • Cell monolayers were co-transfected with the expression plasmids encoding the different E2 mutants and pAdVAntage plasmid (Promega) at 1:1 ratio by polyethylenimine (Boxxssif et al., Proc Natl Acad Sd USA 92:7297-7301 (1995)).
  • Cell supernatants were collected 3 days post-transfection and were clarified by centrifugation.
  • MAbs monoclonal antibodies
  • HCV co-receptor CD81 HCV co-receptor CD81
  • Mutants with correctly folded antibody epitopes or CD81 -binding sites were captured by the corresponding reagents and the captured mutants were detected with a mouse anti-FLAG tag MAb (Sigma), followed by a secondary antibody (Peroxidase- conjugated AffiniPure Goat Anti-mouse IgG from Jackson ImmunoResearch Laboratories) and the colorimetic peroxidase substrate TMB (Pierce). The peroxidase reaction was stopped by adding sulfuric acid.
  • E2 mutants Specific binding of the E2 mutants to the capturing reagents were detected by measuring the absorbances of the samples at 450 nm using a microplate reader (Molecular Devices). The results are summarized in FIG.8.
  • the CD81 -binding sites and AR3 are presented well on the E2 mutants E2 ⁇ TM, E2flrl, E2flr2, E2flr2a, E2 ⁇ N5 and E2 ⁇ N9.
  • the mutant E2 ⁇ N5N9 was captured by MAbs AR3A or AR3C at a comparable level to the above mutants but at a much reduced level by CD81-LEL, MAbs AR3B or AR3D.
  • mutants E2flr3, E2flr4, E2flr5, E2f2r2a and E2f3r2a were captured by the non-neutralizing MAbs ARIA and ARlB but not CD81-LEL or AR3-specific MAbs, suggesting that the CD81 -binding sites and the broadly neutralizing epitopes in AR3 are not present or folded correctly in these mutants.
  • fragments E2flrl and E2flr2abind to the conformation-dependent, broadly neutralizing MAb AR3A and CD81-LEL indicates that the E2 residues 412-645 and cysteines 1-16 are important for correct folding of AR3 (within this region, residues 460-485 and 570-580 are not required).
  • E2 ⁇ TM binds all Abs recognizing ARl , 2 and 3, but weakly to CD81-LEL.
  • the HCV envelope El and E2 glycoproteins are technically challenging to produce as El does not fold properly in the absence of E2 (Michalak et al, J Gen Virol 78:2299-2306 (1997) and Patel et al, Virology 279:58-68 (2001)) and efficient production of E2 is influenced by El (Cocquerel et al., J Virol 77:10677- 10683 (2003), Brazzoli et al, Virology 332:438-453 (2005)).
  • E2661 A truncated version of E2 (known as E2661) can be expressed independently and retain its function in binding to the co-receptor CD81 (Michalak et al., J Gen Virol 78:2299-2306 (1997); Flint et al., J Virol 73 :6235-6244 (1999); Flint et al, J Virol 74:702-709 (2000)).
  • This truncated E2 has not been shown to be produced in a highly purified form suitable for biochemical analysis and crystallization attempts (Flint et al., J Virol 74:702-709 (2000)).
  • E2 displaying correctly folded AR3 epitopes a protein production and purification method was developed.
  • the plasmids encoding the E2 mutants pE2 ⁇ TM and pE2flr2a were co-transfected with pAdVAntage plasmid (Promega) at 1:1 ratio into FreeStyle 293 cells (Invitrogen) using 293fectin Transfection Reagent (Invitrogen). Cell supernatants were collected twice at 3-day and 5-day post- transfection.
  • kifunensine (at 7.5 ⁇ M, Cayman Chemical) (Elbein et al., J Biol Chem 265:15599-15605 (1990); Chang et al., Structure 15:267-273 (2007)) was added to cell culture media to improve glycan homogeneity on E2.
  • the E2 mutants were purified by antibody affinity chromatography.
  • the MAb AR3A which can distinguish folded from misfolded protein, was used.
  • the MAb AR3A recognizes a conformation-dependent epitope on E2, neutralizes HCV in vitro and offers protection against HCV infection in vivo as shown above.
  • MAb AR3A binds natively folded E2 at high affinity but not denatured and reduced E2.
  • MAb AR3A-affinity matrix MAb AR3A was first captured by Protein A-Sepharose (GE Healthcare) at a ratio of 10 mg MAb per mL Sepharose beads. After overnight incubation, the beads were washed 3 times with 0.2 M sodium borate buffer (pH 9). MAb AR3A was then crosslinked chemically to the Protein A-beads using dimethyl pimelimidate (Thermo Scientific). The reaction was stopped after lhour incubation at room temperature by pelleting the beads and washing the beads 3 times with 0.2 M ethanolamine (pH 8).
  • the MAb-conjugated beads were packed into an Econo-Column (Bio-Rad) and the beads were rinsed once with 0.2 M glycine (pH 2.2) followed by PBS to equilibrate the column for affinity purification of the E2 mutants.
  • Cell supernatants containing the E2 mutants were clarified by low-speed centrifugation and filtration through a 0.22- ⁇ m filter before loading onto the affinity columns by gravity flow. The flow-through solutions were collected and the columns were washed with PBS. Bound proteins were released from the affinity columns using different elution conditions and the antigenicity of the eluted proteins were investigated (see below).
  • the eluants were concentrated and monomers of the E2 mutants were purified by size-exclusion chromatography using a Superdex 75 column (Amersham Biosciences).
  • E2flr2a Three batches of E2flr2a were produced by transient transfection of 293T cells ( ⁇ 5 x 10 8 cells per batch) with the corresponding expression plasmid.
  • Batch 3 was produced in the presence of 10 ⁇ M kifunesine (BIOMOL), a potent inhibitor of the glycoprotein processing ⁇ -mannosidase I and is used to improve glycan homogeneity in the glycoproteins.
  • Cell supernatants were loaded onto an antibody- affinity column (MAb AR3A, 5 mL) by gravity flow and bound proteins were eluted with a low pH buffer (0.2 M glycine, pH 2.7). Batches 2 and 3 was purified twice to monitor purification efficiency.
  • E2flr2a was further purified and analyzed by size-exclusion chromatography.
  • E2flr2a purified by MAb AR3A affinity column was concentrated to 0.5 mL using an ultra-centrifugal filter device with a 30 kDa nominal molecular weight limit (Millipore).
  • the concentrated proteins were loaded onto a Sephadex 75 size- exclusion column (GE Healthcare) using a AKTA Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare).
  • the proteins were separated in Tris buffer (0.1 M Tris-HCl pH 7.4 and 150 mM NaCl) and elution fractions of 0.5 mL were collected by an automatic fractionator.
  • the chromatogram of E2flr2a was compared to the chromatogram of protein standards, including: (A) blue dextran 2000, (B) bovine serum albumin 67 kDa, (C) ovalbumin 43 kDa, and (D) chymotrypsinogen 25 kDa (GE Healthcare). Fractions 14-22 were analyzed by non-reducing SDS- PAGE (4-15% gradient, BIO-RAD). The results showed that the high molecular weight impurities eluted from the MAb AR3A-afflnity column were separated from the glycoforms of E2flr2a, which appear to be monomers of size between 43-67 kDa in gel filtration.
  • Reaction conditions included (1) E2flr2a eluted with low pH buffer as a control; (2) E2flr2a eluted with 0.5 M NaSCN; (3) 1 M NaSCN; and (4) 2 M NaSCN.
  • the purified proteins were analyzed by non-reducing SDS- PAGE (4-15% gradient, BIO-RAD). The results show that neutral pH elution conditions can be used to purify the E2flr2a glycoforms, with the exception of elution with 0.5 M NaSCN, which led to high molecular weight impurities.
  • E2flr2a was produced in the presence of kifunensine, loaded onto a MAb AR3A-affinity column and eluted with a step-gradient of buffers with increasing pH.
  • the eluents were collected into tubes with 0.1 volume of neutralizing buffer (2 M T ⁇ s-HCl, pH 7.4).
  • Reaction conditions included (1) E2flr2a eluted with 2 M NaSCN as a control; (2) E2flr2a eluted with 0.2 M glycine pH 9.5; (3) pH 10.5; (4) pH 11.5; (5) pH 12.5; and (6) pH 11.5 sample filtered through an ultra-centrifugal filter device with a 100 kDa nominal molecular weight limit (Milhpore).
  • the results show that both 2 M NaSCN and 0 2 M glycine pH 11.5 elution conditions led to pure E2flr2a protein.
  • E2 ⁇ TM was purified using a MAb AR3A-conjugated affinity column with a 2M NaSCN, pH 7.4 elution and analyzed by SDS-PAGE.
  • High molecular weight impurities were removed by filtering through an ultracentrifugal filter device with a 100 kDa nominal molecular weight limit (Milhpore).
  • the purified proteins were analyzed by 4-15% gradient non-reducing SDS-PAGE (BIO-RAD). The results show that ultracentrifugal filtration removed high molecular weight impurities in samples produced in both the absence and presence of kifunensine. Protein concentration was quantified by the Bradford method (Bradford et al.,
  • the recombinant E2 fragment E2flr2a can be purified to greater than 90 % by a single affinity chromatography step.
  • the purification method is applicable to E2flr2a produced in the presence of the plant alkaloid kifunensine, a potent inhibitor of the glycoprotein processing ⁇ -mannosidase I.
  • N-glycans on recombinant proteins produced in the presence of kifunensine are almost exclusively high-mannose type oligosaccharides, which can be readily trimmed by endoglycosidase H digestion to improve protein homogeneity.
  • E2 mutants E2 ⁇ TM and E2flr2a can be purified as monomers.
  • the recombinant E2 fragments purified by the above method adopt a native fold as found on viral surface.
  • the purified E2 mutants will be extremely useful in research and discovery of anti-viral drugs and HCV vaccines.

Abstract

Provided herein are mutant E2 polypeptides for therapeutic and diagnostic uses, the amino acid sequence of which comprises, from the amino terminus to the carboxy terminus: (1) a first segment that corresponds to amino acid residues 412 to 459 of the E2/ polypeptide of a select hepatitis C vims (HCV), (2) a second segment that corresponds to amino acid residues 486 to 569 of Ε2 polypeptide the select hepatitis C virus, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the E2 polypeptide of the select hepatitis C virus, wherein: the segments are linked directly or via a polypeptide linker; with the proviso. that the mutant polypeptide does not include a contiguous sequence of amino acids corresponding to all of amino acid residues 411 to 662 of the E2 polypeptide of the select hepatitis C virus; the mutant polypeptide has deletions of amino add residues corresponding to amino acids 384 to 411 and amino acids 718 to 746 of the full-length E2 polypeptide of the select hepatitis C virus and the mutant polypeptide can specifically bind an antibody that binds to a conformational epitope on the E2 polypeptide that contains at least amino acids 411 to 462. Tne mutant E2 polypeptide provided can be used for the production of cross- neutralizing antibodies against hepatitis C virus (HCV). Also provided herein are preparations and pharmaceutical compositions containing mutant E2 polypeptides and methods for their preparation, purification and use. Also provided are methods of using the mutant E2 polypeptides for the prevention and treatment of HCV infection.

Description

MUTANT HEPATITIS C VIRUS E2 POLYPEPTIDES FOR HCV TREATMENT
RELATED APPLICATIONS
Benefit of priority is claimed to U.S. Provisional Application Serial No. 61/197,273, filed October 24, 2008, entitled "MUTANTHCV E2 POLYPEPTIDES," to Mansun Law and Dennis R. Burton.
This application is related to International Application No. PCT/US09/05786, filed October 23, 2009, entitled "AGENTS FOR HCV TREATMENT," to Mansun Law and Dennis R. Burton, which claims priority to U.S. Provisional Application Serial Nos. 61/197,292 and 61/200,347 filed October 24, 2008 and November 26, 2008, respectively. This application also is related to U.S. Application Serial No.
12/290,017, filed October 24, 2008, entitled "HCV NEUTRALIZING EPITOPES," to Mansun Law, Toshiaki Maruyama, Dennis R. Burton, Jonathan K. Ball and Norman M. Kneteman. This application also is related to International Application No. PCT/US02/02303 filed January 25, 2002 (published as WO 02/059340 on August 1, 2002), and to U.S. Provisional Application Serial Number 60/264,451, filed January 26, 2001.
Where permitted, the subject matter of each of the above-referenced applications is incorporated by reference in its entirety. FIELD OF THE INVENTION Provided herein are mutant hepatitis C virus (HCV) E2 polypeptides and nucleic acid molecules encoding the polypeptides. The mutant E2 polypeptides and encoding nucleic acid molecules can be used for diagnosis and therapy of hepatitis C virus (HCV) infection and can be employed as vaccines for the prevention and treatment of HCV infection. The mutant E2 polypeptides also can be employed for the production of antibodies against HCV. BACKGROUND
It is estimated that hepatitis C virus (HCV) infects about 2-3 % of the world population, i.e. 120 to 170 million people worldwide. HCV infection predisposes the patient to chronic liver cirrhosis, cancer and liver failure. About 85 % of individuals initially infected with HCV become chronically infected. Once established, chronic HCV infection causes an inflammation of the liver, and this can progress to scarring and eventually, liver cirrhosis. Some patients with cirrhosis will go on to develop liver failure or liver cancer. In the United States and Western Europe, the complications of chronic hepatitis and cirrhosis are the most common reasons for liver transplantation. In addition, liver disease caused by HCV is the leading cause of death in patients co-infected with human immunodeficiency virus. Given the large number of infected people worldwide, HCV infection can be a burden on health care systems worldwide.
Accordingly, there is a need for therapeutic agents and methods for the treatment of new and recurring hepatitis C viral infections. SUMMARY Provided herein are mutant E2 polypeptides, compositions containing mutant
E2 polypeptides, and methods for their production, purification and use. The polypeptides can be employed for the prophylaxis and therapy of hepatitis C virus infection. In some examples, the polypeptides can be used as immunogens to elicit antibodies that can protect against infection by a hepatitis C virus (HCV). The mutant HCV E2 polypeptides provided herein display conserved neutralizing AR3 epitopes recognized by conformation-dependent cross-neutralizing anti-HCV antibodies. Also provided herein are nucleic acid molecules encoding mutant HCV E2 polypeptides and expression vectors containing the nucleic acid molecules for their production. Provided herein are cells that contain nucleic acid molecules encoding mutant HCV E2 polypeptides and cells that contain expression vectors containing the nucleic acid molecules. Also provided herein are preparations and pharmaceutical compositions containing a mutant HCV E2 polypeptide. Also provided herein are methods of eliciting an immune response in a mammal comprising administering a mutant HCV E2 polypeptide provided herein, methods for determining whether a mammal has been infected with an HCV, and methods for identifying an anti-HCV agent.
In some examples, a mutant HCV E2 polypeptide contains the amino acid sequence which includes, from the amino to the carboxy termini: (1) a first segment that corresponds to amino acid residues 412 to 459 of a select HCV, (2) a second segment that corresponds to amino acid residues 486 to 569 of the select HCV, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the select HCV. The segments of the mutant HCV E2 polypeptide can be linked directly or via a linker, typically a polypeptide linker. The mutant HCV E2 polypeptides provided herein contain deletions of amino acid residues corresponding to amino acids 384 to 411 and amino acids 718 to 746 of the fiill-length E2 polypeptide of a select hepatitis C virus and does not include a contiguous sequence of amino acids that corresponds to amino acid residues 411 to 662 of the E2 polypeptide of the select hepatitis C virus. Among the mutant E2 polypeptides provided herein are polypeptides that can specifically or selectively bind to an antibody that is immunospecific for a conformational epitope on the E2 polypeptide that contains at least amino acids 411 to 462. hi some examples, the conformational epitope contains amino acids corresponding to amino acids 412 to 424, 436 to 447 and 523 to 540 of the select hepatitis C virus.
In particular examples, the mutant E2 polypeptides provided herein contain (1) a first segment that corresponds to amino acid residues 412 to 459 of the E2 polypeptide of a select hepatitis C virus, (2) a second segment that corresponds to amino acid residues 486 to 569 of the E2 polypeptide of the select hepatitis C virus, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the E2 polypeptide of the select hepatitis C virus, where the segments are linked directly or via a polypeptide linker, with the proviso that the mutant polypeptide does not include a contiguous sequence of amino acids corresponding to all of amino acid residues 411 to 662 of the E2 polypeptide of the select hepatitis C virus; the mutant polypeptide has deletions of amino acid residues corresponding to amino acids 384 to 411 and amino acids 718 to 746 relative to the full-length E2 polypeptide of the select hepatitis C virus. In some examples, the mutant E2 polypeptides provided herein do not contain the contiguous amino acid residues that correspond to amino acid residues 460 to 485 and/or 570 to 580 of the E2 polypeptide of the select hepatitis C virus. The segments of the mutant E2 polypeptides provided herein can be linked directly or via a linker, such as a polypeptide linker provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope. In some examples, the linker located between the first and second segments contains 10, 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, or more amino acid residues provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope. In some examples, the linker located between the second and third segments contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more amino acid residues provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope. In some examples, the linker located between the first and second segments contains about 26 amino acid residues. In some examples, the linker located between the second and third segments contains about 12 amino acid residues. In some examples, the first and second segments are linked via a linker that comprises amino acids corresponding to amino acids 460 to 485 of the full-length E2 polypeptide of a select hepatitis C virus. In some examples, the second and third segments are linked via a linker that comprises amino acids corresponding to amino acids 570 to 580 of the full-length E2 polypeptide of a select hepatitis C virus.
In some examples, the segments of the mutant E2 polypeptide correspond to amino acid segments in E2 polypeptides in select hepatitis C virus such as subtypes Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p or 6t. In some examples, the select hepatitis C virus is a subtype 1 a, Ib, or 1 c virus. In some examples, the select hepatitis C virus is H77, HCV-L2, or HCV-G9.
In some examples, the first segment of the mutant E2 polypeptide provide herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %,
98 %, or 99 % identical to amino acids 412 to 459 of hepatitis C virus H77. In some examples, the second segment of the mutant E2 polypeptide provide herein is 65 %,
70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 % 95 %, 96 %, 97 %, 98 %, or
99 % identical to amino acids 486 to 569 of hepatitis C virus H77. In some examples, the third segment of the mutant E2 polypeptide provided herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 581 to 645 of hepatitis C virus H77.
In some examples, the first amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 888-912 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith. In some examples, the second amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS : 913-937 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith. In some examples, the third amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 938-962 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith. In some examples, the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 727-730 and 740-742 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
In some examples, the mutant E2 polypeptide further includes a polypeptide tag, such as amino or carboxy terminal tag. In some examples, the tag is an N- terminal ubiquitin signal, a poly-histidine sequence, a FLAG (DYKDDDDK) sequence(SEQ ID NO:607), an HA sequence, a myc sequence, a V5 sequence, a chitin binding protein sequence, a maltose binding protein sequence or a glutathione- S-transferase sequence.
Provided herein are isolated nucleic acid molecules that encode a mutant E2 polypeptide provided herein. Also provided herein are expression vectors that contain a nucleic acid molecule sequence that encodes a mutant E2 polypeptide provided herein. In some examples, the nucleic acid molecule encoding a polypeptide provided herein is operably linked to an expression control sequence or regulatory sequence. In some examples, the expression control sequence or regulatory sequence is a promoter. In some examples, the promoter is a viral promoter, a bacterial promoter or a mammalian promoter. In some examples, the promoter is a SV40 promoter, a Rous Sarcoma Virus promoter, or a cytomegalovirus immediate early promoter.
Provided herein are cells that contain a mutant E2 polypeptide provided herein. Also provided herein are cells that contain a nucleic acid molecule encoding a mutant E2 polypeptide provided herein. Also provided herein are cells that contain an expression vector that contains a nucleic acid molecule sequence encoding a mutant E2 polypeptide provided herein. In some examples, the cell is a bacterial cell, a mammalian cell, such as, for example, a Chinese hamster ovary cell.
Provided herein are methods of eliciting an immune response in a subject that involves administering to the mammal a mutant E2 polypeptide provided herein. In some examples, the subject is a mammal, such as, but not limited to, mouse, sheep, goat, horse, rabbit, hamster, rat or human. In particular examples, the subject is a human. In some examples, the method also involves obtaining a sample from the mammal, such as a blood, lymph, or urine sample. In some examples, the method includes administering to the subject a second dose of the polypeptide at a selected time after the first administration.
The methods provided herein further can involve isolating an antibody or antibody-producing cell from the mammal. In some examples, the antibody is a cross-neutralizing antibody. In some examples, the antibody is a conformation- dependent antibody. In some examples, the method further includes fusing the antibody-producing cells from the mammal with a myeloma cell to obtain an antibody-producing hybridoma. In some examples, the polypeptide is in a pharmaceutical composition that includes a pharmaceutically acceptable carrier. In some examples, the polypeptide is in an amount effective to prevent or treat HCV infection in the mammal.
Provided herein are antibodies isolated using methods of production provided herein. The antibody can be a full length antibody (e.g. an IgG) or an antibody fragment (e.g. a Fab or F(ab')2). In some examples, the antibody is a monoclonal antibody, a single chain variable fragment (scFv), or other antigen binding fragment. In some examples, the antibody is a murine antibody. In some examples, the antibody is a conformation dependent antibody. In some examples, the antibody is a cross- neutralizing antibody. Provided herein are methods of eliciting an immune response in a subject that involves administering to the subject a nucleic acid molecule that encodes a mutant E2 polypeptide provided herein. In another aspect, the method involves administering to the mammal an expression vector that includes a nucleic acid molecule sequence encoding a polypeptide provided herein. In some examples, the nucleic acid molecule can include a sequence encoding a polypeptide of SEQ ID NO: 866, 867, 868, 869 or 870. The nucleic acid molecule sequence can include the sequence of SEQ ID NO: 874, 875, 876, 877, 878, 879, 880 or 881. In some examples, the nucleic acid molecule is operably linked to an expression control sequence. The expression control sequence can be a viral, phage, bacterial, or mammalian promoter. In some examples, the promoter can be a SV40 promoter, a Rous Sarcoma Virus promoter, or a cytomegalovirus immediate early promoter. Provided herein are pharmaceutical compositions that include (1) a mutant E2 polypeptide provided herein, (2) a nucleic acid molecule that encodes a mutant E2 polypeptide provided herein, (3) a vector, such as an expression vector, that includes a nucleic acid molecule sequence encoding a mutant E2 polypeptide provided herein, or (4) an antibody that binds to a mutant E2 polypeptide provided herein, and a pharmaceutically acceptable carrier. Also provided herein are immunostimulatory or immunogenic compositions and vaccines that include a mutant E2 polypeptide provided herein or a nucleic acid molecule that encodes a mutant E2 polypeptide provided herein. Provided herein are uses of a mutant E2 polypeptide provided herein or a nucleic acid molecule that encodes a mutant E2 polypeptide provided herein for the treatment or prevention of HCV infection in a subject. Provided herein are uses of a mutant E2 polypeptide provided herein or a nucleic acid molecule that encodes a mutant E2 polypeptide for the preparation of a pharmaceutical composition for the treatment or prevention of HCV infection in a subject.
Provided herein are purified preparations of the polypeptides provided herein in which at least 80 % of the polypeptides are in a conformation capable of binding to a conformation-dependent cross-neutralizing antibody.
Provided herein are purified preparations of the antibody provided herein in which the antibody is at least 5 % of the antibodies in the preparation.
Provided herein are methods for purifying the polypeptide provided herein that involves contacting a sample containing the polypeptide with a conformation- dependent antibody that binds specifically with a polypeptide provided herein under conditions effective for formation of a polypeptide-antibody complex and separating the polypeptide-antibody complex from unrelated polypeptides in the sample. The sample can be a cell lysate, for example, a cell lysate from a bacterial, yeast, insect, or mammalian cell.
In some examples, the method also involves separating the polypeptide from the antibody to obtain a preparation that has at least 50 % polypeptides. In some examples, the polypeptide is separated from the antibody by elution with 0.2 M glycine at pH 2.2; 2M sodium thiocyanate at pH 7.4; or 0.2 M glycine at pH 11.5. In some examples, the method further involves purifying the polypeptide using size- exclusion chromatography.
Provided herein are preparations obtained by a method provided herein in which at least 50 %, at least 75 %, at least 85 %, at least 90 %, or at least 95 % of the polypeptides in the preparation are in a conformation capable of binding to a conformation dependent antibody. In some examples, the conformation-dependent antibody binds specifically with a conformational epitope that includes: (1) amino acids 396-424 having the sequence TAGLVGLLTPGAKQNIQLINTNGSWHINS (SEQ ID NO: 694); (2) amino acids 436-447 having the sequence GWLAGLFYQHKF (SEQ ID NO: 695), and (3) amino acids 523-540 having the sequence GAPTYSWGANDTDVFVLN (SEQ ID NO: 696). In other examples, the conformation-dependent antibody binds specifically with a conformational epitope that includes: (1) amino acids 412-424 having the sequence QLINTNGSWHINS (SEQ ID NO: 688); (2) amino acids 436-447 having the sequence GWLAGLFYQHBCF (SEQ ID NO: 695), and (3) amino acids 523-540 having the sequence GAPTYSWGANDTDVFVLN (SEQ ID NO: 696). In yet other examples, the conformation-dependent antibody binds specifically with a conformational epitope on the HCV E2 polypeptide.
Provided herein are methods for determining whether a mammal has been infected with a HCV that involves contacting a sample, such as for example, a blood sample, from the mammal with a polypeptide provided herein and determining whether the polypeptide binds specifically with an antibody from the sample (e.g., a blood sample) of the mammal to form a polypeptide-antibody complex, wherein the presence of the complex indicates that the mammal has been infected with an HCV and the absence of the complex indicates that the mammal has not been infected with the virus.
Provided herein are methods for identifying an anti-HCV agent that involves contacting a candidate agent with the E2 polypeptide provided herein, identifying the candidate agent as an anti-HCV agent if the candidate agent binds to the E2 polypeptide and prevents its binding with a cell receptor such as CD81 or prevents its ability to inhibit viral replication. Provided herein are mutant HCV E2 polypeptides correctly presenting a conserved HCV E2 conformational epitope and a method for purifying HCV E2 polypeptides that correctly present the conserved E2 conformational epitope. The purified polypeptides are useful as immunogens for raising broadly neutralizing antibodies against HCV, as reagents for diagnosis of HCV infection and for screening of new anti-HCV antiviral agents. The polypeptides and corresponding coding nucleic acid molecules can be used as polypeptide- and nucleic acid-based vaccines to elicit a protective immune response against HCV. Thus, provided herein are methods of immunizing a mammal against HCV that involves administering a polypeptide- or nucleic acid-based vaccine.
Any feature or combination of features described herein are included within the scope provided herein provided that the features included in any such combination are not mutually inconsistent, as will be apparent from the context, this specification and the knowledge of one of ordinary skill in the art. Other features and advantages provided herein will be apparent from the following detailed description and from the claims. DESCRIPTION OF THE FIGURES
FIGS IA-C illustrate properties of anti-HCV E2 Fabs isolated as described herein. FIG. IA illustrates the specificity of anti-E2 Fabs. The binding of Fabs to GST-E1E2 complex and E2 is compared. The GST-E 1E2 fusion protein was captured by a goat anti-GST antibody while soluble E2 and ovalbumin were coated directly onto ELISA plates. Fabs were added to the antigens and subsequently detected with phosphatase-conjugated goat anti-human F(ab)'2 IgG. Recombinant Fabs were produced in cleared lysate of E. coli transformed with the corresponding phagemids. FIG. IB illustrates competition between MAb AR3A and anti-E2 Fabs. Vaccinia-expressed El E2 was captured onto ELISA wells by lectin and preincubated with saturating concentration of soluble Fabs before the addition of MAb AR3A. Binding of MAb AR3A was detected with a goat anti-human IgG Fc antibody and the % reduction of binding compared to that in the absence of a Fab is shown. Lightly-shaded bars indicate that Fabs bind E2 better than El E2; while bars of medium shading indicate that Fabs bind E1E2 better than E2. FIG. 1C illustrates the inhibition of anti-E2 Fab binding to El E2 by mouse MAb H53. El E2 was captured onto ELISA wells in the same manner as shown for FIG. IB and was pre-incubated with a saturating concentration of MAb H53 before the addition of soluble Fabs. Binding of Fabs was detected with a goat anti-human IgG F(ab)'2 antibody and the % reduction of binding compared to that without MAb H53 is shown. FIG. 2 shows neutralization of HCVpp by human Fabs. Infectivity in Relative
Light Units (RLU) is shown for infection of pseudotype virus particles generated with viral Env gene from murine leukemia virus (MLV) (FIG. 2A), H77 (GT Ia) (FIG. 2B), OH8 (GT Ib) (FIG. 2C), CONl (GT Ib) (FIG. 2D) or J6 (GT 2a) (FIG. 2E) in the presence of 10 μg/mL Fabs. ARl-Fabs: B2, Dl & E; AR2-Fabs: F & G; AR3- Fabs: Cl, J2, J3 & L4. Control, anti-HIV gpl20 Fab bl2; Empty, background infectivity from pseudotype virus generated without Env gene. Dotted lines indicate HCVpp infectivity in the absence of any antibody. Error bars represent SEM calculated from three experiments performed in the same manner.
FIG. 3 is a schematic diagram of E2 regions important for binding of AR3- specific antibodies. E2 (residues 384-746) is a transmembrane glycoprotein, and a truncated form of E2 (residues 384-661) can be expressed as a soluble protein that retains its ability to bind cell lines expressing HCV receptors and CD81 -LEL (Michalak, J.P. et al. J. Gen. Virol. 78, 2299-2306 (1997)). The regions that were investigated by antibody competition and alanine mutagenesis are indicated by dotted and solid boxes, respectively. The AR3 discontinuous epitopes include: (1) amino acids 396-424 having the sequence TAGLVGLLTPGAKQNIQLINTNGSWHINS (SEQ ID NO: 694); (2) amino acids 436-447 having the sequence GWLAGLFYQHKF (SEQ ID NO: 695), and (3) amino acids 523-540 having the sequence GAPTY S WGANDTD VF VLN (SEQ ID NO: 696). Important residues in these regions include S424, G523, P525, G53O, D535, V538 and N540. The locations of the N-linked glycans are indicted by branched forks. The hypervariable regions (see Troesch, M. et al. Virology 352, 357-367 (2006) and the transmembrane regions are indicated by the designation HVRs and TM.
FIG. 4 illustrates the levels of human MAb in human liver-chimeric mice 24 hours post-administration. Human liver-chimeric mice (n=6) were injected intraperitoneally with a dose of 200 mg/kg of the isotype control mAb b6, AR3A or AR3B and blood samples were collected at 24 hours before challenging with a genotype Ia HCV-infected human serum KP (100 μL) by intrajugular venous injection. Intravenous administration of human serum is the most reliable way to assure delivery of human serum but a stressful procedure: 5 of 18 treated mice did not recover after the procedure. Human IgG in mouse sera were quantified by a quantitative sandwich ELISA using conjugated and unconjugated goat anti-human F(ab)'2 antibody. Filled bars, mice that died after intrajugular injection of KP serum; Open bars, mice survived the procedure and used in the protection experiments. The mean serum human IgG levels ± s.d. in the surviving mice of group b6, AR3A and AR3B are 2.5 ± 0.3, 3.1 ± 0.5 and 2.6 ± 0.3 mg/mL, respectively. Note that decay of the human MAbs following virus challenge, which may be an explanation for the infection of several antibody-treated mice at later time points, could not be determined because the infected human serum contains normal human antibodies which interfere with IgG quantification. In a control experiment using transplanted mice with low level of human chimerism, human IgG antibodies were readily detected in the mice challenged with 100 μL of the infected serum (n=5, Day 1 mean ± s.d. mouse serum human IgG concentration: 2.6 ± 0.5 mg/mL) (data not shown).
FIG. 5 demonstrates passive antibody protection against an HCV population. Human liver-chimeric mice (n = 6) injected intraperitoneally with 200 mg/kg of the isotype control mAb b6 (left), mAb AR3A (middle) or AR3B (right), were challenged 24 hours later by intrajugular venous injection of genotype 1 a HCV-infected human serum (~2 x 10 HOV RNA copies). One or two mice per group did not recover from anesthesia after intrajugular injection. Data shown are serum viral load in mice quantified by real-time TaqMan PCR. Owing to morbidity, mice N680 and N672 were killed on days 41 and 45, respectively. IU, international units; ID, identification number; i.p., intraperitoneally; i.v., intravenously. In mice injected with mAb AR3A and mAb AR3B, no serum HCV RNA was detected 6 days after viral challenge. FIG. 6 is a sequence comparison of a viral quasispecies population in the HCV genotype la-infected human serum. Partial E2 amino acid sequences (residues 384-622) of a total of 40 clones (represented by KP S9 (SEQ ID NO: 701), KP R14 (SEQ ID NO: 702), KP S6 (SEQ ID NO: 703), KP S 18 (SEQ ID NO: 704), KP S 16 (SEQ ID NO: 705), KP R8 (SEQ ID NO: 706), KP S20 (SEQ ID NO: 707), KP S4 (SEQ ID NO: 708), KP R3 (SEQ ID NO: 709), KP S3 (SEQ ID NO: 710), KP S 12 (SEQ ID NO: 711), KP S15 (SEQ ID NO: 712), KP S5 (SEQ ID NO: 713), KP R7 (SEQ ID NO: 714), KP Rl 1 (SEQ ID NO: 715), KP Rl (SEQ ID NO: 716), KP R12 (SEQ ID NO: 717), KP S7 (SEQ ID NO: 718), KP Rl 5 (SEQ ID NO: 719), KP Rl 8 (SEQ ID NO: 720), KP Sl 1 (SEQ ID NO: 721) and KP R20 (SEQ ID NO: 722)) randomly selected from two independent RT-PCR cloning are shown. See also Table 15. The top sequence, clone KP S9, represents the consensus and dominant sequence in this infectious serum. The periods indicate regions of amino acid sequence identity. The frequency of each clone is bracketed. Hypervariable regions (HVRs) are within the dashed-line boxes. Regions important for binding of AR3-antibodies are within the solid-line boxes. The corresponding sequences of isolates H77 (SEQ ID NO: 723) and UKNlbl2.16 (SEQ ID NO: 724), sharing 87% and 75% amino acid identity with KP S9, respectively, are shown for comparison.
FIG. 7 is a schematic illustration of a panel of recombinant E2 fragments. Full length E2 (residues 384-746) is shown at the top and the relative locations of N- gl yeans and cysteines are marked by light and darker vertical lines, respectively. The hypervariable region 1 (HVRl) at the N-terminus and transmembrane region at the C- terminus of E2 are shaded. The positions of N- or C-terminal truncation in the mutants are indicated, and the Flag tags are indicated by boxes at the C-termini. Fragments are named according to the primer sets used in gene amplification. According to the E2 model proposed by Yagnik et ai, Proteins 40, 355-66 (2000), disulfide bridges are predicted to form between C1-C16 (i.e. residues C429-C644), C2-C4 (C452-C486), C8-C9 (C552-C564), C13-C14 (C597-C607), and C7-C11 (C508-581) or C7-C12 (C581-585).
FIG. 8A-H illustrate the binding properties of E2 fragments. 293T cells were transfected with DNA plasmids encoding the E2 fragments depicted in FIG. 7 and the expression of the corresponding proteins was assayed by sandwich ELISA. ELISA wells were pre-coated with MAbs specific to the 3 different E2 antigenic regions (ARl, AR2 and AR3), or CD81-LEL, to capture the recombinant proteins in serially diluted cell supernatants. The reagents used in the capture are indicated on the left of the bar charts. Bound E2 fragments were detected using the mouse anti-Flag tag M2 MAb (Sigma). Data shown are means of duplicate measurements. FIG. 9A-E are graphs illustrating the antigenic properties of E2flr2a. E2flr2a produced in the presence of kifunensine was purified using a MAb AR3A- affinity column and was eluted with low pH (0.2 M glycine pH 2.2), 2 M NaSCN (pH 7.4) or high pH (0.2 M glycine pH 11.5) buffer. The different purified E2flr2a monomers were titrated from 4 μg/mL (~145 nM, 5-fold serial dilution) to investigate their antigenicities. To study their binding to anti-E2 antibodies, the purified proteins were captured onto microwells precoated with Galanthus nivalis lectins (5 μg/mL) and the captured proteins detected with the indicated human anti-E2 monoclonal antibodies (MAbs). To study their binding to CD81-LEL, microwells coated with maltose binding protein (MBP)-fused CD81-large extracellular loop (LEL) (10 μg/mL) were used to capture the purified proteins and bound proteins were detected with the mouse anti-FLAG tag MAb M2. Bound human or mouse MAbs were detected with peroxidase-conjugated anti-human or anti-mouse secondary antibodies and TMB substrate. The results show that E2flr2a monomers eluted by buffers with different pH are similar antigenically.
FIG. 10A-E are graphs illustrating the antigenic properties of E2ΔTM. E2ΔTM produced in the presence of kifunensine was purified using a MAb AR3A- affinity column and was eluted with 2 M NaSCN buffer (pH 7.4). The effect of pH on the antigenicity of the protein was investigated. Purified E2ΔTM monomers were exposed briefly to low or high pH by adding 10-fold excess volume of 0.2M glycine pH 2.2 or pH 11.5, respectively. After 10 minutes, the solutions were neutralized by adding equal volume of 2M Tris-HCl pH 7.4 and treated and untreated E2ΔTM monomers were titrated from 5.5 μg/mL (~145 nM, 5-fold serial dilution). To study their binding to anti-E2 antibodies, the purified proteins were captured onto microwells precoated with Galanthus nivalis lectins (5 μg/mL) and the captured proteins detected with the indicated human anti-E2 monoclonal antibodies (MAbs). To study their binding to CD81-LEL, microwells coated with maltose binding protein (MBP)-fused CD81 -large extracellular loop (LEL) (10 μg/mL) were used to capture the purified proteins and bound proteins were detected with the mouse anti-FLAG tag MAb M2. Bound human or mouse MAbs were detected with peroxidase-conjugated anti-human or anti-mouse secondary antibodies and TMB substrate. The results show that pH does not have a significant effect on the antigenicity of E2ΔTM. DETAILED DESCRIPTION
A. OVERVIEW
B. Hepatitis C Virus
C. Mutant E2 Polypeptides D. Nucleic Acids Encoding Mutant E2 Polypeptides
E. Cross-neutralizing Antibodies
F. Diagnostic Uses
G. Development of Anti-HCV Therapeutic Agents H. Therapeutic or Prophylactic Uses I. Preparations and Compositions
J. Articles of Manufacture and Other Compositions
K. EXAMPLES
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information is known and can be readily accessed, such as by searching the internet and/or appropriate databases. Reference thereto evidences the availability and public dissemination of such information.
As used herein, the term "hepatitis C virus," "HCV," or "HCVs" includes different viral genotypes, subtypes, quasispecies and isolates. It includes any noncytopathic RNA virus that has a single and positive-stranded RNA genome belonging to the Hepacivirus genus of the Flaviviridae family. The term includes different isolates of HCV such as, without limitation, those having polyprotein sequences and accession numbers shown above, as well as any others in the NCBI database. Examples of different genotypes encompassed by this term include, without limitation, genotype 1, 2, 3, 4, 5 and 6, as described in Simmonds et al. (Hepatology 42:962-973). Reference to HCV also includes those of any additional genotypes that are established. Examples of different subtypes include, without limitation, Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p and 6t . The term also includes cell culture HCVs (HCVcc) and pseudotype HCVs (HCVpp), as well as HCV quasispecies. Various HCVs are described by Simmonds P. in Genetic diversity and evolution of hepatitis C virus— 15 years on, J Gen Virol 85:3173-3188 (2004) and Simmonds et al. in Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes, Hepatology 42:962-973 (2005). HCV nucleotide sequences are known in the art. For example, see the Viral Bioinfomatics Research Center (hcvdb.org) and the Hepatitis C Virus database (hcv.lanl.gov/). An "E2" polypeptide is the HCV viral envelope protein that forms a heterodimer with the El glycoprotein through non-covalent interactions. HCV El and E2 envelope glycoproteins are exposed on the viral surface where they function in viral attachment and fusion to target cells. In the prototype HCV strain H77 (shown herein as SEQ ID NO: 763), the E2 glycoprotein is residues 384 to 746. As used herein, the term "mutant" as used in reference to an HCV E2 polypeptide provided herein means that the polypeptide is modified by one or more substitutions or deletions relative to a naturally occurring HCV E2 polypeptide. Typically, the mutant polypeptide provided herein is free of sequences in the hypervariable region of the HCV E2 polypeptide, in particular, sequences that correspond to the segment defined by amino acid residues 384 to 395. Generally mutant E2 polypeptides provided herein that contain one or more deletions relative to a naturally occurring E2 are able to fold into a conformation that preserves a conformational epitope, such as for example, a conformational epitope recognized by an AR3A, AR3B, AR3C, or AR3D antibody. Exemplary mutant E2 polypeptides provided herein differ from the corresponding naturally-occurring E2 amino acid sequence in that the mutant E2 polypeptide provided herein does not include one or more segments defined by (1) amino acid residues 384 to 411; (2) amino acid residues 460 to 485; (3) amino acid residues 570-580, (4) amino acid residues 646-661, (5) amino acid residues 662-717 or 718-746, or any combination thereof, relative to the naturally-occurring E2 polypeptide. The mutant E2 polypeptides provided herein also can have one or more amino acid modifications that decrease the immunogenicity of particular immunodominant epitopes, such as for example epitopes recognized by the ARIA and ARlB antibodies. Such modifications include, but are not limited to deletion or substitution of amino acids at positions 416, 417, 483, 484, 485, 538, 540, 544, 545, 547, 549 or any combinations thereof relative to the E2 polypeptide sequence of HCV (e.g. HCV strain H77). As used herein, the recitation that a sequence of amino acids "corresponds to" particular amino acids from a selected isolate of HCV, means the corresponding amino acids in another isolate. Since E2 and other proteins may include slight amino acid variations from isolate-to-isolate, the exact amino acid residues or positions may vary by one or two residues. One of skill in the art readily can align, using standard programs, or by eye, since the amount of variation is minimal, polypeptides from different isolates to identify corresponding segments or residues.
As used herein, a "modification" is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods for modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies or direct synthesis.
As used herein, "deletion," when referring to a nucleic acid molecule or polypeptide sequence, refers to the deletion of one or more nucleotides or amino acids compared to a sequence, such as a target polynucleotide or polypeptide or a native or wild-type sequence.
As used herein, "insertion," when referring to a nucleic acid molecule or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids, within a target, native, wild-type or other related sequence. Thus, a nucleic acid molecule that contains one or more insertions compared to a wild-type sequence, contains one or more additional nucleotides within the linear length of the sequence.
As used herein, "additions," to nucleic acid and amino acid molecules describe addition of nucleotides or amino acids onto either termini compared to another nucleic acid or amino acid molecule.
As used herein, "substitution" refers to the replacing of one or more nucleotides or amino acids in a native, target, wild-type or other nucleic acid molecule or polypeptide with an alternative nucleotide or amino acid, without changing the length (as described in numbers of residues) of the molecule. Thus, one or more substitutions in a molecule does not change the number of amino acid residues or nucleotides of the molecule. Substitution mutations compared to a particular polypeptide can be expressed in terms of the number of the amino acid residue along the length of the polypeptide sequence. For example, a modified polypeptide having a modification in the amino acid at the 19l position of the amino acid sequence that is a substitution of Isoleucine (He; I) for cysteine (Cys; C) can be expressed as Il 9C, Ilel9C, or simply C 19, to indicate that the amino acid at the modified 19th position is a cysteine. In this example, the molecule having the substitution has a modification at He 19 of the unmodified polypeptide. As used herein, the term "polypeptide" refers to a polymer of three or more amino acids, regardless of post-translational modifications such as methylation, glycosylation or phosphorylation.
As used herein, a "peptide" refers to a polypeptide that is from 2 to about or 40 amino acids in length. As used herein, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids contained in the antibodies provided include the twenty naturally-occurring amino acids (Table 1), non-natural amino acids, and amino acid analogs (e.g., amino acids wherein the α-carbon has a side chain). As used herein, the amino acids, which occur in the various amino acid sequences of polypeptides appearing herein, are identified according to their well-known, three- letter or one-letter abbreviations (see Table 1). The nucleotides, which occur in the various nucleic acid molecules and fragments, are designated with the standard single- letter designations used routinely in the art. As used herein, "amino acid residue" refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are generally in the "L" isomeric form. Residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968) and adopted at 37 CF. R.. §§. 1.821 - 1.822, abbreviations for amino acid residues are shown in Table IA:
Figure imgf000019_0001
All sequences of amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl- terminus. In addition, the phrase "amino acid residue" is defined to include the amino acids listed in the Table of Correspondence (Table 1), modified, non-natural and unusual amino acids. Furthermore, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.
In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224).
Such substitutions can be made in accordance with those set forth in Table IB as follows:
TABLE IB
Figure imgf000020_0001
Other substitutions also are permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.
As used herein, "naturally occurring amino acids" refer to the 20 L-amino acids that occur in polypeptides.
As used herein, the term "non-natural amino acid" refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non- naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids. Exemplary non-natural amino acids are known to those of skill in the art, and include, but are not limited to, 2- Aminoadipic acid (Aad), 3-Aminoadipic acid (Baad), β-alanine/β -Amino-propionic acid (BaIa), 2-Aminobutyric acid (Abu), 4-Aminobutyric acid/piperidinic acid (4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe), 2-Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm), 2,4- Diaminobutyric acid (Dbu), Desmosine (Des), 2,2'-Diaminopimelic acid (Dpm), 2,3- Diaminopropionic acid (Dpr), N-Ethylglycine (EtGIy), N-Ethylasparagine (EtAsn), Hydroxylysine (HyI), allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3 Hyp), 4- Hydroxyproline (4Hyp), Isodesmosine (Ide), allo-Isoleucine (AiIe), N-Methylglycine, sarcosine (MeGIy), N-Methylisoleucine (MeIIe), 6-N-Methyllysine (MeLys), N- Methylvaline (MeVaI), Norvaline (Nva), Norleucine (NIe), Ornithine (Orn).
As used herein, a "native polypeptide" or a "native nucleic acid" molecule is a polypeptide or nucleic acid molecule, respectively, that can be found in nature. A native polypeptide or nucleic acid molecule can be the wild-type form of a polypeptide or nucleic acid molecule. A native polypeptide or nucleic acid molecule can be the predominant form of the polypeptide, or any allelic or other natural variant thereof. The variant polypeptides and nucleic acid molecules provided herein can have modifications compared to native polypeptides and nucleic acid molecules.
As used herein, the wild-type form of a polypeptide or nucleic acid molecule is a form encoded by a gene or by a coding sequence encoded by the gene. Typically, a wild-type form of a gene, or molecule encoded thereby, does not contain mutations or other modifications that alter function or structure. The term wild-type also encompasses forms with allelic variation as occurs among and between species and virus genotype and subtype variations. As used herein, a predominant form of a polypeptide or nucleic acid molecule refers to a form of the molecule that is the major form produced from a gene. A "predominant form" varies from source to source. For example, different cells or tissue types can produce different forms of polypeptides, for example, by alternative splicing and/or by alternative protein processing. In each cell or tissue type, a different polypeptide can be a "predominant form." As used herein, numeric terms identifying amino acid residues or positions in a polypeptide, i.e. the protein or polypeptide "coordinates," for example, the term "residues 396 to 424," "residue 416," or "amino acid 416," are based on the absolute amino acid numbering system for HCV described by Kuiken et al. in Hepatology 44: 1355-1361 (2006), which is incorporated herein by reference in its entirety. Briefly, the polyprotein sequence of HCV strain WIl is used as a reference in the numbering system, and the first amino acid of the core protein is amino acid residue number 1. Other HCV polyprotein sequences are compared with the H77 polyprotein sequences by sequence alignment. Insertions in other non-H77 sequences are identified using a residue number/alphabet designation relative to the H77 reference. For example, three inserted amino acids in a non-H77 polyprotein sequence inserted between amino acid residues 396 and 397 of the reference H77 sequence would be identified as follows: residue 396a, 396b and 396c. Insertions longer than the length of the alphabet would be identified as follows: ...396x, 396y, 396z, 396aa, 396ab, 396ac, ... 396ax, 396ay, 396az, 396ba, 396bb ... . Deletions in a non-H77 sequence relative to the H77 reference sequence can be indicated by identifying the residue deleted. For example, a missing residue, i.e. a "deletion", in a non-H77 sequence relative to the H- 77 reference sequence identified in a sequence alignment such as a deletion of amino acid residue 396 is indicated by the term "del 396". Thus, according to the numbering system used herein, a polypeptide coordinate or coordinates, such as "amino acid 396," "residue 396," or "amino acids 396 to 424," refer to analogous residues or segments in HCV polyproteins from different isolates, strains, subtypes or genotypes. Analogous residues or segments can be identified by sequence alignment as described below. A similar system is used for identifying HCV nucleotide sequence.
Generally, the amino acid sequences of two or more HCV E2 polypeptides can be compared by alignment using methods known in the art including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York ( 1991 ); and Carillo et al., Applied Math . 48 : 1073 ( 1988), the teachings of which are incorporated herein by reference. Two HCV polyprotein sequences can be compared by sequence alignment in a manner to produce the highest degree of sequence similarity or identity. Upon such alignment, sequence identity is determined on a position-by-position basis, e.g., the sequences are "identical" at a particular position if at that position, the amino acid residues are identical. Exemplary methods to determine sequence identity between two sequences are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs. Examples of such programs include, but are not limited to, the GCG program package (Devereux, et ah, Nucleic Acids Research, 12:387 (1984)), BLASTP, BLASTN and FASTA (Altschul et al., J. Molec. Biol., 215:403 (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul et ah, NCVI NLM NIH
Bethesda, MD 20894, Altschul et al, J. Molec. Biol., 215:403 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between sequences. A variety of HCV sequence analysis tools are known and available in the art, such as for example, but not limited to, online tools available from the Hepatitis C Virus (HCV) Database Project provided by the Los Alamos National laboratory (hcv.lanl.gov) and the European HepCVax Cluster provided by the Leiden University Medical Center, The Netherlands (euhcvdb.ibcp.fr).
Once an HCV amino acid sequence is optimally aligned with that of the HCV strain H77, the E2 amino acid sequence can be identified based on its correspondence with the HCV strain H77 amino acid residues 384 to 746. Accordingly, the term "amino acid 396" or "amino acids 396 to 424" refers to analogous residues in different HCVs including, for example, HCVs of different isolates, strains, species, quasispecies, subtypes or genotypes. As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantly, produced, including any fragment thereof containing at least a portion of the variable region of the immunoglobulin molecule that retains the binding specificity ability of the full-length immunoglobulin. Hence, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). Antibodies include antibody fragments, such as anti-RSV antibody fragments. As used herein, the term antibody, thus, includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b).
As used herein, an "antibody fragment" refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody that binds antigen (e.g. one or more CDRs and/or one or more antibody combining sites) and thus retains the binding specificity, and at least a portion of the specific binding ability of the full-length antibody; antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically, e.g. recombinantly produced derivatives. An antibody fragment is included among antibodies. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fvs (scFv), Fv, dsFv, diabody, Fd and Fd' fragments other fragments, including modified fragments (see, for example, Methods in Molecular Biology, VoI 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1 ; p 3-25, Kipriyanov). The fragment can include multiple chains linked together, such as by disulfide bridges and/or by peptide linkers. An antibody fragment generally contains at least or about 50 amino acids and typically at least or about 200 amino acids.
As used herein, an Ig domain is a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond. Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.
As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. The variable domains provide antigen specificity, and thus are responsible for antigen recognition. Each variable region contains CDRs that are part of the antigen-binding site domain and framework regions (FRs).
As used herein, "antigen-binding domain," "antigen-binding site," "antigen combining site" and "antibody combining site" are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with cognate antigen. A native conventional full-length antibody molecule has two conventional antigen-binding sites, each containing portions of a heavy chain variable region and portions of a light chain variable region. A conventional antigen-binding site contains the loops that connect the anti-parallel beta strands within the variable region domains. The antigen combining sites can contain other portions of the variable region domains. Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions also are called complementarity-determining regions (CDRs).
As used herein, framework regions (FRs) are the domains within the antibody variable region domains that are located within the beta sheets; the FRs are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved than that of the variable region domain. In conventional full-length antibody molecules, each light chain has a single light chain constant region (CL) domain and each heavy chain contains one or more heavy chain constant region (CH) domains, which include, CHI, CH2, CH3 and CH4. Full-length IgA, IgD and IgG isotypes contain CHU CH2, CH3 and a hinge region, while IgE and IgM contain CHI, CH2, CH3 and CH4. CHI and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms. Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g. through interactions with various cells, biomolecules and tissues. As used herein, the term "binds specifically" or "specifically binds," with reference to an antibody/antigen interaction, means that the antibody binds with a particular antigen without substantially binding to other unrelated antigens. For example, the antibody has at least 50 % or greater affinity, such as about 75 % or greater affinity, such as, for example, about 90 % or greater affinity, to a particular polypeptide than to other unrelated polypeptides. In addition, typically, an antibody that specifically binds (or that immunospecifically binds) to a virus antigen or virus is one that binds to a virus antigen (or to the antigen in the virus or to the virus) with an affinity constant (K3) of about or IxIO7M'1 or Ix 108M' 'or greater (or a dissociation constant (Kd) of Ix 10"7M or IxIC8M or less). Affinity and dissociation constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11 :54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336
(1989); see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of anti-HCV E2 antibodies). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g. , BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).
As used herein, "neutralize," as used herein with reference to an antibody, means that the antibody can prevent or reduce HCV infection or replication in a cell culture or in a mammal, as well as alleviate one or more symptoms associated with HCV infection in a mammal. The term "reduce," as used herein, means a decrease in any amount such as a 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 % or more than 65 %. HCV infection or replication can be detected by examining HCV RNA levels, virus particles count or clinical symptoms associated with HCV infection using methods known to those of skill in the art. Whether an antibody will prevent or reduce HCV infection or replication or alleviate associated symptoms can be determined using methods known in the art, as well as the methods described herein, including determining the level of HCV RNA in a sample from a mammal that has been infected with HCV or detecting reduction of signals from a reporter gene encoded by the virus such as, for example, the relative light unit (RLU) for luciferase or the mean fluorescence intensity (MFI) of green fluorescent protein (GFP). Thus, whether an antibody will bind selectively to HCV and neutralize it can be determined using methods known in the art, as well as the methods described herein, including determining the level of HCV RNA or detecting reduction of signals from a reporter gene encoded by the virus such as, for example, the relative light unit (RLU) for luciferase or the mean fluorescence intensity (MFI) of green fluorescent protein (GFP). As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically comprise chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three dimensional structural characteristics, as well as specific charge characteristics. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds, and is made up of one or more segments of amino acids. An epitope can be a linear or conformational epitope, and can be continuous or discontinuous. Typically, linear epitopes are continuous, i.e. made up of one continuous stretch of amino acids. Conformational epitopes can be discontinuous i.e. made up of two or more discontinuous segments of amino acids that come together to form an epitope when the antigen is folded. Methods for determining whether antibodies bind to the same epitope are known in the art. Epitopes can be defined or mapped by standard methods well known in art. For example, epitopes can be mapped using assays, such as ELISA assays, utilizing peptide libraries or site-directed mutagenesis of the antigen (such as alanine-scanning of the antigen).
As used herein, "binds to the same epitope" with reference to two or more antibodies means that the antibodies compete for binding to an antigen and bind to the same, overlapping or encompassing continuous or discontinuous segments of amino acids. Those of skill in the art understand that the phrase "binds to the same epitope" does not necessarily mean that the antibodies bind to exactly the same amino acids. The precise amino acids to which the antibodies bind can differ. For example, a first antibody can bind to a segment of amino acids that is completely encompassed by the segment of amino acids bound by a second antibody. In another example, a first antibody binds one or more segments of amino acids that significantly overlap the one or more segments bound by the second antibody. For the purposes herein, such antibodies are considered to "bind to the same epitope."
Antibody competition assays can be used to determine whether an antibody "binds to the same epitope" as another antibody. Such assays are well known in the art and are described herein (see, e.g. Examples 1-4). Typically, competition of 70 % or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more, of an antibody known to interact with the epitope by a second antibody under conditions in which the second antibody is in excess and the first saturates all sites, is indicative that the antibodies "bind to the same epitope." To assess the level of competition between two antibodies, for example, radioimmunoassays or assays using other labels for the antibodies, such as biotin (see, e.g., Example 1) can be used. For example, an HCV antigen, such as the E1E2 complex, can be incubated with a saturating amount of a first anti-HCV antibody or antigen-binding fragment thereof conjugated to a labeled compound (e.g., 3H, 125I or biotin) in the presence of the same amount of a second unlabeled anti-HCV antibody. The amount of labeled antibody that is bound to the antigen in the presence of the unlabeled blocking antibody is then assessed and compared to binding in the absence of the unlabeled blocking antibody. Competition is determined by the percentage change in binding signals in the presence of the unlabeled blocking antibody compared to the absence of the blocking antibody. Thus, if there is a 70% inhibition of binding of the labeled antibody in the presence of the blocking antibody compared to binding in the absence of the blocking antibody, then there is competition between the two antibodies of 70%. Thus, reference to competition between a first and second antibody of 70 % or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more, means that the first antibody inhibits binding of the second antibody (or vice versa) to the antigen by 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more (compared to binding of the antigen by the second antibody in the absence of the first antibody). Thus, inhibition of binding of a first antibody to an antigen by a second antibody of 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more indicates that the two antibodies bind to the same epitope.
As used herein, the term "bind selectively" or "selectively binds," in reference to a polypeptide or an antibody provided herein, means that the polypeptide or antibody binds with a selected epitope without substantially binding to another epitope. Typically, an antibody or fragment thereof that selectively binds to a selected epitope specifically binds to the epitope, such as with an affinity constant (Ka) of about or IxIO7M"1 or 1 x 108M-Or greater, as defined below. As used herein, "specifically binds" or "immunospecifically binds," with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of the antibody and the antigen. The antigen can be an isolated antigen or presented in a virus. Typically, an antibody that specifically binds to a virus antigen or virus is one that binds to the virus antigen (or to the antigen in the virus or to the virus) with an affinity constant (Ka) of about or IxIO7M"1 or Ix 108M'1 or greater (or a dissociation constant (K<0 of 1 x 10'7M or 1 x 10"8M or less). Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of anti-HCV antibodies). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335). An antibody that specifically binds to a virus antigen (or virus) can bind to other peptides, polypeptides, or proteins or viruses with equal or lower binding affinity. Typically, an antibody or antigen-binding fragment thereof provided herein that binds specifically binds to a HCV E2 protein does not cross-react with other antigens or cross reacts with substantially (at least 10-100 fold) lower affinity for such antigens. Similarly, an antibody or antigen-binding fragment thereof provided herein that binds specifically to a HCV E1E2 complex does not cross-react with other antigens or cross reacts with substantially (at least 10-100 fold) lower affinity for such antigens. An antibody or antigen-binding fragment thereof provided herein that binds selectively to E2 typically also binds to the El E2 complex. Antibodies or antigen- binding fragments that selectively bind to a particular virus antigen (e.g. a HCV E2 or El E2) can be identified, for example, by immunoassays, such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISAs), surface plasmon resonance, or other techniques known to those of skill in the art. An antibody or antigen-binding fragment thereof that specifically binds to an epitope on a HCV E2 or El E2 typically is one that binds to the epitope (presented in the protein or virus) with a higher binding affinity than to any cross-reactive epitope as determined using experimental techniques, such as, but not limited to, immunoassays, surface plasmon resonance, or other techniques known to those of skill in the art. Specifically binding to an isolated HCV protein (i.e., a recombinantly produced protein), such as HCV E1E2, does not necessarily mean that the antibody will exhibit the same immunospecific binding and/or neutralization of the virus. Such measurements and properties are distinct. The affinity for the antibody or antigen-binding fragments for virus or the antigen as presented in the virus can be determined. For purposes herein, when describing an affinity or related term, the target, such as the isolated protein or the virus, will be identified.
As used herein, the term "conformation-dependent," in reference to an antibody, means that the antibody recognizes and binds specifically with discontinuous epitopes composed of amino acid residues that are located at some distance from each other, i.e. the residues are discontinuous in the polypeptide sequence. The discontinuous epitopes come together through proper folding of the polypeptide to form a binding site, i.e. a conformational epitope that is recognized by a conformation-dependent antibody.
As used herein, the term cross-neutralizing means the ability to neutralize at least two HCV strains, isolates, species, quasispecies, subtypes or genotypes. The term "neutralize," as used herein in reference to an antibody, means that the antibody can prevent or reduce HCV infection or replication in a cell culture or in a mammal, as well as alleviate one or more symptoms associated with HCV infection in a mammal. The term "reduce," as used herein, means a decrease in any amount such as a 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 % or more than 65 %. HCV infection or replication can be detected by examining HCV RNA levels, virus particles count or clinical symptoms associated with HCV infection. Whether an antibody will prevent or reduce HCV infection or replication or alleviate associated symptoms can be determined using methods known in the art, as well as the methods described herein, including determining the level of HCV RNA in a sample from a mammal that has been infected with HCV or detecting reduction of signals from a reporter gene encoded by the virus such as, for example, the relative light unit (RLU) for luciferase or the mean fluorescence intensity (MFI) of green fluorescent protein (GFP). Examples of cross-neutralizing antibodies that bind specifically with the discontinuous epitopes provided herein include AR3A, AR3B, AR3C and AR3D. As used herein, "linker" or "spacer" peptide refers to short sequences of amino acids, such as 2, 5 or 10 to 20, 30, 40, 50, 60, 70 or 80, that join two polypeptide sequences (or nucleic acid encoding such an amino acid sequence). "Peptide linker" refers to the short sequence of amino acids joining the two polypeptide sequences. Linkers are well-known and any known linkers can be used in the provided methods. Exemplary of polypeptide linkers are (Gly-Ser)n amino acid sequences, with some GIu or Lys residues dispersed throughout to increase solubility. Other exemplary linkers are described herein; any of these and other known linkers can be used with the provided compositions and methods. Linkers with reference to the mutant E2 polypeptides, also include all or part of the native sequence joining the segments in the native E2 polypeptide, as long as the resulting polypeptide does not correspond to amino acids about 412-462 of the E2 polypeptide of the particular HCV isolate.
As used herein, a "tag" or an "epitope tag" refers to a sequence of amino acids, typically added to the N- or C- terminus of a polypeptide, such as the polypeptides provided herein. The inclusion of tags fused to a polypeptide can facilitate polypeptide purification and/or detection. Typically, a tag or tag polypeptide refers to a polypeptide that has enough residues to provide an epitope recognized by an antibody or can serve for detection or purification, yet is short enough such that it does not interfere with activity of the chimeric polypeptide to which it is linked. The tag polypeptide typically is sufficiently unique so an antibody that specifically binds thereto does not substantially cross-react with epitopes in the polypeptide to which it is linked. Suitable tag polypeptides generally have at least 5 or 6 amino acid residues and usually between about 8-50 amino acid residues, typically between 9-30 residues. The tags can be linked to one or more chimeric polypeptides in a multimer and permit detection of the multimer or its recovery from a sample or mixture. Such tags are well known and can be readily synthesized and designed. Exemplary tag polypeptides include those used for affinity purification and include, histidine (His) tags, the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5, (Field et al. (1988) MoI. Cell. Biol. 5:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (see, e.g., Evan et al. (1985) Molecular and Cellular Biology S :3610-3616); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. (1990) Protein Engineering 3:547-553 (1990). An antibody used to detect an epitope-tagged antibody is typically referred to herein as a secondary antibody.
As used herein, the term "nucleic acid" refers to a polymer of deoxyribose nucleic acids (DNA), as well as ribose nucleic acids (RNA). The term includes linear molecules, as well as covalently closed circular molecules. It includes single stranded molecules, as well as double stranded molecules. Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid). The term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is undine. Nucleic acids can contain nucleotide analogs, including, for example, mass modified nucleotides, which allow for mass differentiation of nucleic acid molecules; nucleotides containing a detectable label such as a fluorescent, radioactive, luminescent or chemiluminescent label, which allow for detection of a nucleic acid molecule; or nucleotides containing a reactive group such as biotin or a thiol group, which facilitates immobilization of a nucleic acid molecule to a solid support. A nucleic acid also can contain one or more backbone bonds that are selectively cleavable, for example, chemically, enzymatically or photolytically cleavable. For example, a nucleic acid can include one or more deoxyribonucleotides, followed by one or more ribonucleotides, which can be followed by one or more deoxyribonucleotides, such a sequence being cleavable at the ribonucleotide sequence by base hydrolysis. A nucleic acid also can contain one or more bonds that are relatively resistant to cleavage, for example, a chimeric oligonucleotide primer, which can include nucleotides linked by peptide nucleic acid bonds and at least one nucleotide at the 3' end, which is linked by a phosphodiester bond or other suitable bond, and is capable of being extended by a polymerase. Peptide nucleic acid sequences can be prepared using well-known methods (see, for example, Weiler et al. (1997) Nucleic Acids Res. 25:2792-2799).
As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), joined together, typically by phosphodiester linkages. Polynucleotides also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid). Polynucleotides (nucleic acid molecules), include single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) as well as analogs or derivatives of either RNA or DNA. The term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is undine. Polynucleotides can contain nucleotide analogs, including, for example, mass modified nucleotides, which allow for mass differentiation of polynucleotides; nucleotides containing a detectable label such as a fluorescent, radioactive, luminescent or chemiluminescent label, which allow for detection of a polynucleotide; or nucleotides containing a reactive group such as biotin or a thiol group, which facilitates immobilization of a polynucleotide to a solid support. A polynucleotide also can contain one or more backbone bonds that are selectively cleavable, for example, chemically, enzymatically or photolytically cleavable. For example, a polynucleotide can include one or more deoxyribonucleotides, followed by one or more ribonucleotides, which can be followed by one or more deoxyribonucleotides, such a sequence being cleavable at the ribonucleotide sequence by base hydrolysis. A polynucleotide also can contain one or more bonds that are relatively resistant to cleavage, for example, a chimeric oligonucleotide primer, which can include nucleotides linked by peptide nucleic acid bonds and at least one nucleotide at the 3' end, which is linked by a phosphodiester bond or other suitable bond, and is capable of being extended by a polymerase. Peptide nucleic acid sequences can be prepared using well-known methods (see, for example, Weiler et al. (1997) Nucleic Acids Res. 25:2792-2799). Exemplary of the nucleic acid molecules (polynucleotides) provided herein are oligonucleotides, including synthetic oligonucleotides, oligonucleotide duplexes, primers, including fill- in primers, and oligonucleotide duplex cassettes.
As used herein, a "DNA construct" is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
As used herein, a "DNA segment" is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5' to 3' direction, encodes the sequence of amino acids of the specified polypeptide.
As used herein, a positive strand polynucleotide refers to the "sense strand" of a polynucleotide duplex, which is complementary to the negative strand or the "antisense" strand. In the case of polynucleotides which encode genes, the sense strand is the strand that is identical to the mRNA strand that is translated into a polypeptide, while the antisense strand is complementary to that strand. Positive and negative strands of a duplex are complementary to one another. The term "isolated," as used herein with reference to a nucleic acid molecule, means that the nucleic acid molecule is free of unrelated nucleic acid sequences, i.e. nucleic acid sequences encoding other genes or non-E2 polypeptide sequences, or those involved in the expression of such other genes, that flank it's 5' and 3' ends in the naturally-occurring genome of the organism from which the nucleic acid provided herein is derived. Accordingly, an "isolated nucleic acid" provided herein has a structure that is different from that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. Thus, the term "isolated nucleic acid molecule" includes, for example, (1) a DNA molecule that has the sequence of part of a naturally occurring genomic DNA molecule, but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (2) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally-occurring vector or genomic DNA; (3) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (4) a recombinant nucleotide sequence that is part of a hybrid gene, i.e. a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (1) DNA molecules, (2) transfected cells, and (3) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
As used herein, a regulatory region or an "expression control sequence" of a nucleic acid molecule means a cis-acting nucleotide sequence that influences expression, positively or negatively, of an operatively linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.
Particular examples of gene regulatory regions or expression control sequences are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or as part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.
Regulatory regions or expression control sequences also include, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and stop codons, leader sequences and fusion partner sequences, internal ribosorne binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector. As used herein, the term "operably linked" means that a nucleic acid and an expression control sequence are positioned in such a way that the expression control sequence promoter regulates or mediates the transcription of the nucleic acid. As used herein, a "host cell" is a cell that is used to receive, maintain, reproduce and amplify a vector. A host cell also can be used to express the polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids. In one example, the host cell is a genetic package, which can be induced to express the variant polypeptide on its surface. In another example, the host cell is infected with the genetic package. For example, the host cells can be phage-display compatible host cells, which can be transformed with phage or phagemid vectors and accommodate the packaging of phage expressing fusion proteins containing the variant polypeptides. As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation. Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression/display of the polypeptide encoded by the nucleic acid. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
As used herein, a vector also includes "virus vectors" or "viral vectors." Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.
An "expression vector" is a nucleic acid molecule capable of transporting and/or allowing for the expression of another nucleic acid to which it has been linked. Expression vectors contain appropriate expression control sequences that direct expression of a nucleic acid that is operably linked to the expression control sequence to produce a transcript. The product of that expression is referred to as a messenger ribose nucleic acid (mRNA) transcript. The expression vector also can include other sequences such as enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modified polyadenylation and transcriptional termination sequences, e.g. bovine growth hormone or rabbit beta-globulin polyadenylation sequences, to improve expression of the nucleic acid encoding the E2 polypeptide.
As used herein, "expression" refers to the process by which polypeptides are produced by transcription and translation of polynucleotides. The level of expression of a polypeptide can be assessed using any method known in art, including, for example, methods of determining the amount of the polypeptide produced from the host cell. Such methods can include, but are not limited to, quantitation of the polypeptide in the cell lysate by ELISA5 Coomassie blue staining following gel electrophoresis, Lowry protein assay and Bradford protein assay.
As used herein, the term "purified" with reference to a polypeptide or antibody preparation means that the polypeptide or antibody in the preparation is substantially free of naturally-associated components, i.e. components that accompany it in its natural state. A chemically synthesized polypeptide, one produced using recombinant DNA technology, or one produced in a cellular system different from the cell system from which the polypeptide provided herein naturally originates, is substantially free from its naturally associated components. The term "purified" also encompasses a biological sample such as a blood sample that has been subject to at least one separation step, for example, centrifugation to separate cellular components from non- cellular components. In this case, both fractions of the original blood sample are encompassed by the term "purified." The term "purified" does not encompass a polypeptide or antibody separated in a lane of a protein gel in which multiple unrelated polypeptides or antibodies have been separated. In general, a polypeptide provided herein can constitute at least about 25 % by weight of a sample containing the polypeptide provided herein, and usually constitutes at least about 50%, at least about 75 %, at least about 85 %, at least about 90 % of a sample, particularly at least about 95 % of the sample or 99 % or more. As used herein, "prevent," "preventing" or "prevention" or prophylaxis, and grammatically equivalent forms thereof, refers to use in a prophylactic manner that includes, for example, preventing a new infection or viral replication or reducing the probability of infection, as well as preventing the onset of symptoms and/or their underlying cause. The terms "treat," "treating" and "treatment," include reducing viral replication, reducing the severity and/or frequency of symptoms, eliminating the symptoms and/or underlying cause or improving or remediating damage associated with the infection. The term "reduce" or "reduction" means a decrease in any amount, for example, a decrease of 5 %, 10 %, 20 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more than 90 %.
As used herein, a "pharmaceutically effective agent" includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.
As used herein, a "therapeutic effect" means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition.
As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.
As used herein, "therapeutic efficacy" refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, or composition containing a compound has been administered.
As used herein, a "prophylactically effective amount" or a "prophylactically effective dose" refers to the quantity of an agent, compound, material, or composition containing a compound that when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset, or reoccurrence, of disease or symptoms, reducing the likelihood of the onset, or reoccurrence, of disease or symptoms, or reducing the incidence of viral infection. The full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations. As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic. As used herein, the term "diagnostically effective" amount refers to the quantity of an agent, compound, material, or composition containing a detectable compound that is at least sufficient for detection of the compound following administration to a subject. Generally, a diagnostically effective amount of an anti- RSV antibody or antigen-binding fragment thereof, such as a detectably-labeled antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment thereof that can be detected by a secondary agent, administered to a subject for detection is the quantity of the antibody or antigen-binding fragment thereof which is sufficient to enable detection of the site having the HCV antigen for which the antibody or antigen-binding fragment thereof is specific. In using the antibodies provided herein for the in vivo detection of antigen, a detectably labeled antibody or antigen-binding fragment thereof is given in a dose which is diagnostically effective. As used herein, a label or detectable moiety is a detectable marker (e.g., a fluorescent molecule, chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product) that can be attached or linked directly or indirectly to a molecule or associated therewith and can be detected in vivo and/or in vitro. The detection method can be any method known in the art, including known in vivo and/or in vitro methods of detection (e.g., imaging by visual inspection, magnetic resonance (MR) spectroscopy, ultrasound signal, X-ray, gamma ray spectroscopy (e.g., positron emission tomography (PET) scanning, single-photon emission computed tomography (SPECT)), fluorescence spectroscopy or absorption). Indirect detection refers to measurement of a physical phenomenon, such as energy or particle emission or absorption, of an atom, molecule or composition that binds directly or indirectly to the detectable moiety.
As used herein, the term "subject" refers to an animal, including a mammal, such as a human being. As used herein, a patient refers to a human subject.
As used herein, animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and other animals. Non-human animals exclude humans as the contemplated animal. The enzymes provided herein are from any source, animal, plant, prokaryotic and fungal. Most enzymes are of animal origin, including mammalian origin. As used herein, "pharmaceutically acceptable" it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof, for example, a buffered aqueous, oil or organic medium containing optional stabilizing agents and adjuvants for stimulation of immune binding.
As used herein, a "unit dose form" refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.
As used herein, a "single dosage formulation" refers to a formulation for direct administration. As used herein, an "article of manufacture" is a product that is made and sold.
As used throughout this application, the term is intended to encompass any of the compositions provided herein contained in articles of packaging.
As used herein, a "fluid" refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
As used herein, a "composition" refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.
As used herein, a "combination" refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.
As used herein, combination therapy refers to administration of two or more different therapeutics, such as two or more different anti-RSV antibodies and/or anti- RSV antibodies and antigen-binding fragments thereof. The different therapeutic agents can be provided and administered separately, sequentially, intermittently, or can be provided in a single composition.
As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof, for a purpose including, but not limited to, activation, administration, diagnosis, and assessment of a biological activity or property. As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide, comprising "an immunoglobulin domain" includes polypeptides with one or a plurality of immunoglobulin domains. As used herein, the term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Hence "about 5 amino acids" means "about 5 amino acids" and also "5 amino acids." As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non- variant. As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732). A. OVERVIEW Provided herein are mutant E2 polypeptides, compositions containing them, as well as methods for their production, purification and use. The polypeptides provided herein can be used as immunogens to elicit antibodies that can protect against infection by a hepatitis C virus (HCV). The mutant HCV E2 polypeptides provided herein display the conserved neutralizing AR3 epitopes recognized by conformation- dependent cross-neutralizing anti-HCV antibodies. Thus, provided herein are mutant E2 polypeptides displaying conserved neutralizing epitopes, nucleic acids encoding these polypeptides and expression vectors for their production. Provided herein are cells comprising such nucleic acids or expression vectors, a preparation or pharmaceutical composition comprising a mutant HCV E2 polypeptide, as well as (1) a method of eliciting an immune response in a mammal comprising administering a mutant HCV E2 polypeptide, (2) a method for determining whether a mammal has been infected with an HCV, and (3) a method for identifying an anti-HCV agent. B. Hepatitis C Virus
Hepatitis C virus (HCV) is a noncytopathic, positive-stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family that causes acute and chronic hepatitis and hepatocellular carcinoma (Hoofhagle, J. H. (2002) Hepatology 36, S21-29). The hepatocyte is the primary target cell, although various lymphoid populations, especially B cells and dendritic cells also can be infected at lower levels (Kanto et al, Immunol. 162, 5584-5591 (1999); Auffermann-Gretzinger et al, Blood 97, 3171-3176 (2001); Hiasa et al. (1998) Biochem. Biophys. Res. Commun. 249, 90-95). A striking feature of HCV infection is its tendency towards chronicity with at least 70 % of acute infections progressing to persistence (Hoofhagle, J. H. (2002) Hepatology 36, S21-29). HCV chronicity is often associated with significant liver disease, including chronic active hepatitis, cirrhosis and hepatocellular carcinoma (Alter, H. J. & Seeff, L. B. (2000) Semin. Liver Dis. 20, 17-35). With over 170 million people currently infected (id.), HCV represents a growing public health concern.
HCV viruses can be categorized into several genotypes and subtypes. Exemplary HCV genotypes include, but are not limited to, genotype 1, 2, 3, 4, 5 and 6. Exemplary of HCV subtypes include, but are not limited to, Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p and 6t .
The single stranded HCV RNA genome has a single open reading frame (ORF) encoding a large polyprotein. The polyprotein has about 3010-3033 amino acids (Q.-L. Choo, etal. Proc. Natl. Acad. ScL USA 88, 2451-2455 (1991); N. Kato et al., Proc. Natl. Acad. Sci. USA 87, 9524-9528 (1990); A. Takamizawa et al., J. Virol. 65, 1105-1113 (1991)). Nucleic acid and amino acid sequences for different isolates of HCV can be found in the art, for example, in the National Center for Biotechnology Information (NCBI) database (see ncbi.nlm.nih.gov).
An example of an HCV subtype 1 a is strain H77, which can be found in the NCBI database as accession number AF009606. Its polyprotein sequence (AAB66324) is as follows:
1 MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRG
61 RRQPI PKARRPEGRTWAQPGYPWPI1YGNEGCGWAGWLLS PRGSRPSWGPTDPRRRSRNLG
121 KVI DTLTCGFADLMGYI PLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSI FLLA 181 LLSCLTVPASAYQVRNSSGLYHVTNDCPNSSIVYEAADAILHTPGCVPCVREGNASRCWV
241 AVTPTVATRDGKLPTTQLRRHIDLLVGSATLCSALYVGDLCGSVFLVGQLFTFSPRRHWT
301 TQDCNCSIYPGHI TGHRMAWDMMMNWS PTAALWAQLLRI PQAIMDMIAGAHWGVLAGIA
361 YFSMVGNWAKVLVVLLLFAGVDAETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSW 421 HINSTALNCNESLNTGWLAGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPI SYANGSGL
481 DERPYCWHYPPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLN
541 NTRPPLGNWFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLLCPTDCFRKHPEATYSRCGSG
601 PWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRS
661 ELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQNIVDVQYLYGVGSSIASWAI KWEYVV 721 LLFLLLADARVCSCLWMMLLI SQAEAALENLVILNAASLAGTHGLVSFLVFFCFAWYLKG
781 RWVPGAVYAFYGMWPLLLLLLALPQRAYALDTEVAASCGGVVLVGLMALTLSPYYKRYIS
841 WCMWWLQYFLTRVEAQLHVWVPPLNVRGGRDAVILLMCVVHPTLVFDITKLLLAIFGPLW
901 ILQASLLKVPYFVRVQGLLRICALARKIAGGHYVQMAI IKLGALTGTYVYNHLTPLRDWA
961 HNGLRDLAVAVEPVVFSRMETKLITWGADTAACGDIINGLPVSARRGQEILLGPADGMVS 1021 KGWRLLAPITAYAQQTRGLLGCI ITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWT
1081 VYHGAGTRTIAS PKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVI
1141 PVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVEN
1201 LETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAA
1261 TLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDI I ICDECHS 1321 TDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGKAI
1381 PLEVI KGGRHLI FCHSKKKCDELAAKLVALGINAVAYYRGLDVSVI PTSGDVVVVSTDAL
1441 MTGFTGDFDSVI DCNTCVTQTVDFSLDPTFTI ETTTLPQDAVSRTQRRGRTGRGKPGIYR 1501 FVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEG
1561 VFTGLTHI DAHFLSQTKQSGENFPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHG 1621 PTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCV
1681 VIVGRIVLSGKPAII PDREVLYQEFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTAS
1741 RQAEVITPAVQTNWQKLEVFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSP
1801 LTTGQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVA 1861 GALVAFKIMSGEVPSTEDLVNLLPAILS PGALVVGVVCAAILRRHVGPGEGAVQWMNRLI 1921 AFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDI
1981 WDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVK
2041 NGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYKFALWRVSAEEYVEIRRVGDFH
2101 YVSGMTTDNLKCPCQI PSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQL
2161 PCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSMASSSASQLSAPSLKATCTANHD 2221 SPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREVSVPAEILRKSRR
2281 FARALPVWARPDYNPPLVETWKKPDYEPPVVHGCPLPPPRSPPVPPPRKKRTVVLTESTL
2341 STALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDVESYSSMPPLEGEPGDPDL
2401 SDGSWSTVSSGADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYST
2461 TSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSK 2521 FGYGAKDVRCHARKAVAHINSVWKDLLEDSVTPI DTTIMAKNEVFCVQPEKGGRKPARLI
2581 VFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYD
2641 TRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCR
2701 ASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGDDLVVICESAGVQEDAASLRAFT
2761 EAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETA 2821 RHTPVNSWLGNI IMFAPTLWARMILMTHFFSVLIARDQLEQALNCEIYGACYSIEPLDLP
2881 PI IQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAI 2941 CGKYLFNWAVRTKLKLTPIAAAGRLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLA 3001 AGVGIYLLPNR(SEQ ID NO: 763)
An example of an HCV subtype Ib is strain HCV-L2, which can be found in the NCBI database as accession number UOl 214 (gi 437107). Its polyprotein sequence (AAA75355 ) is as follows:
1 MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRG
61 RRQPIPKARQPEGRAWAQPGYPWPLYANEGLGWAGWLLSPRGSRPSWGPTDPRRRSRNLG 121 KVI DTPTCGFADLMGYIPLVGAPLGGVARALAHGVRVLEDSVNYATGNLPGCSFSI FLLA 181 LLSCLTVPASAYEVRNVSGIYHVTNDCSNSSIVYEAADLIMHTPGCVPCVREANSSRCWV
241 ALTPTLAARDSSIPTATIRRHVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTFSPRLHQT
301 VQDCNCSIYPGHLTGHRMAWDMMMNWSPTAALVVSQLLRIPQAIVDMVAGAHWGVLAGLA
361 YYPMVGNWAKVLIVMLLFAGVDGTTVTMGGTVARTTYGFTGLFRPGASQKIQLINTNGSW 421 HINRTALNCNDSLNTGFLAALFYTHRFNASGCPERMASCQSIDKFVQGWGPITYAENGSS
481 DQRPYCWHYAPRRCGIVPASQVCGPVYCFTPSPVVVGTTDRSGAPTYSWGENETDVLLLN
541 NTRPPQGNWFGCTWMSSTGFTKTCGGPPCNIGGAGNNTLTCPTDCFRKHPEATYTKCGSG
601 PWLTPRCLVDYPYRLWHYPCTVNFTTFKVRMYVGGVEHRLIAACNWTRGERCNLEDRDRS
661 ELSPLLLSTTEWQILPCSYTTLPALSTGLIHLHQNIVDVQYLYGIGSAVVSFVIKWEYVL 721 LFFLLLADARVCACLWMILLIAQAEAALENLVVLNAASVAGAHGILSFLVFFCAAWYIKG
781 RLVPGAAYASYGVWPLLLLLLALPPRAYAMDQGMAASSGGTVLVGLMLLTLSPYYKVVLA
841 RLIWWLQYFITRAEAHLQVWVPPLNVRGGRDAVILLTCAVYPELVFDITKLLLAIFGPLM
901 VLQAGI IKMPYFVRAQGLIRACMLVRKVAGGHYVQMAFMKLAALTGTYVYDHLTPLRDWA
961 HTGLRDLAVAVEPVVFSDMETKIITWGADTAECGDIILGYRSSARRGREILLGPADSLEG 1021 QGWRLLAPITAYAQQTRGLLGC11TSLTGRDKNQVEGEVQVVSTATQSFLATCVNGVCWT
1081 VFHGAGSKTLAGPKGPITQMYTNVDQDLVGWQAAPGMRSLTPCTCGSSDLYLVTRHADVI
1141 PVRRRGDGRGSLLSPRPVSYLKGSSGGPLLWPSGHAVGIFRAAVCTRGVAKAVDFVPVES
1201 METTMRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAA
1261 TLGFGAYMSKAHGTDPNIRTGARTITTGAPITYSTYGKFFADGGCSGGAYDII ICDECHS 1321 TDSTTILGIGTVLDRAETAGARLVVLATATPPGSTTVPHPNIEEVALPNTGEIPFYGRAI
1381 PIEFIKGGRHLI FCPSKKKCDELAAKLSALGINAVAYYRGLDVΞVI PTSGDVVVVATDAL
1441 MTGYTGDFDSVI DCNTCVTQTVDFSLDPTFTIETTTVPQDAVSRTQRRGRTGRGRGGIYR 1501 FVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYLNTPGLPVCQDHLEFWES
1561 VFTGLNHIDAHFLSQTKQAGDNFPYLVAYQATVCARAQAPPPSWDQMWKCLIWLKPVLHG 1621 PTPLLYRLGAVQNEITLTHPITKLIMASMSADLEVVTSTWVLVGGVLAALAAYCLTTGSV
1681 VIVGRI ILSGRPAVIPDREVLYREFDEMEECASHLPYIEQGVQLAEQFKQKALGLLQTAT
1741 KQAEAAAPVVESKWRALETFWAKHMWNFISGIQYLAALSTLPGNPAIASLMAFTASITSP 1801 LTTQNTLLFNILGGWVAAQLAPASAASAFVGAGSAGAAIGTIGLGKVLVDILAGYGAGVA
1861 GALVAFKVMSGEMPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLI 1921 AFASRGNHDSPTHYVPESDAAARVTQILSSLTITQLLKRLHQWINEDCSTPCSGSWLRDV
1981 WDWICTVLTDFKTWLQSKLLPRLPGVPFFSCQRGYKGVWRGDGIMQTTCPCGAQITGHVK 2041 NGSMRIVGPKTCSNTWHGTFPINAYTTGPCTPAPTPNYSRALWRVAAEEYVEVTRVGDFH 2101 YVTGMTTDNVKCPCQVPAPEFFTEVDGVRLHRYAPACKTLLREEVTFQVGLNQYLVGSQL 2161 PCEPEPDVAVLTSMLTDPSHITAETAKRRLARGSPPSLASSSASQLSAPSLKATCTTHHD 2221 SPDADLIEANLLWRQEMGGNITRVESESKVVILDSFDPLRAEEGEGEVSVAAEILRKSKK 2281 FPPALPEWARPDYNPPLLESWKDPDYVPPVVHGCPLPPAKAPPIPPPRRKRTVVLTESTV 2341 SSALAELAVKTFGSSESSAVDSGTATAPPDQVSDNGDKGSDAESYSSMPPLEGEPGDPDL 2401 SDGSWSTVSEEASEDVVCCSMSYSWTGALITPCAAEESKLPINALSNSLLRHHNMVYATT 2461 SRSAGLRQKKVTFDRLQVLDDHYRDVLKEMKAKASTVKAKLLSVEEACKLTPPHSAKSKF 2521 GYGAKDVRNLSSRAVNHIRSVWKDLLEDTETPIDTTIMAKSEVFCVQPEKGGRKPARLIV 2581 FPDLGVRVCEKMALYDVVSTLPQAVMGPSYGFQYSPGQRVEFLVNAWKSKKCPMGFSYDT 2641 RCFDSTVTESDIRTEESIYQCCDLAPEAKQAIKSLTERLYIGGPLTNSKGQNCGYRRCRA 2701 SVVLTTSCGNTLTCYLKASAACRAAKLQDCTMLVNGDDLVVICESAGTQEDAANLRAFTE 2761 AMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETAR 2821 HTPVNSWLGNIIMYAPTLWARMILMTHFFSILLAQEQLEKALECQIYGACYSIEPLDLPQ 2881 IIERLHGLSAFSLHSYSPGEINRVASCLRKLGVPPLRVWRHRARRVRAKLLSQGGRAATC 2941 GKYLFNWAVRTKLKLTPI PAASRLDLSSWFVAGYSGGDIYHSVSHARPRWFMLCLLLLSV 3001 GVGIYLLPNR(SEQIDNO: 764) An example of an HCV subtype Ic strain HC-G9 can be found in the NCBI database as accession number D14853 (gi 464177). The polyprotein sequence (BAA03581.1) is as follows:
1 MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRVGVRATRKTSERSQPRG
61 RRQPI PKARRPEGRSWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPSDPRRRSRNLG 121 KVIDTLTCGFADLMGYI PLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSI FLLA 181 LLSCLTVPASAVGVRNSSGVYHVTNDCPNASVVYETENLIMHLPGCVPYVREGNASRCWV 241 SLSPTVAARDSRVPVSEVRRRVDSIVGAAAFCSAMYVGDLCGSIFLVGQIFTFSPRHHWT 301 TQDCNCSIYPGHVTGHRMAWDMMMNWSPTGALVVAQLLRIPQAIVDMIAGAHWGVLAGLA 361 YYSMVGNWAKVVVVLLLFAGVDAETRVTGGAAGHTAFGFASFLAPGAKQKIQLINTNGSW 421 HINRTALNCNESLDTGWLAGLLYYHKFNSSGCPERMASCQPLTAFDQGWGPITHEGNASD 481 DQRPYCWHYALRPCGIVPAKKVCGPVYCFTPSPVVVGTTDRAGVPTYRWGANETDVLLLN 541 NSRPPMGNWFGCTWMNSSGFTKTCGAPACNIGGSGNNTLLCPTDCFRKHPDATYSRCGSG 601 PWLTPRCLVDYPYRLWHYPCTVNYTIFKIRMFVGGVEHRLDAACNWTRGERCDLDDRDRA 661 ELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQNIVDVQYLYGLSSAVTSWVIKWEYVV 721 LLFLLLADARICACLWMMLLISQVEAALENLIVLNAASLVGTHGIVPFFIFFCAAWYLKG
781 KWAPGLAYSVYGMWPLLLLLLAIiPQRAYALDQELAASCGATVFICLAVLTLSPYYKQYMA
841 RGIWWLQYMLTRAEALLQVWVPPLNARGGRDGVVLLTCVLHPHLLFEITKIMLAILGPLW 901 ILQASLLKVPYFVRAHGLIRLCMLVRKTAGGQYVQMALLKLGAFAGTYIYNHLSPLQDWA 961 HSGLRDLAVATEPVIFSRMEIKTITWGADTAACGDIINGLPVSARRGREVLLGPADALTD 1021 KGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCVNGVCWT 1081 VYHGAGSRTIASASGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGASDLYLVTRHADVI 1141 PVRRRGDNRGSLLSPRPISYLKGSSGGPLLCPMGHAVGIFRAAVCTRGVAKAVDFVPVES 1201 LETTMRSPVFTDNSSPPTVPQSYQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAA 1261 TLGFGAYMSKAHGIDPNVRTGVRTITTGSPITHSTYGKFLADGGCSGGAYDIIICDECHS 1321 VDATSILGIGTVLDQAETAGVRLTILATATPPGSVTVPHSNIEEVALΞTEGEIPFYGKAI 1381 PLNYIKGGRHLIFCHSKKKCDELAAKLVGLGVNAVAFYRGLDVSVIPTTGDVVVVATDAL 1441 MTGYTGDFDSVIDCNTCVVQTVDFSLDPTFSIETSTVPQDAVSRSQRRGRTGRGKHGIYR 1501 YVSPGERPSGMFDSVVLCECYDAGCAWYELTPAETTVRLRAYLNTPGLPVCQDHLEFWES 1561 VFTGLTHIDAHFLSQTKQSGENFPYLVAYQATVCARAKAPPPSWDQMWKCLIRLKPTLTG 1621 ATPLLYRLGGVQNEITLTHPITKYIMACMSADLEVVTSTWVLVGGVLAALAAYCLSTGSV 1681 VIVGRIILSGKPAVIPDREVLYREFDEMEECAAHIPYLEQGMHLAEQFKQKALGLLQTAS 1741 KQAETITPAVHTNWQKLESFWAKHMWNFVSGIQYLAGLSTLPGNPAIASLMSFTAAVTSP 1801 LTTQQTLLFNILGGWVAAQLAAPAAATAFVGAGITGAVIGSVGLGKVLVDILAGYGAGVA 1861 GALVAFKIMSGEAPTAEDLVNLLPAILSPGALWGVVCAAILRRHVGPGEGAVQWMNRLI 1921 AFASRGNHVSPTHYVPESDASVRVTHILTSLTVTQLLKRLHVWISSDCTAPCAGSWLKDV 1981 WDWICEVLSDFKSWLKAKLMPQLPGIPFVSCQRGYRGVWRGEGIMHARCPCGADITGHVK 2041 NGSMRIVGPKTCSNTWRGSFPINAHTTGPCTPSPAPNYTFALWRVSAEEYVEVRRLGDFH 2101 YITGVTTDKIKCPCQVPSPEFFTEVDGVRLHRYAPPCKPLLRDEVTFSIGLNEYLVGSQL 2161 PCEPEPDVAVLTSMLTDPSHITAETAARRLNRGSPPSLASSSASQLSAPSLKATCTTHHD 2221 SPDADLITANLLWRQEMGGNITRVESENKIVILDSFDPLVAEEDDREISVPAEILLKSKK 2281 FPPAMPIWARPDYNPPLVEPWKRPDYEPPLVHGCPLPPPKPTPVPPPRRKRTWLDESTV 2341 SSALAELATKTFGSSTTSGVTSGΞAAESSPAPSCDGELDSEAESYSSMPPLEGEPGDPDL 2401 SDGSWSTVSSDGGTEDVVCCSMSYSWTGALITPCAAEETKLPINALSNSLLRHHNLVYST 2461 TSRSAGQRQKKVTFDRLQVLDDHYRDVLKEAKAKASTVKAKLLSVEEACSLTPPHSARSK 2521 FGYGAKDVRSHSSKAIRHINSVWQDLLEDNTTPIDTTIMAKNEVFCVKPEKGGRKPARLI 2581 VYPDLGVRVCEKRALYDVVKQLPIAVMGTSYGFQYSPAQRVDFLLNAWKSKKNPMGFSYD 2641 TRCFDSTVTEADIRTEEDLYQSCDLVPEARAAIRSLTERLYIGGPLTNSKGQNCGYRRCR 2701 ASGVLTTSCGNTITCYLKASAACRAAKLRDCTMLVCGDDLVVICESAGVQEDAANLRAFT 2761 EAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPETPLARAAWETA 2821 RHTPVNSWLGNIIMFAPTLWVRMVLMTHFFSILIAQEHLEKALDCEIYGAVHSVQPLDLP 2881 EIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRATLLSQGGRAAI 2941 CGKYLFNWAVKTKLKLTPLPSASQLDLSNWFTGGYSGGDIYHSVSHVRPRWFFWCLLLLS 3001 VGVGIYLLPNR(SEQIDNO: 765)
Other HCV polyprotein sequences are known in the art and can be used to generate the mutant HCV E2 polypeptide provided herein (see for example, hcvdb.org/viruses.asp; .ncbi.nlm.nih.gov; and hcv.lanl.gov and HCV sequence databases referenced in Kuiken et al. (2005) Bioinformatics 21 (3):379-84 and Yusim et al. (2005) Applied Bioinformatics 4(4)). Additional examples include a Taiwan isolate of hepatitis C virus available in the NCBI database at accession number P29846 (gi: 266821). Other examples of HCV polyprotein sequences include, but are not limited to, NCBI accession number AF009606, AY734971, AJ238799, AY545953, AY734974, AB047639, AFl 77036, AY734977, AY734982, AY734984, AY734987, EF427672, and AY736194. C. Mutant E2 Polypeptides
Provided herein are mutant HCV E2 polypeptides. A mutant E2 polypeptide provided herein differs from a naturally-occurring E2 polypeptide of HCV in that the mutant E2 polypeptide provided herein has amino acid deletions relative to the naturally-occurring E2 polypeptide. Exemplary regions of an E2 polypeptide that can be removed include, for example, the region defined by amino acid residues corresponding to 384-411, 460 to 485, 570-580, 646-647, 648-661, 662-717 or 718- 746, (the amino acid positions corresponding to the amino acid positions in an HCV polyprotein) or any combination thereof can be deleted from a naturally-occurring E2 polypeptide of HCV to generate a mutant E2 polypeptide provided herein.
A mutant E2 polypeptide provided herein thus has an amino acid sequence that comprises, from the amino to the carboxy termini: (1) a first segment that corresponds to amino acid residues 412 to 459 of a select hepatitis C virus polyprotein, (2) a second segment that corresponds to amino acid residues 486 to 569 of the select hepatitis C virus polyprotein, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the select hepatitis C virus polyprotein.
In a mutant E2 polypeptide provided herein, one or more immunodominant epitopes in the naturally-occurring E2 polypeptide are eliminated or its immunogenicity to particular epitopes attenuated, while the immunogenicity of conserved or cross-neutralizing epitopes are augmented. For example, when the naturally-occurring E2 polypeptide is used an immunogen, greater than half of antibodies generated are directed against immunodominant epitopes such as, for example, the hypervariable region 1 (amino acid residues 384 to 412) or the epitopes recognized by the ARIA and ARlB antibodies that include the residues T416, T416, N417, R483, P484, Y485, V538, N540, P544, P545, G547 and W549. Accordingly, the mutant E2 polypeptide provided herein also can have one or more amino acid substitutions or deletions at positions 416, 417, 483, 484, 485, 538, 540, 544, 545, 547, 549 or any combinations thereof relative to the E2 polypeptide sequence of HCV (e.g. corresponding to the HCV strain H77).
The segments of the mutant E2 polypeptide can be linked directly or indirectly, in any order, via a linker provided that the polypeptide properly folds to present the conserved HCV E2 conformational epitope. Various linkers are known in the art and include, for example, polypeptide linkers. In some examples, the polypeptide linker can link the first and second segments, and the polypeptide linker is at least 10 amino acids, e.g. 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, or more than 48 amino acid residues provided that the polypeptide properly folds to present the conserved HCV E2 conformational epitope. In some examples, the polypeptide linker can link the second and third segments, and the polypeptide linker is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more than 28 amino acid residues in length provided that the polypeptide properly folds to form the conserved HCV E2 conformational epitope. In some examples, the first and second segments are linked via a polypeptide linker which is about 26 amino acid residues, and the second and third segments are linked via a polypeptide linker which is about 12 amino acid residues in length.
Any HCV E2 polypeptide can be modified to produce the mutant E2 polypeptides provided herein. For example, the amino acid segments can be derived for an HCV E2 polypeptide of any genotype (e.g. 1, 2, 3, 4, 5, or 6) or subtype (e.g. Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p or 6t). Exemplary naturally-occurring HCV E2 sequences that can be modified include, but are not limited to, HCV E2 polypeptides shown in Table 2 below.
Table 2
HCV origin
(accession Amino acid sequence number)
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Examples of amino acid sequence segments from select hepatitis C viruses that can be employed to produce the mutant E2 polypeptides provided herein include, but are not limited, to the segments that correspond to the polypeptide segments shown in Table 3.
Table 3
Figure imgf000052_0002
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
In some examples, the first segment of the mutant E2 polypeptide provide herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino.acids 412 to 459 of a hepatitis C virus E2 polypeptide, such as H77. In some examples, the second segment of the mutant E2 polypeptide provide herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 486 to 569 of a hepatitis C virus E2 polypeptide, such as H77. In some examples, the third segment of the mutant E2 polypeptide provided herein is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 581 to 645 of a hepatitis C virus E2 polypeptide, such as H77.
In some examples, the first amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 888-912 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to any one of SEQ ID NOS: 888-912. Tn some examples, the second amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 913-937 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to any one of SEQ ID NOS: 913- 937. In some examples, the third amino acid segment of the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 938-962 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity to any one of SEQ ID NOS: 938-962. In some examples, the mutant E2 polypeptide has the sequence of any one of SEQ ID NOS: 727 '-730 and 740-742 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %,
97 %, 98 %, or 99 % sequence identity to any one of SEQ ID NOS: 727-730 and 740-742.
Exemplary mutant E2 polypeptides provided herein differ from the corresponding naturally-occurring E2 amino acid sequence in that the mutant E2 polypeptide provided herein does not include one or more segments defined by (1 ) amino acid residues 384 to 411 of the hypervariable region 1 of the naturally- occurring E2 polypeptide; (2) amino acid residues 460 to 485; (3) amino acid residues 570-580, (4) amino acid residues 647-661, (5) amino acid residues 662-717 or (6) 718-746, or any combination thereof.
The mutant E2 polypeptide provided herein also can differ from the corresponding naturally-occurring E2 amino acid sequence in that the mutant E2 polypeptide can have at least one amino acid substitution at position 416, 417, 483, 484, 485, 538, 540, 544, 545, 547, 549 or any combinations thereof. In some examples, a mutant E2 polypeptide provided herein can have at least two amino acid substitutions at these positions, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions.
The amino acid that can be substituted at these positions can be one that has a different chemical or physical property from the naturally-occurring residue. For example, the proline residues at position 484, 544 or 545 can be substituted with an amino acid residue that enable the polypeptide to be more flexible such as for example an alanine, valine or other non-cyclic residues. The glycine residue at position 547 can be substituted with an amino acid residue that has a bulkier side chain such as, for example, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine or glutamine, while the tryptophan residue at position 549 can be substituted with an amino acid residue that has a less bulky side chain, for example, glycine, alanine, valine, serine, cysteine, or threonine. The threonine residue at position 416 can be substituted with a residue that does not have a hydroxyl or sulfur-containing side chain. The acidic asparagine residue at position 417 or 540 can be substituted with, for example, a basic amino acid residue such as histidine, lysine or arginine, while the basic arginine residue at position 483, for example, can be substituted with, for example, an acidic residue such as aspartic acid, glutamic acid, asparagine or glutamine. The aromatic amino acid tyrosine at position 485 can be substituted with, for example, a non-aromatic residue, while the valine at position 538 can be substituted with a residue having a bulkier side chain, a basic or acidic residue, or one with an aromatic, hydroxyl or sulfur-containing side chain. An exemplary substitution or combination of substitutions is one that decreases the immunogenicity or function of epitopes recognized by the ARl antibodies such as ARIA and ARlB. The mutant E2 polypeptide provided herein also can have one or more other substitutions, insertions or deletions relative to a naturally-occurring E2 polypeptide as long as the mutant E2 polypeptide sequence includes the discontinuous epitopes described herein that come together to form a conformational epitope recognized by a conformation-dependent cross-neutralizing antibody such as the AR3A, AR3B, AR3C or AR3D antibody.
The mutant E2 polypeptide provided herein contain one or more discontinuous epitopes of an E2 polypeptide. For example, the polypeptides provided herein include the following amino acid regions: (1) amino acid residues 412 to 424; (2) amino acid residues 436 to 447; and (3) amino acid residues 523 to 540 relative to the naturally- occurring E2 polypeptide (e.g. HCV strain H77 polypeptide and other HCV strains, isolates, species, quasispecies, subtypes or genotypes). Sequences of the discontinuous epitopes can be determined based on sequence alignment of the HCV E2 or HCV polyprotein sequence with the sequence of strain H77 using the methods described above.
Examples of the amino acid sequences of discontinuous epitopes of select E2 polypeptides provided herein and their HCV origin are shown in Table 4 below. Table 4
Figure imgf000058_0001
Figure imgf000059_0001
Non-limiting examples of mutant E2 polypeptides provided herein include the mutant E2 polypeptides shown in Table 5 below.
Table 5
Figure imgf000059_0002
Figure imgf000060_0001
Figure imgf000061_0001
A polypeptide provided herein also can include non-E2 sequences at the N or C terminus. Non-E2 sequences can be, for example, a tag such as an N-terminal ubiquitin signal, a poly-histidine sequence (SEQ ID NO:685), a FLAG sequence (SEQ ID NO:607), an HA sequence (SEQ ID NO:608), a myc sequence (SEQ ID NO:609), a V5 sequence (SEQ ID NO:610), a chitin binding protein sequence, a maltose binding protein sequence (SEQ ID NO:687) or a glutathione-S-transferase sequence. D. Nucleic Acids Encoding Mutant E2 Polypeptides Provided herein are isolated nucleic acids encoding modified E2 polypeptides.
Nucleic acids encoding mutant E2 polypeptides provided herein can be generated from nucleic acids encoding the naturally-occurring HCV polyprotein using methods known to those skilled in the art. For example, nucleic acids encoding mutant E2 polypeptides containing various amino acid substitutions can be produced by site- specific mutagenesis and polymerase chain reaction (PCR) amplification from the nucleic acids encoding the naturally-occurring HCV polyprotein. Nucleic acids encoding mutant E2 polypeptides, i.e. polypeptides that do not include amino acid residues 384 to 410 of the hypervariable region of the naturally occurring E2 protein, can be produced by PCR using primers that do not encompass the nucleotides coding for amino acid residues 384 to 410. Nucleic acid sequences encoding the naturally- occurring HCV polyproteins are disclosed at the NCBI website (ncbi.nlm.nih.gov). Selected accession numbers for nucleic acids encoding the naturally-occurring HCV polyproteins are as follows: AF009606; D10749; U01214; AY051292; AY746460; AY232731; D50409; DQ155561; AB031663; DQ437509; D49374; D63821; Yl 1604; DQ516083; EF589160; AF064490; AY859526; NC009827; EF420130; DQ314805 ; DQ835764; D63822; D84264; DQ835763; and DQ278894.
Methods for isolating nucleic acids encoding the naturally-occurring HCV polyprotein, as well as technologies for generation of nucleic acids encoding E2 polypeptides provided herein are known to those of skill in the art. See for example, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubβl et al. edts. (John Wiley & Sons, Inc., 1999) or MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al. (Cold Spring Harbor Laboratory Press, New York, 1989).
Nucleic acids encoding a polypeptide provided herein can be used for recombinant expression of the E2 polypeptide provided herein. Nucleic acids encoding a polypeptide provided herein also can be used in a nucleic acid-based vaccine to elicit an immune response against an HCV.
Nucleic acid encoding a polypeptide provided herein can be operably-linked to an expression control sequence in an expression vector, which can be introduced into a host cell for expression of the encoded polypeptide or administered to a mammal to elicit an immune response against the polypeptide.
Examples of nucleic acid sequences encoding mutant E2 polypeptides provided herein are shown in Table 6 below.
Table 6
Mutant E2 Polypeptides Nucleic Acid Sequences
E2(4l2.74s) CAACTGATCAACACCAACGGCAGTTGGCACATCAATAGCACGGCCTTGAACTGC
(Δ384-395) AATGAAAGCCTTAACACCGGCTGGTTAGCAGGGCTCTTCTATCAGCACAAATTC AACTCTTCAGGCTGTCCTGAGAGGTTGGCCAGCTGCCGACGCCTTACCGATTTT GCCCAGGGCTGGGGTCCTATCAGTTATGCCAACGGAAGCGGCCTCGACGAACGC CCCTACTGCTGGCACTACCCTCCAAGACCTTGTGGCATTGTGCCCGCAAAGAGC GTGTGTGGCCCGGTATATTGCTTCACTCCCAGCCCCGTGGTGGTGGGAACGACC GACAGGTCGGGCGCGCCTACCTACAGCTGGGGTGCAAATGATACGGATGTCTTC GTCCTTAACAACACCAGGCCACCGCTGGGCAATTGGTTCGGTTGTACCTGGATG AACTCAACTGGATTCACCAAAGTGTGCGGAGCGCCCCCTTGTGΓCATCGGAGGG GTGGGCAACAACACCTTGCΓCTGCCCCACTGATTGTTTCCGCAAGCATCCGGAA GCCACATACTCTCGGTGCGGCTCCGGTCCCTGGATTACACCCAGGTGCATGGTC GACTACCCGTATAGGCTTTGGCACTATCCTTGTACCATCAATTACACCATATTC AAAGTCAGGATGTACGTGGGAGGGGTCGAGCACAGGCTGGAAGCGGCCTGCAAC TGGACGCGGGGCGAACGCTGTGATCTGGAAGACAGGGACAGGTCCGAGCTCAGC CCATTGCTGCTGTCCACCACACAGTGGCAGGTCCTTCCGTGTTCTTTCACGACC CTGCCAGCCTTGTCCACCGGCCTCATCCACCTCCACCAGAACATTGTGGACGTG CAGTACTTGTACGGGGTAGGGTCAAGCATCGCGTCCTGGGCCATTAAGTGGGAG TACGTCGTTCTCCTGTTCCTCCTGCTTGCAGACGCGCGCGTCTGCTCCTGCTTG TGGATGATGTTACTCATATCCCAAGCGGAGGCG (SEQ ID NO: 874)
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Nucleic acids encoding E2 polypeptides provided herein can be incorporated into viral, bacterial, insect, yeast or mammalian expression vectors. As such, nucleic acids encoding E2 polypeptides can be operably-linked to expression control sequences such as viral, bacterial, insect, yeast or mammalian promoters and enhancers. Examples of expression control sequences such as enhancers and promoters include viral promoters such as SV 40 promoter, Rous Sarcoma Virus (RSV) promoter, and cytomegalovirus (CMV) immediate early promoter. Examples of viral vectors include retrovirus-based vectors, e.g. lentiviruses, adenoviruses and adeno-associated viruses. These are particularly useful as DNA-based vaccines. The nucleic acid encoding an E2 polypeptide provided herein also can be linked to nucleic acid sequences that code for unrelated amino acid sequences such as N-terminal ubiquitin signals to improve antigen targeting, a poly-histidine sequence, a FLAG (DYKDDDDK, SEQ ID NO:607) sequence, an HA sequence, a myc sequence, a V5 sequence, a chitin binding protein sequence, a maltose binding protein sequence or a glutathione-S-transferase sequence.
Expression vectors containing nucleic acids encoding E2 polypeptides can be introduced into bacterial, insect, yeast or mammalian host cells (e.g. CHO, Balb/3T3, HeLa3 MT2, mouse NSO (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293 (e.g. 293T), 293S, 2B8, and HKB cells) for expression using conventional methods including, without limitation, transformation, transduction and transfection. Expression vectors containing nucleic acids encoding E2 polypeptides, in saline for example, can be introduced into a mammal, e.g. mammalian tissues, using standard methods including, for example, injection using a standard hypodermic needle, by a gene gun delivery, jet injection or liposome-mediated delivery. Injection can be intramuscular or intradermal. Electroporation, myotoxins such as bupivacaine or hypertonic solutions of saline or sucrose also can aid in delivery.
When expressed in bacterial, yeast, insect or mammalian host cells, E2 polypeptides provided herein can be purified using a method provided herein. Specifically, E2 polypeptides provided herein are purified by affinity chromatography using a cross-neutralizing antibody such as, for example, AR3A, AR3B, AR3C or AR3D in combination with size exclusion chromatography. More specifically, an E2 polypeptide provided herein can be separated from unrelated proteins by affinity chromatography using a conformation-dependent antibody provided herein such as AR3A. The E2 polypeptide can be eluted at acidic, neutral or basic pH using: (1) 0.2M glycine pH 2.2, (2) 2M sodium thiocyanate (pH adjusted to pH 7.4 with 5OmM Tris-HCl); or (3) 0.2M glycine pH 11.5, and then further purified by size-exclusion chromatography. The method provided herein for purifying E2 polypeptide allows for the purification of E2 polypeptides that properly fold to form the conformational epitope described herein. As described herein in the Examples, the methods of purification using a conformation dependent antibody, such as, but not limited to AR3A, provide for the purification of E2 polypeptide monomers that properly fold to form the E2 conformational epitope. When introduced into a mammal or mammalian tissue, nucleic acids encoding
E2 polypeptides, incorporated in a viral vector, for example, can be used as a nucleic acid-based vaccine to elicit an immune response against HCV. E. Cross-neutralizing Antibodies
Provided herein are antibodies that bind specifically with a mutant E2 polypeptide provided herein. The antibody is a cross-neutralizing antibody, i.e. one that neutralizes at least two HCV strains, isolates, species, quasispecies, subtypes or genotypes.
An antibody provided herein can be a polyclonal or monoclonal antibody. Polyclonal antibodies can be obtained by immunizing a mammal with a mutant polypeptide provided herein, and then isolating antibodies from the blood of the mammal using standard techniques including, for example, enzyme linked immunosorbent assay (ELISA) to determine antibody titer and protein A chromatography to obtain the antibody-containing IgG fraction.
A monoclonal antibody is a population of molecules having a common antigen binding site that binds specifically with a particular antigenic epitope. A monoclonal antibody can be obtained by selecting an antibody-producing cell from a mammal that has been immunized with a mutant polypeptide provided herein and fusing the antibody-producing cell, e.g. a B cell, with a myeloma to generate an antibody- producing hybridoma. A monoclonal antibody provided herein also can be obtained by screening a recombinant combinatorial library such as an antibody phage display library using, for example, a mutant polypeptide provided herein. See, for example, PHAGE DISPLAY - A LABORATORY MANUAL, Barbas, et al., eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001 ; and Kontermann & Dϋbel, ANTIBODY ENGINEERING, Heidelberg: Springer-Verlag. Berlin, 2001. An immunologically-active fragment of an antibody is the biologically active fragment of an immunoglobulin molecule, for example, the F(ab) or F(ab')2 fragment generated by cleavage of the antibody with an enzyme such as pepsin. An antibody provided herein also can be a murine, chimeric, humanized or fully human antibody. A murine antibody is an antibody derived entirely from a murine source, for example, an antibody derived from a murine hybridoma generated from the fusion of a mouse myeloma cell and a mouse B-lymphocyte cell. A chimeric antibody is an antibody that has variable regions derived from a non-human source, e.g. murine or primate, and constant regions derived from a human source. A humanized antibody has antigen-binding regions, e.g. complementarity-determining regions, derived from a mouse source, and the remaining variable regions and constant regions derived from a human source. A fully human antibody is an antibody from human cells or derived from transgenic mice carrying human antibody genes.
Methods to generate antibodies are well known in the art. For example, a polyclonal antibody provided herein can be prepared by immunizing a suitable mammal with a mutant polypeptide provided herein. The mammal can be, for example, a rabbit, goat, sheep, rabbit, hamster, cow, or mouse. At the appropriate time after immunization, antibody molecules can be isolated from the mammal, e.g. from the blood or other fluid of the mammal, and further purified using standard techniques that include, without limitation, precipitation using ammonium sulfate, gel filtration chromatography, ion exchange chromatography or affinity chromatography using protein A. In addition, an antibody-producing cell of the mammal can be isolated and used to prepare a hybridoma cell that secretes a monoclonal antibody provided herein. Techniques for preparing monoclonal antibody-secreting hybridoma cells are known in the art. See, for example, Kohler and Milstein, Nature 256:495-97 (1975) and Kozbor etai, Immunol Today 4: 72 (1983). A monoclonal antibody provided herein also can be prepared using other methods known in the art, such as, for example, expression from a recombinant DNA molecule, or screening of a recombinant combinatorial immunoglobulin library using a mutant polypeptide provided herein.
Methods to generate chimeric and humanized monoclonal antibodies are also well known in the art and include, for example, methods involving recombinant DNA technology. A chimeric antibody can be produced by expression from a nucleic acid that encodes a non-human variable region and a human constant region of .an antibody molecule. See, for example, Morrison et al., Proc. Nat. Acad. ScL U.S.A. 86: 6851 (1984). A humanized antibody can be produced by expression from a nucleic acid that encodes non-human antigen-binding regions (complementarity-determining regions) and a human variable region (without antigen-binding regions) and human constant regions. See, for example, Jones et al, Nature 321 :522-24 (1986); and Verhoeven et al, Science 239:1534-36 (1988). Completely human antibodies can be produced by immunizing engineered transgenic mice that express only human heavy and light chain genes. In this case, therapeutically useful monoclonal antibodies can then be obtained using conventional hybridoma technology. See, for example, Lonberg & Huszar, Int. Rev. Immunol. 13:65-93 (1995). Nucleic acids and techniques involved in design and production of antibodies are well known in the art. See, for example, Batra et al, Hybridoma 13:87-97 (1994); Berdoz et al, PCR Methods Appl 4: 256-64 (1995); Boulianne et al, Nature 312:643-46 (1984); Carson et al, Adv. Immunol 38 :274-311 (1986); Chiang et al, Biotechniques 7 :360-66 (1989); Cole et al, MoI. Cell. Biochem. 62 :109-20 (1984); Jones et al, Nature 321 : 522-25 (1986); Larrick et al, Biochem Biophys. Res. Commun. 160 : 1250-56 (1989); Morrison, Annu. Rev. Immunol 10 :239-65 (1992); Morrison et al, Proc. Nat'lAcad. ScL USA 81 : 6851-55 (1984); Orlandi et al, Pro. Nat 'I Acad. ScL U.S.A. 86:3833-37 (1989); Sandhu, Crit. Rev. Biotechnol. 12:437-62 (1992); Gavilondo & Larrick, Biotechniques 29: 128-32 (2000); Huston & George, Hum. Antibodies. 10:127-42 (2001); Kipriyanov & Le GaIl5 Mo/. Biotechnol. 26: 39-60 (2004). F. Diagnostic Uses
A mutant HCV E2 polypeptide or cross-neutralizing antibody provided herein can be used to detect the presence of HCV in a sample obtained from a subject, such as a mammal. Such a diagnostic use is based on the detection of antibodies generated by a subject (e.g. a mammal) that has been infected with HCV. Diagnostic use also can be based on detection of HCV antigens. Detection of an antibody-antigen complex indicates that the mammal has been exposed to or infected with HCV.
Thus, provided herein are methods for determining whether a mammal such as a human has been or is infected with an HCV. To determine whether a mammal has been infected with HCV, a mutant polypeptide provided herein can be used to detect the presence of anti-HCV antibodies in a sample from the mammal. Alternatively, a cross-neutralizing antibody provided herein can be used to detect HCV particles or antigens in the sample.
The sample from the mammal can be a biological fluid such as blood or a cell or tissue sample. The mutant E2 polypeptides or antibodies provided herein can be labeled with a detectable label. Thus, to facilitate detection, the polypeptide or cross-neutralizing antibody provided herein can be labeled with a detectable molecule, which can be an enzyme such as, but not limited to, alkaline phosphatase, acetylcholinesterase, β- galactosidase or horseradish peroxidase; a prosthetic group such as, but not limited to, streptavidin, biotin, or avidin; a fluorescent group such as dansyl chloride, dichlorotriazinylamine, dichlorotriazinylamine fluorescein, fluorescein, fluorescein isothiocyanate, phycoerythrin, rhodamine, umbelliferone; a luminescent group such as luminal; a bioluminescent group such as aequorin, luciferase, and luciferin; or a radioisotope such as, but not limited to, ^H, ^5js 131^ 35g. The formation of an antibody-antigen complex indicates that the mammal has been or is infected with HCV. The presence of HCV particles or antigens in the sample indicates that that mammal is infected with HCV. The presence of HCV antibodies in the sample indicates that the mammal has been or is infected with HCV. G. Development of Anti-HCV Therapeutic Agents A polypeptide provided herein can be used to generate cross-neutralizing antibodies against HCV. For example, a polypeptide provided herein can be used to elicit an immune response in a subject, such as a mammal. Antibodies that bind specifically with the mutant E2 polypeptide provided herein can be isolated using known methods as described above. A mutant polypeptide provided herein is particularly useful to focus the immune response to the conserved AR3 neutralizing epitopes as the immunogenicity of the hypervariable regions and the ARl residues are dampened by deletion of a large portion of the hypervariable region and substitution of important selected ARl residues.
Thus, provided herein are methods of eliciting an immune response in a subject, such as a mammal, comprising administering to the subject a mutant E2 polypeptide provided herein and then isolating antibodies or antibody producing cells from the subject using methods known to those of skilled in the art. The subject can be a rabbit, rat, mouse, sheep, cow, monkey, horse, goat or a pig. The method is particularly useful to generate antibodies against conserved HCV epitopes. Thus, the method can be used to develop passive vaccines containing one or more anti-HCV antibodies provided herein. A polypeptide provided herein also can be used to screen for anti-HCV agents, such as those that block viral entry into target cells. Since the discontinuous epitopes of the E2 polypeptide described herein are involved in binding to cell receptors, an E2 polypeptide provided herein can be used to screen for agents that bind to an E2 polypeptide provided herein and prevent binding of the E2 polypeptide with a cell receptor.
H. Therapeutic or Prophylactic Uses
A polypeptide or cross-neutralizing antibody provided herein can be used to prevent or treat a new or recurring HCV infection, or prevent or reduce HCV replication, as well as treat the associated disease condition or clinical symptoms. The mutant HCV polypeptide provided herein can be used to provide immune protection against HCV. The immune protection against HCV provided by the immunogenic polypeptide can be any immune response, cellular or humoral, that either inhibits or helps to prevent HCV infection. For example, the immunogenic polypeptide of the present invention can bind to CD81, induce antibodies associated with resolving HCV infection, induce production of cytokines, induce antibodies that can neutralize HCV binding to host cells, or prime an immune system against secondary HCV infection or exposure. The immune protection provided by the immunogenic polypeptide of the present invention can be protective against more than one of the HCV genotypes. HCV infection or replication is indicated by the amount of HCV particles or the amount of HCV RNA in a sample from the subject determined using methods known in the art and also those described herein. HCV infection is also indicated by clinical symptoms described further below.
Thus, the E2 polypeptide provided herein, corresponding nucleic acid or cross- neutralizing antibody provided herein can be used to prevent or reduce transmission, to prevent or treat disease progression, and to prevent or reduce HCV replication or reduce viral load. Treatment includes the alleviation or diminishment of at least one symptom typically associated with the infection. Ideally, the treatment cures, e.g., substantially inhibits viral infection and/or eliminates the symptoms associated with the infection. Symptoms of HCV exposure or infection include, without limitation, inflammation of the liver, decreased appetite, fatigue, abdominal pain, jaundice, flu- like symptoms, itching, muscle pain, joint pain, intermittent low-grade fevers, sleep disturbances, nausea, dyspepsia, cognitive changes, depression headaches and mood changes.
Subjects (e.g. mammals) that can benefit from the polypeptide, nucleic acid or antibody provided herein can be identified using the diagnostic and screening techniques discussed above. Thus, HCV infection can be diagnosed by detecting antibodies to the virus using the mutant E2 polypeptide provided herein, detecting the HCV itself using a cross-neutralizing antibody provided herein, detecting liver inflammation by biopsy, liver cirrhosis, portal hypertension, thyroiditis, cryoglobulinemia and glomerulonephritis. In addition, diagnosis of exposure or infection or identification of one who is at risk of exposure to HCV can be based on medical history, abnormal liver enzymes or liver function tests during routine blood testing. Generally, infection can be diagnosed using polymerase chain reaction (PCR) for detecting viral nucleic acids, enzyme linked immunosorbent assay (ELISA) for detecting viral antigens or anti-viral antibodies, and immunofluorescence for detecting viral antigens. For example, a polypeptide or antibody provided herein can be combined with an appropriate sample from the patient to produce a complex. The complex in turn can be detected with a marker reagent for binding with such a complex. Typical marker reagents include secondary antibodies selective for the complex, secondary antibodies selective for certain epitopes of the polypeptide or antibody or a label attached to the polypeptide or antibody itself. In particular, radioimmunoassay (RIA), radioallergosorbent test (RAST), radioimmunosorbent test (RIST), immunoradiometric assay (IRMA), Fair assay, fluorescence immunoassay (FIA), sandwich assay, enzyme linked immunosorbent assay (ELISA), northern or southern blot analysis, and color activation assay can be used following protocols well known for these assays. See for example Immunology, An Illustrated Outline by
David Male, CV. Mosby Company, St Louis, MO, 1986 and the Cold Spring Harbor Laboratory Manuals cited above. Labels including radioactive labels, chemical labels, fluorescent labels, luciferase and the like also can be directly attached to the polypeptide according to the techniques described in U.S. Patent No. (BN patent cite), the disclosure of which is incorporated herein by reference.
A subject (e.g. a mammal) that can benefit from a polypeptide, nucleic acid or cross-neutralizing antibody provided herein includes one who is likely to be or has been exposed to HCV. Exemplary subjects include, without limitation, someone present in an area where HCV is prevalent or commonly transmitted, e.g., Africa, Southeast Asia, China, South Asia, Australia, India, the United States, Russia, as well as Central and South American countries. A subject who is likely to be or has been exposed to HCV also includes a recipient of donated body tissues or fluids including, for example, a recipient of blood or one or more of its components such as plasma, platelets, or stem cells and an organ or cell transplant recipient such as a liver transplantee. A subject (e.g., a mammal), who is likely to be or has been exposed to HCV also can include medical, clinical or dental personnel handling body tissues and fluids. A subject (e.g., a mammal), who has been exposed to HCV includes, without limitation, someone who has had contact with the body tissue or fluid, e.g. blood, of an infected person or otherwise have come in contact with HCV. A subject (e.g., a mammal), who can benefit from a polypeptide or cross-neutralizing antibody provided herein includes one who is susceptible to HCV infection or one who has recurring HCV infection.
Thus, provided herein are methods for preventing a new or recurring HCV infection and its associated symptoms and/or complications such as by preventing or reducing HCV replication in a subject (e.g. a mammal) infected with HCV. A polypeptide, nucleic acid or cross-neutralizing antibody provided herein can be used prophylactically to prevent a susceptible individual from being infected with HCV or to prevent recurring HCV infection, for example, in an individual who has received a liver transplant.
A polypeptide or cross-neutralizing antibody provided herein can be used to prevent or treat infection of a cell, e.g. a mammalian cell, such as a human cell. A polypeptide, nucleic acid or cross-neutralizing antibody provided herein can be used to prevent or treat acute or chronic HCV infection, or prevent or reduce HCV replication, in a subject, e.g. a mammal such as a human. Thus, an E2 polypeptide or a nucleic acid encoding an E2 polypeptide provided herein can be used as an active vaccine, a nucleic acid or DNA-based vaccine, or be incorporated into vaccine carriers, to elicit a protective immune response in a subject. Exemplary vaccines include vaccines that are effective for the prevention of HCV infection by one or more HCV genotypes.
Methods of preventing or treating HCV infection include contacting a cell with an effective amount of an antibody provided herein; mixing biological fluids, cells or tissues to be administered or transplanted into a subject with a polypeptide, nucleic acid or antibody provided herein prior to the administration or transplant; or administering to a subject such as a human a therapeutically effective amount of a polypeptide, nucleic acid or antibody provided herein. Thus, provided herein are in vitro methods of preventing HCV infection or transmission by contacting biological samples such as fluids, cells or tissues containing the virus with an effective amount of the polypeptide, nucleic acid or antibody provided herein, as well as in vivo methods of treating or preventing HCV infection by administering the polypeptide, nucleic acid or antibody to the subject.
A polypeptide, nucleic acid or antibody provided herein can be administered in a variety of ways. Routes of administration include, without limitation, oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, vaginal, dermal, transdermal (topical), transmucosal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. The means of administration can be by injection, using a pump or any other appropriate mechanism.
A polypeptide, nucleic acid or antibody provided herein can be administered in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the polypeptide, nucleic acid or antibody provided herein can be essentially continuous over a pre-selected period of time or can be in a series of spaced doses. For example, provided herein are methods of eliciting an immune response in a subject (e.g. a mammal) that involves administering a modified polypeptide, nucleic acid or antibody provided herein at a select time and then administering a second, third, fourth or additional doses at select times after the first administration. Both local and systemic administrations are contemplated.
The dosage to be administered to a subject (e.g. a mammal) can be any amount appropriate to reduce or prevent viral infection or to treat at least one symptom associated with the viral infection. Some factors that determine appropriate dosages are well known to those of ordinary skill in the art and can be addressed with routine experimentation. For example, determination of the physicochemical, toxicological and pharmacokinetic properties can be made using standard chemical and biological assays and through the use of mathematical modeling techniques known in the chemical, pharmacological and toxicological arts. The therapeutic utility and dosing regimen can be extrapolated from the results of such techniques and through the use of appropriate pharmacokinetic and/or pharmacodynamic models. Other factors will depend on individual patient parameters including age, physical condition, size, weight, the condition being treated, the severity of the condition, and any concurrent treatment. The dosage will also depend on the polypeptide or antibody chosen and whether prevention or treatment is to be achieved, and if the polypeptide or antibody is chemically mutant. Such factors can be readily determined by the clinician employing viral infection models such as in vitro HCV infection system described herein, or other animal models or test systems that are available in the art. The precise amount to be administered to a subject (e.g. a mammal) such as a human will be the responsibility of the attendant physician. The amount useful to establish treatment of HCV can be determined by diagnostic and therapeutic techniques well known to those of ordinary skill in the art. The dosage can be determined by titrating a sample of the patient's blood sera with the polypeptide or antibody to determine the end point beyond which no further immunocomplex is formed. Such titrations can be accomplished by the diagnostic techniques discussed below. Available dosages include administration of from about 1 to about 1 million effective units of antibody per day, wherein a unit is that amount of polypeptide, which will provide at least 1 microgram of antigen-polypeptide complex. In some examples, about 10 to about 100,000 units of antibody per day can be administered.
To achieve the desired effect(s), one or more mutant polypeptides or antibody provided herein can be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500, 750 or 1000 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages can provide beneficial results. For post-exposure prophylactic use, the one or more polypeptide or antibody provided herein can be administered as soon as possible, e.g. within 24 hours if possible, after exposure to HCV. To prevent recurrent HCV infection, e.g. in a transplant recipient such as a liver transplant recipient, a mutant polypeptide or antibody provided herein can be administered prior to and after transplantation. For example, the polypeptide or antibody provided herein can be administered during the anhepatic phase, as well as during the post-operative phase. The polypeptide, nucleic acid or antibody provided herein can be administered daily, biweekly or monthly after the transplant. The polypeptide, nucleic acid or antibody provided herein can be administered daily for the first week after transplant, weekly for two, three or more weeks after the transplant and then monthly. The absolute weight of a polypeptide or antibody included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one polypeptide, nucleic acid or antibody provided herein, or a plurality of polypeptides, nucleic acids or antibodies can be administered. Alternatively, the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.
The daily dose of a polypeptide, nucleic acid or antibody provided herein can vary as well. Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.
A polypeptide, nucleic acid or antibody provided herein can be used alone or in combination with a second medicament. The second medicament can be another polypeptide or antibody provided herein, a known antiviral agent such as, for example, an interferon-based therapeutic or another type of antiviral medicament such as ribavirin. A polypeptide, nucleic acid or antibody provided herein also can be used in combination with one or more agents to enhance the immune response in a subject. In some examples, the polypeptide, nucleic acid or antibody provided herein can be administered in combination with other therapeutic agents including, without limitation, immunoregulatory agents, immunoglobulin, cytokines, lymphokines, and chemokines, e.g., IL-2, modified IL-2 (e.g. Cl 25S), GM-CSF, IL-12, gamma- interferon, IP-IO, MIPl β, or RANTES.
The second medicament also can be an anticancer, antibacterial, or another antiviral agent. The antiviral agent can act at any step in the life cycle of the virus from initial attachment and entry to egress. Thus, the second antiviral agent can interfere with attachment, fusion, entry, trafficking, translation, viral polyprotein processing, viral genome replication, viral particle assembly, egress or budding.
Stated another way, the antiviral agent can be an attachment inhibitor, entry inhibitor, a fusion inhibitor, a trafficking inhibitor, a replication inhibitor, a translation inhibitor, a protein processing inhibitor, an egress inhibitor, in essence an inhibitor of any viral function. The effective amount of the second medicament will follow the recommendations of the manufacturer of the second medicament, as well as the judgment of the attending physician, and will be guided by the protocols and administrative factors for amounts and dosing as indicated in the PHYSICIAN'S DESK REFERENCE.
In some examples, the polypeptide, nucleic acid or antibody provided herein can be administered in combination with an adjuvant. The polypeptide, nucleic acid or antibody provided herein can be administered with any suitable adjuvant for stimulating immune response, e.g., providing immune protection. For example, it can be a particulate or a non-particulate adjuvant. A particulate adjuvant usually includes, without limitation, aluminum salts, calcium salts, water-in-oil emulsions, oil-in water emulsion, immune stimulating complexes (ISCOMS) and ISCOM matrices (U.S. Pat. No. 5,679,354), liposomes, nano- and microparticles, proteosomes, virosomes, stearyl tyrosine, and gamma-inulin. A non-particulate adjuvant usually includes, without limitation, muramyl dipeptide (MDP) and derivatives, e.g., treonyl MDP or murametide, non-ionic block copolymers, saponins, e.g., Quil A and QS21, lipid A or its derivative 4' monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), various cytokines including gamma-interferon and interleukins 2 or 4, carbohydrate polymers, derivatized polysaccharides, e.g., di ethyl aminoethyl dextran, and bacterial toxins, e.g., cholera toxin or E. coli labile toxin. In one embodiment, the polypeptide, nucleic acid or antibody provided herein is administered with adjuvant QS-21.
To determine the effectiveness of a polypeptide, nucleic acid or antibody provided herein for inhibition and treatment of HCV infection, methods available in the art and those described herein can be used. The effectiveness of the method of treatment can be assessed by monitoring the patient for signs or symptoms of the viral infection as discussed above, as well as determining the presence and/or amount of viral particle or viral RNA present in the blood, e.g. the viral load, using methods known in the art. Viral infection or replication in the presence or absence of a polypeptide or antibody provided herein can be evaluated, for example, by determining intracellular viral RNA levels or the number of viral foci by immunoassays using antibodies against viral proteins as described herein. A polypeptide or antibody is effective for treatment and inhibition of HCV if it can inhibit or reduce viral infection or replication by any amount, for example, by 2 fold or more than 2 fold. For example, a polypeptide or antibody provided herein can inhibit or reduce HCV infection by 2-5 folds, 5-10 folds, or more than 10 folds.
A polypeptide, nucleic acid or antibody provided herein also can be used to increase the safety of blood and blood products, to increase the safety of clinical laboratory samples and to increase the safety of biological tissues as well as articles, devices, or instruments intended for preventative or therapeutic use. For example, a polypeptide, nucleic acid or antibody provided herein can be added to blood or blood products such as plasma, platelets, and blood or marrow cells prior to use. A polypeptide, nucleic acid or antibody provided herein can be combined with cells or tissues prior to use or administration. It can be coated on articles, devices or instruments such as, for example, valves, bags and stents. I. Preparations and Compositions
Provided herein are purified preparations containing a mutant polypeptide provided herein or a preparation containing a cross-neutralizing antibody provided herein. In a purified preparation of a mutant polypeptide provided herein, at least 50 % of the mutant polypeptides in the preparation are folded in a conformation such that the discontinuous epitopes (i.e. amino acid segments corresponding to amino acids 412 to 424, amino acids 436 to 447 and amino acids 523 to 540 of HCV strain H77) come together to form a conformational epitope that can bind with a conformation-dependent antibody such as a cross-neutralizing antibody, for example, AR3A, AR3B, AR3C or AR3D. In such a polypeptide preparation, at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 % or 95 % of the mutant polypeptides are folded as described above. For example, in such a polypeptide preparation provided herein, about 85 %, 88 %, 90 %, 92 %, 94 %, 95 %, 96 %, 97 %,
98 %, or 99 % of the mutant polypeptides are folded as described.
In a preparation of a cross-neutralizing antibody provided herein, a larger proportion of the antibodies are cross-neutralizing antibodies. For example, such an antibody preparation can be a biological sample such as blood or plasma obtained from a subject (e.g. a mammal) that has been immunized with a mutant polypeptide provided herein. In this case, the blood sample contains a larger proportion of cross- neutralizing antibodies than a blood sample obtained from a similar animal that has been immunized with a naturally-occurring E2 polypeptide. Such a cross-neutralizing antibody preparation can be a partially purified or purified polypeptide preparation, i.e. a preparation resulting from one or more protein purification steps known in the art as well as those discussed herein. Such cross- neutralizing antibody preparation provided herein contains at least about 2 %, 5%, 10 %, 20 %, 30 %, 40 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, or 80 % cross- neutralizing anti-HCV antibodies. For example, such cross-neutralizing antibody preparation provided herein can contain about 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, 50 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %, 58 %, 59 %, 60 %, 62 %, 63 %, 64 %, 65 %, 66 %, 67 %, 68 %, 69 %, 70 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %,
99 % cross-neutralizing anti-HCV antibodies.
Methods of preparing mutant polypeptides and cross-neutralizing antibodies provided herein are described above. Preparations of these can be obtained using protein purification procedures known to those skilled in the art. See, for example, CURRENT PROTOCOLS IN PROTEIN SCIENCE, Coligan et al, eds., John Wiley & Sons, Inc., 1997.
Provided herein are pharmaceutical compositions comprising a mutant polypeptide, nucleic acid or antibody provided herein. To prepare such a pharmaceutical composition, a mutant polypeptide or antibody provided herein is obtained, e.g. by expression in a host cell or using polymerase chain reaction, purified as necessary or desired and then lyophilized and stabilized. The polypeptide, nucleic acid or antibody can then be adjusted to the appropriate concentration and then combined with other agent(s) or pharmaceutically acceptable carrier(s). A pharmaceutical formulation containing therapeutic amounts of one or more polypeptides, nucleic acids or antibodies provided herein can be prepared by procedures known in the art using well-known and readily available ingredients. For example, one or more polypeptides, nucleic acids or antibodies can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents also can be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone.
Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution also can be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds also can be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols also can be included. Preservatives also can be added. The compositions provided herein also can contain thickening agents such as cellulose and/or cellulose derivatives. They also can contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like. For oral administration, one or more polypeptides, nucleic acids or antibodies can be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The active polypeptide also can be presented as a bolus, electuary or paste. The formulations can, where appropriate, be conveniently presented in discrete unit dosage forms and can be prepared by any of the methods well known to the pharmaceutical arts including the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
One or more polypeptides, nucleic acids or antibodies provided herein also can be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. A pharmaceutical formulation containing one or more therapeutic polypeptides, nucleic acids or antibodies provided herein also can take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.
Thus, one or more polypeptides, nucleic acids or antibodies can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelf life of the dosage form. The polypeptides, nucleic acids or antibodies and other ingredients can form suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the polypeptides, nucleic acids or antibodies and other ingredients can be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
These formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol," polyglycols and polyethylene glycols, C1-C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol," isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.
It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol and its derivatives can be added.
In some examples the one or more polypeptides, nucleic acids or antibodies are formulated as a microbicide, which is administered topically or to mucosal surfaces such as the vagina, the rectum, eyes, nose and the mouth. For topical administration, the therapeutic agents can be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap. Thus, in one example, an agent provided herein can be formulated as a vaginal cream or a microbicide to be applied topically. Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the one or more polypeptides, nucleic acids or antibodies provided herein can be delivered via patches or bandages for dermal administration. Alternatively, the polypeptides, nucleic acids or antibodies can be formulated to be part of an adhesive polymer, such as polyacrylate or acryl ate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns. Ointments and creams can, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions can be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active agents also can be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051 ,842. The percent by weight of one or more polypeptides, nucleic acids or antibodies provided herein present in a topical formulation will depend on various factors, but generally will be from 0.01 % to 95 % of the total weight of the formulation, and typically 0.1-85 % by weight.
Drops, such as eye drops or nose drops, can be formulated with one or more of the polypeptides, nucleic acids or antibodies in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
The one or more polypeptides, nucleic acids or antibodies further can be formulated for topical administration in the mouth or throat. For example, the active ingredients can be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition provided herein in a suitable liquid carrier.
The pharmaceutical formulations provided herein can include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations provided herein include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0.
The polypeptides, nucleic acids or antibodies provided herein also can be administered to the respiratory tract. Thus, provided herein are aerosol pharmaceutical formulations and dosage torms tor use in the methods provided herein. In general, such dosage forms comprise an amount of at least one of the polypeptides, nucleic acids or antibodies provided herein effective to treat or prevent the clinical symptoms of the viral infection. Any statistically significant attenuation of one or more symptoms of the infection that has been treated pursuant to the method provided herein is considered to be a treatment of such infection within the scope provided herein.
Alternatively, for administration by inhalation or insufflation, the composition can take the form of a dry powder, for example, a powder mix of one or more polypeptides, nucleic acids or antibodies and a suitable powder base such as lactose or starch. The powder composition can be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder can be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung. Clarke. S. W. and Davia, D. eds., pp. 197-224, Butterworths, London, England, 1984).
The one or more polypeptides, nucleic acids or antibodies provided herein also can be administered in an aqueous solution when administered in an aerosol or inhaled form. Thus, other aerosol pharmaceutical formulations can comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/mL and about 100 mg/mL of one or more of the polypeptides, nucleic acids or antibodies provided herein specific for the indication or disease to be treated. Dry aerosol in the form of finely divided solid polypeptide, nucleic acid or antibody particles that are not dissolved or suspended in a liquid are also useful in the practice provided herein. Polypeptides, nucleic acids or antibodies provided herein can be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 μm, alternatively between 2 and 3μm. Finely divided particles can be prepared by pulverization and screen filtration using techniques well known in the art. The particles can be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount can be achieved using less than the dose in the dosage form, either individually, or in a series of administrations. For administration to the upper (nasal) or lower respiratory tract by inhalation, the one or more polypeptides, nucleic acids or antibodies provided herein are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs can comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those described in U.S. Patent Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co., (Valencia, CA). For intra-nasal administration, the therapeutic agent also can be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker). An exemplary formulation involves lyophilized polypeptides, nucleic acids or antibodies and separate pharmaceutical carrier. Immediately prior to administration, the formulation is constituted by combining the lyophilized polypeptides, nucleic acids or antibodies and pharmaceutical carrier. Administration by a parenteral or oral regimen will deliver the polypeptides, nucleic acids or antibodies to the desired site of action. Pharmaceutical formulations of the polypeptides, nucleic acids or antibodies provided herein can be prepared as liquids, gels and suspensions. Exemplary formulations are suitable for injection, insertion or inhalation. Injection can be accomplished by needle, cannula, catheter and the like. Insertion can be accomplished by lavage, trochar, spiking, surgical placement and the like. Inhalation can be accomplished by aerosol, spray or mist formulation. The polypeptides, nucleic acids or antibodies provided herein also can be administered topically such as to the epidermis, the buccal cavity and instillation into the ear, eye and nose. The polypeptides, nucleic acids or antibodies can be present in the pharmaceutical formulation at concentrations ranging from about 1 percent to about 50 percent, such as about 1 percent to about 20 percent, such as, for example, about 2 percent to about 10 percent by weight relative to the total weight of the formulation. A polypeptide, nucleic acid or antibody provided herein also can be used in combination with one or more known therapeutic agents, for example, a pain reliever; an antiviral agent such as an anti-HBV, other anti-HCV (HCV inhibitor, HCV protease inhibitor) or an anti-herpetic agent; an antibacterial agent; an anti-cancer agent; an anti-inflammatory agent; an antihistamine; a bronchodilator; an immunomodulatory agent; and appropriate combinations thereof, whether for the conditions described or some other condition. J. Articles of Manufacture and Other Compositions
Provided herein are articles of manufacture that include a pharmaceutical composition containing one or more polypeptides, nucleic acids or antibodies provided herein for controlling microbial infections. Such articles can be a useful device such as a vaginal ring, a condom, a bandage or a similar device. The device holds a therapeutically effective amount of a pharmaceutical composition for controlling viral infections. The device can be packaged in a kit along with instructions for using the pharmaceutical composition for control of the infection. The pharmaceutical composition includes at least one polypeptide, nucleic acid or antibody provided herein, in a therapeutically effective amount such that viral infection is controlled.
An article of manufacture also can be a vessel or filtration unit that can be used for collection, processing or storage of a biological sample containing a polypeptide or antibody provided herein. The vessel can be evacuated. Vessels include, without limitation, a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter. The filtration unit can be part of another device, for example, a catheter for collection of biological fluids. Moreover, the one or more polypeptides or antibodies provided herein also can be adsorbed onto or covalently attached to the article of manufacture, for example, a vessel or filtration unit. Thus, when material in the article is decanted therefrom or passed through, the material will not retain substantial amounts of the polypeptides or antibodies. Adsorption or covalent attachment of the one or more polypeptides or antibodies to the article kills viruses or prevents their transmission, thereby helping to control viral infection. Thus, for example, the one or more polypeptides or antibodies provided herein can be in filtration units integrated into biological collection catheters and vials, or added to collection vessels to remove or inactivate viral particles that can be present in the biological samples collected, thereby preventing transmission of the disease.
Provided herein are compositions comprising one or more polypeptides, nucleic acids or antibodies provided herein and one or more clinically useful agents such as a biological stabilizer. Biological stabilizer includes, without limitation, an anticoagulant, a preservative and a protease inhibitor. Anticoagulants include, without limitation, oxalate, ethylene diamine tetraacetic acid, citrate and heparin. Preservatives include, without limitation, boric acid, sodium formate and sodium borate. Protease inhibitors include inhibitors of dipeptidyl peptidase IV. Compositions comprising an agent provided herein and a biological stabilizer can be included in a collection vessel such as a capillary tube, a vacutainer, a collection bag for blood or other body fluids, a cannula, a catheter or any other container or vessel used for the collection, processing or storage of biological samples.
Provided herein are compositions comprising one or more polypeptides, nucleic acids or antibodies provided herein and a biological sample such as blood, semen or other body fluids that is to be analyzed in a laboratory or introduced into a recipient subject (e.g. a mammal). For example, one or more polypeptides, nucleic acids or antibodies provided herein can be mixed with blood prior to laboratory processing and/or transfusions. The one or more polypeptides, nucleic acids or antibodies are present in at least about 0.15 mg/mL of the sample, e.g. 0.16 mg/mL, 0.17 mg/mL, 0.18 mg/mL, 0.19 mg/mL, 0.2 mg/mL, 0.22 mg/mL, 0.24 mg/mL, 0.25 mg/mL, 0.27 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL or more than 0.4 mg/mL of sample.
In another example, the one or more polypeptides, nucleic acids or antibodies provided herein can be included in physiological media used to store and transport biological tissues, including transplantation tissues. Thus, for example, liver, heart, kidney and other tissues can be bathed in media containing the present agents to inhibit viral transmission to transplant recipients. In this case, the one or more polypeptides, nucleic acids or antibodies are present in at least about 1.5 mg/kg of the sample, e.g. 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg or more than 4 mg/kg. K. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1. Materials and Methods
Cells, antibodies and viruses.
Huh-7 (Zhong, J. et al., Proc. Natl. Acad. ScL U.S.A. 102, 9294-9299 (2005)) and 293T cells were grown in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% fetal calf serum (FCS) (Invitrogen). The human MAbs CBH- 2, CBH-5, CBH-4B and CBH-7, Mouse MAbs A4 , H53, AP33 , AP320 and ALP98, and rat MAbs 7/59, 9/27, 3/11, 1/39, 2/69A, 7/16B, 11/20, 9/75 and 6/53 have been described elsewhere. Keck, Z. Y. etal,. J. Virol. 78, 9224-9232 (2004); Keck, Z. Y. et al., J. Virol. 81, 1043-1047 (2007); Keck, Z.Y. et al, J. Virol. 79, 13199-13208 (2005); Dubuisson, J. etal, J. Virol. 68, 6147-6160 (1994); Clayton, R.F. et al, J. Virol 76, 7672-7682 (2002); Deleersnyder, V. et al, J. Virol. 71, 697-704 (1997) ; Owsianka, A. et al, J. Virol. 79, 11095-11104 (2005) ; Tarr, A.W. et al, Hematology 43, 592-601 (2006) ; Flint, M. et al, J. Virol 73, 6235-6244 (1999); Hsu, M. et al, Proa Natl Acad. ScL U.S.A. 100, 7271-7276 (2003); Maruyama, T. et al., Am. J. Pathol. 165, 53-61 (2004). The panel of linear epitope-specific MAbs covers known linear regions. The generation of HCVpp has been described below. Phage display antibody library construction.
In a study of autoantibodies in patients with Sjogren's syndrome, bone marrow mononuclear cell RNA from a 35-year-old female patient with Sjogren's syndrome and chronic HCV infection was used as source material for an IgGl Fab phage display library (Maruyama, T. etal, Am. J. Pathol. 165, 53-61 (2004)). The donor was diagnosed with HCV in 1991 and developed mixed cryoglobulinemia, symptoms of Sjogren's syndrome and tested positive for antinuclear antibody in 1994. The donor was treated with interferon-α with initial decrease in viral load but the treatment was stopped due to severe drop in platelet count (idiopathic thrombocytopenic purpura). Bone marrow samples were collected for the evaluation of neutropenia as an outpatient clinical procedure at Scripps Clinic. After meeting the needs of clinical pathology, a fraction of the biopsy was used to construct the antibody library. The human subjects protocol was approved by the Human Subjects Committee for General Clinical Research Center of Scripps Clinic and informed consent was obtained from the donor. Due to subsequent relapse of HCV, the donor underwent a liver transplant in 2000 and has been maintained on anti-rejection medications since. The viral genotype in this donor was not determined at the time of tissue donation but was found to be genotype 1 a seven years later. The bone marrow (~2 ml) was separated by Histopaque-1077 gradient (Sigma- Aldrich) and RNA was extracted from mononuclear cells (7 x 107 cells) homogenized in 10 mL of TRI reagent (Sigma- Aldrich). First-strand cDNA was synthesized using Superscript First-Strand Synthesis Kit (Invitrogen), and the light chain and IgGl heavy chain fragments were amplified by PCR using gene-specific primers and were sequentially cloned into the SacVXbal anάXhollSpel sites of a phagemid vector, pComb3H, as described previously (Maruyama, T. et ai, J. Infect. Dis. 179 Suppl 1, S235-239 (1999)). The Fab heavy chains were expressed as a fusion protein with the phage gene HI surface protein for display. The library was amplified in XL-I Blue cells (Stratagene) using 0.3% SeaPrep agarose (BioWhittaker) in SuperBroth (SB) Medium by a semisolid phase amplification method.
Library panning on HCV E2 glycoprotein
The phagemid library was transformed into E. coli (XL-I Blue) (Stratagene) by electroporation and the phage was propagated overnight with VCS-Ml 3 helper phage (Stratagene). Recombinant E2 glycoprotein (genotype Ia, amino acids 388- 644; Lesniewski, R. et al., J. Med. Virol. 45, 415-422 (1995)) was coated directly onto a microtiter plate overnight at 40C (Costar). The wells were washed and then blocked with 4% non-fat dry milk in phosphate-buffered saline (PBS). The phage library was added to the wells and incubated for 1-2 hours at 37°C and unbound phage washed away with PBS. Bound phage were eluted and used to infect freshly grown E. coli (XLl -Blue) (Stratagene) for titration on LB agar plates with carbenicillin. The phage libraries were panned for four consecutive rounds with increasing washing stringency.
Library panning by an epitope masking strategy. In order to broaden the diversity of antibody specificities selected, library panning was repeated using recombinant E1E2 fused to glutathione S transferase (GST-E1E2; Chan-Fook, C. et al., Virology TTb, 60-66 (2000)) pre-incubated with Fabs obtained above. GST-E1E2 was first captured with goat anti-GST antibody (Amersham Biosciences) and the wells were washed and blocked with 4% non-fat dry milk in PBS. Fabs obtained from the panning using E2 antigen above were added to the captured antigens to mask corresponding specific epitopes. The epitope-masked GST-El E2 was used to pan the phage library as described above. It is important to note that, highly isolate-specific antibodies, e.g. those against HVRl, were not selected due to the use of heterologous antigens in panning. Screening of Fab displayed phage. Single individual colonies were isolated from titration plates after the 2nd, the
3rd, and the 4th round. The colonies were grown in SB medium with carbenicillin and tetracycline and Fab-phage production was induced with the addition of helper phage overnight at 30°C. The specificities of the Fab-phage were assessed by ELISA and the DNA sequences of the Fab-phage that bound with high specificity were determined. To produce soluble Fabs, the phage gene III surface protein in fusion with the Fab heavy chain was excised by restriction enzymes Spel and Nhel. The cut phagemids were self-ligated and transformed into XLl -Blue cells for the production of soluble Fabs by standard protocols. Barbas III, C.F., Burton, D. R., Scott, J.K. & Silverman, GJ. Phage Display: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, New York, 2001 ). Conversion of Fab into IgGl.
The vectors pDR12 (Burton, D.R. et al., Science 266, 1024-1027 (1994)) and plgGl encoding the leader sequence and constant region of human IgGl gene were used for the cloning and expression of full length IgGl . Vector plgGl is a derivative of pDR12 in which heavy and light chain cloning sites were altered to XhollBstEll and SacllXbal sites to facilitate direct cloning of the antibody gene fragments. For pDR12, the heavy and light chain genes of Fab Cl were amplified by PCR then inserted sequentially into the SacVXbal and Hindlll/EcoRl sites of the vector (Burton, D.R. et al, Science 266, 1024-1027 (1994)). For plgGl, the heavy and light chain gene fragments were excised from the phagemids and inserted sequentially into the XhollBstEll and SacVXbal sites of the vector. The recombinant plasmids were transfected into Chinese hamster ovarian (CHO) cells. Stable cell clones were established by selection with L-methionine sulfoxide (MSX) and by limiting dilution. Cell clones expressing high IgG levels were amplified and the IgGs were purified using a protein A-agarose column (Pharmacia).
ELISA. (i) To study the relative reactivity of Fabs to GST-E1E2 and E2, soluble Fabs were added to ELISA wells coated with soluble E2 (4 μg/mL), with
GST-El E2 (8 μg/mL) captured by pre-coated goat anti-GST-antibody (10 μg/mL), or with ovalbumin (4 μg/mL). Specific binding was detected by alkaline phosphatase (AP)-conjugated goat anti-human IgG F(ab')2 antibody (Pierce) (1 :500) in 1% BSA/PBS and disodium /j-nitrophenyl phosphate (Sigma), (ii) To study the relationship of different ARs to the mouse MAb epitope H53 (Cocquerel et al., J. Virol. 72, 2183-2191 (1998)), a saturating concentration of MAb H53 was added to vaccinia-expressed E1E2 (isolate HCV-I, obtained through the NIH AIDS Research and Reference Reagent Program: rVV El 2 C/B from Chiron Corporation; Cooper, S. et al. Immunity 10, 439-449 (1999); Selby, M. et al. J. Immunol. 162, 669-676 (1999)) captured by pre-coated Galanthus nivalis lectin (5 μg/mL, Sigma) for 30 min before the addition of soluble Fabs (2 μg/mL). Non-fat milk (4%, BioRad) in PBS was used as a blocker in assays using lectin-captured antigens. The ELISA plates were washed after a 1 hour incubation and binding of human Fabs was detected by peroxidase (HRP)-conjugated goat anti-human IgG F(ab')2 antibody (1 :2000) (Pierce) and TMB substrate (Pierce). The level of inhibition by MAb H53 was calculated as the % reduction of optical density signals produced by the human Fabs in the presence of H53. (iii) To study whether the MAbs recognized continuous or discontinuous epitopes, vaccinia-expressed E1E2 was either captured directly onto ELISA wells pre- coated with lectin (folded protein), or unfolded with 0.1% SDS, 50 mM DTT and incubated at 1000C for 5 minutes before capture onto ELISA wells (unfolded protein). Binding of the MAbs to folded and unfolded proteins was detected using the peroxidase system. Mouse MAb A4 (Dubuisson, J. et al., J. Virol. 68, 6147-6160 (1994)), specific for a linear epitope in El, was used as a positive control, (iv) To study the ability of MAb in inhibiting E1E2 binding to CD81, serially diluted MAbs (4-fold dilution from 10 μg/mL) were incubated with E1E2 (isolate H77) for 30 min before adding to ELISA wells pre-coated with the large extracellular loop of CD81 (CD81-LEL). After 1 hour incubation, the plates were washed and binding of E1E2 to CD81-LEL was detected with an anti-El mouse MAb A4 (Dubuisson, J. et al, J. Virol. 68, 6147-6160 (1994)), HRP-conjugated goat anti-mouse Fc antibody (Pierce) (1 :2000) and TMB substrate. Two forms of recombinant CD81-LEL, either in fusion with glutathione S-transferase (GST) (Owsianka, A.M. et al, J. Virol. 80, 8695- 8704 (2006)) or maltose binding protein (MBP) (Chan-Fook, C. et al, Virology 273, 60-66 (2000)), were used and the results were equivalent, (v) To study the apparent affinity of the MAbs, serially diluted MAbs (2-fold dilution from 20 μg/mL) were added to lectin-captured E1E2 antigens for 1 hour. E1E2 antigens were prepared from cell lysates from vaccinia-expressed HCV-I El E2, 293T cells transfected with H77 ElE2-expression plasmid (McKeating, J.A. et al, J. Virol 78, 8496-8505 (2004)). The binding of human MAbs was detected by HRP-conjugated goat anti- human IgG F(ab')2 antibody as above. Non-infected/non-transfected cell lysate were used as negative controls to determine background for each MAb. Apparent affinity was defined by the concentration of MAbs that produced half of the maximal specific binding in the titration curves, (vi) To construct the MAb competition matrix, saturating concentrations of blocking MAbs (typically at 20 μg/mL or undiluted hybridoma supernatants) were added to lectin-captured vaccinia-expressed HCV-I E1E2 for 30 minutes before the addition of an equal volume of biotinylated human MAbs (2 μg/mL). The E1E2 antigens were titrated to ensure that saturating concentrations of the blocking MAbs were used in the assays. It is important to note that, MAbs recognizing linear epitopes bind to both folded and unfolded proteins but the biotinylated human MAbs bind conformational epitopes on folded E2. Consequently, competition is performed with the MAbs to linear epitopes as blocking MAbs to eliminate potential non-specific signals caused by misfolded proteins in the system. After incubation for 1 h, the ELISA plates were washed and binding of biotinylated MAbs was detected with HRP-conjugated streptavidin (1 :2000, Sigma- Aldrich) in PBS with 1% BSA and TMB substrate (Pierce). Competition was determined by the % change in binding signals in the presence of the blocking antibodies, (vii) To study the effect of alanine substitution in E2 on MAb binding, El E2 mutant antigens were produced by transfection of 293T cells with the corresponding expression plasmids and the antigens in clarified cell lysate were captured by lectin as above. A panel of 38 H77 E1E2 mutants having the conserved residues in the putative CD81 binding pocket substituted with alanine was used in this study (Owsianka, A.M. et al, J. Virol. 80, 8695-8704 (2006)). The binding signals of the human MAbs to the alanine mutants were compared to the wild type H77 E1E2 to determine the importance of the residues in the antibody-antigen interaction, (viii) To quantify human IgG in mouse serum, diluted mouse sera in triplicate were added to ELISA wells coated with human goat anti-human IgG F(ab)'2 (10 μg/mL, Pierce) for 1 hour and bound human IgG was detected with AP-conjugated goat anti-human F(ab)'2 (10 μg/mL) (Pierce). Serially diluted MAb AR3A (2-fold dilution starting from 4 g/mL) was used to construct a standard curve in each ELISA plate. The concentration of human IgG in each serum sample was calculated from the 4- parameter fitted standard curve using SOFTmax Pro Software (Molecular Devices). HCV neutralization assays.
The neutralization assays were performed in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% fetal calf serum (FCS) (Invitrogen). For HCVpp neutralization, HCVpp was generated by co-transfection of 293T cells with pNL4-3.1ucR-E- (Connor et al, Virology 206, 935-944 (1995); He, J. et al., J. Virol. 69, 6705-671 1 (1995)) and the corresponding expression plasmids encoding the El E2 genes at 4:1 ratio by polyethylenimine (Boussif, O. et al, Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301 (1995)). Virus infectivity was detected using the firefly luciferase assay system (Promega). Background infectivity of the pseudotype virus was determined using cells transfected with pNL4-3.1ucR-E- only. The El E2 expression plasmids for the isolates H77, H, CH35, OH8, UKNl B 12.16, J6, UKN2A1.2, UKN2B1.1 , UKN3A13.6, UKN3A1.28c, UKN4.21.16, UKN5.15.7 and UKN6.5.8 have been described previously (Owsianka, A. et al., J. Virol. 79, 11095- 11 104 (2005); McKeating, J.A. et al., J. Virol. 78, 8496-8505 (2004); Lavillette, D. et al., Hepatology 41, 265-274 (2005)). The expression plasmids encoding E1E2 of the virus in an infected human serum (KP) used in the protection experiment are described below (see also, FIG. 6). The majority of HCV Envs, except H77, H and OH8, produce only low levels of HCVpp (<5,000 Relative Light Unit, RLU). To ensure the quality of data for determining virus neutralization, 1 HCVpp of low infectivity was concentrated 10-20 fold by centrifugation at 38,000 x g for 1 hour. Serially diluted antibodies were first incubated with the virus for lhour at 37°C before adding to Huh-7 cells in triplicate and the cells were incubated for 3 days. After background subtraction, virus neutralization was determined by the % change of RLU in the presence of antibodies. Virus concentration did not affect the neutralization of the prototype HCVpp-H77 by the MAbs in comparison to unconcentrated virus (data not shown). Although virus concentration improved the signals of several HCV Envs (Table 10), consistent signals were not obtained with HCVpp displaying CH35, UKN3A1.28c, UKN6.5.8 or KP E1E2 in repeated experiments and these Envs were excluded in the analysis.
Cloning of E1E2 from an infected human serum. Total RNA in the HCV GT 1 a-infected human serum KP ( 140 μl) was purified using a QIAamp Viral RNA Mini Kit (Qiagen). First strand cDNA was generated using either a reverse primer specific to HCVIa (HCVlaOuterR, GGGATGCTGCATTGAGTA, (SEQ ID NO: 697); Lavillette, D. et al., Hepatology 41, 265-274 (2005)) or random hexamer using the Superscript III reverse transcriptase (Invitrogen). The GTIa E1E2 genes were amplified by a nested PCR as described previously (Lavillette, D. et al, Hepatology 41, 265-274 (2005)) and the PCR products were cloned using the pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen). An HIV-positive human serum was used as a negative control throughout the experiments and no non-specific product was generated. The sequences of 40 clones were determined by DNA sequencing and analyzed using
VectorNTI software (Invitrogen). Expression of E1E2 proteins was confirmed by the presence of folded E2 proteins in cell lysates, prepared from 293T cells transfected with the corresponding DNA plasmids, by ELISA using MAb AR3A. Antibody protection studies. Human liver-chimeric mice were prepared as described previously. Mercer,
D.F. et al. Nat. Med. 7, 927-933 (2001); Kneteman, N.M. et al., Hepatology A3, 1346-1353 (2006). The animal experiments were approved by the University of Alberta Animal Care and Use Committee for Health Sciences. All human liver biopsies and sera were collected under informed consent and the human subjects protocols were approved by the University of Alberta Health Research Ethics Board (Biomedical Panel). Colonization of human hepatocytes in the livers of AIb- uPA/SCID mice was confirmed by the presence of human alpha- 1-anti-trypsin
(hAAT) in the mice. Only mice with serum levels of hAAT greater than 60 μg/mL at 6 weeks and 100 μg/mL at 8 weeks, an indication for successful transplantation, were used in the protection study (~50% of transplanted mice). Mice with low level of human liver chimerism were used in preliminary experiments to measure the toxicity and kinetics of MAbs in Alb-uPA/SCID mice, and the level of human IgG present in mice injected with a genotype 1 a HCV-infected human serum KP. This serum, serially diluted from 1 : 50 to 1 :4050, did not neutralize HCVpp-H77 (data not shown). Different doses of MAbs, at 10 mg/mL, were injected into the mice via the intraperitoneal route. For virus challenge, the experiments were conducted in blinded fashion; the identity of the MAbs was not provided to the technicians performing the animal procedures and HCV RNA tests. Human liver-chimeric mice were given MAbs by intraperitoneal injection (200 mg/kg) 24 hours before virus challenge. Mice were anaesthetized and injected intrajugularly with 100 μL of infected serum KP (2.3 x 106 IU/mL). Blood was sampled by tail bleed and sera were prepared by centrifugation of clotted blood for ELISA and viral load measurement. HCV RNA quantification.
HCV RNA in mouse serum was quantified by a real-time TaqMan PCR assay. The two primers in the real-time PCR system were designed to produce a 194 bp PCR fragment corresponding to the 5 ' non-coding region with maximum specificity to all HCV genotypes. The forward primer (T-149-F, 5'-
TGCGGAACCGGTGAGTACA, (SEQ ID NO: 698) and reverse primer (T-342-R,
5'-
AGGTTTAGGATTCGTGCTCAT, (SEQ ID NO: 699) were designed with the aid of software Primer Express (PE biosystems) and were purchased from PE Applied Biosystems. To quantify HCV RNA, total RNA in serum was isolated by the guanidinium thiocyanate (GuSCN) and silico method (Boom, R. et al., J. Clin. Microbiol. 28, 495-503 (1990)). Briefly, 30 μL of serum was mixed with 500μl GuSCN lysis buffer and 20 μL size- fractionated silica particles for 15 minutes. The silica particles were pelleted and washed twice with 500 μL washing buffer, twice with 70% ethanol and once with acetone. The pellet was dried for 10 min on heat block, and RNA was eluted in 20 μL distilled water and quantified by optical density. Superscript Il First-Strand Synthesis Kit (Invitrogen) was used to synthesize first- strand cDNA for PCR. Five μL of the serum RNA was mixed with 100 μM of Superscript II reverse transcriptase, 20 μM of RNAseOut and 14 μL reaction cocktail (which includes 1 x first-strand buffer, 5 μM DTT, 375 nM dNTP, 1.25 μM T-342-R primer) and incubated at 420C for 60 min then 70°C for 15 minutes. For quantitative PCR, a 50 μL mixture contained 9 μL of template HCV cDNA, 1 x TaqMan
Universal PCR Master Mixture (Applied Biosystems Inc.), 375 nM dNTP, 400 nM of T-149-F and T-342-R primers and 200 nM TaqMan probe (6-FAM18 CACCCTATCAGGCAGTACCACAAGGCC-TAMRA, (SEQ ID NO: 700). Thermocycling was performed on a Taqman 7300 (Applied Biosystems Inc.) using the default setting program recommended by the manufacturer: 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 600C for 60 s. A serial dilution of HCV cDNA, including 1.5 X lO6, 1.5 x 10s, 1.5 x 104, 1.5 x 103, 1.5 x 102, 1.5 x 101, 1.5 x 10° IU, was used to generate a standard curve for calculation of HCV RNA copy number. The dynamic range of HCV RNA detection for the two step RT-PCR procedure is 6.0 x 102 IU/ml to 3.0 x 108 IU/mL. Each assay run incorporates in duplicate a negative control and an HCV RNA positive control. The positive control is the OptiQual HCV RNA 1 Control purchased from AcroMetrix which has been calibrated to the WHO first International Standard for HCV RNA. Statistical analysis. GraphPad Prism 4 software was used for statistical analysis of the antibody protection experiment. Animals seropositive for HCV RNA by the quantitative PCR assay at or after day 7 post-infection were scored as "infected" subjects and animals seronegative up to week 6 were scored as "censored" subjects. The scores were used to construct the Kaplan-Meier survival (infection in this case) curves to calculate statistical significance between the neutralizing antibody-treated and isotype antibody control groups by a two-tailed log rank test within the experimental period. Motulsky, H. Survival curves, in GraphPad Prism4 Statistics Guide: Statistical analyses for laboratory and clinical researchers 107-117 (GraphPad Software, San Diego, 2005).
Example 2.
Anti-HCV Neutralizing Antibodies The Example describes the identification of human monoclonal antibodies
(mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV can be achievable.
A total of 115 clones that exhibit specific binding to HCV E2 glycoprotein were isolated from an antibody antigen-binding fragment (Fab) phage display library generated from a donor chronically infected with HCV (see Example 1). DNA sequence analysis identified 36 distinct Fabs with 13 unique heavy chain sequences. The sequences of the 36 distinct Fabs belonging to 13 groups based on the heavy chain sequences are also shown in Table 7 below. Fabs with the same designation and * or ** have the same heavy chain but distinct light chains, e.g. Hl, Hl* and Hl** have the same heavy chain, but 3 different light chains.
Table 7: Fab HCDR3 Sequences
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Table 8B: Anti-HCV E2 Fabs (IgG/c, heavy chain) - CON 'T
Figure imgf000100_0002
Figure imgf000101_0001
Table 8C: Anti-HCV E2 Fabs (IgGκ, heavy chain) - CONT
Figure imgf000101_0002
Figure imgf000102_0001
Figure imgf000103_0001
Table 9A: Anti-HCV E2 *abs (IgGκ, Light chain)
Fab FRAMEWORK! CDRl
EL TQSPATLSVSPGESATLSC RASQSVSDN LA
A (SEQ ID NO: 433) (SEQ ID NO: 171)
Figure imgf000104_0001
Figure imgf000105_0001
Table 9B: Anti-HCV E2 Fabs (IgGκ, Light chain) - CON T
Figure imgf000105_0002
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000107_0002
Figure imgf000108_0001
The binding properties of soluble Fabs prepared from the phage-Fab clones were characterized (FIG. 1). This allowed the Fabs to be sorted into three groups recognizing three antigenic regions (AR) of HCV E2 as shown in the table below.
Table 10: Three Distinct Antigenic Regions Defined by the Fab Panel
Figure imgf000108_0002
The numbers in parenthesis denote the percentage of clones recognizing each AR in the phage-display panning. It is important to note that highly isolate-specific antibodies, e.g. those against HVRl, would unlikely be selected in this study due to the use of heterologous antigens in the panning. Fab K was excluded in this table due to its poor signal in FIG. 1.
A total of seven Fabs from different heavy-chain groups recognizing the three different antigenic regions were converted into full-length IgGIs and their binding properties were evaluated (Table 11). In addition, the neutralizing activities of the mAbs were studied using a panel of HCV pseudotype virus particles (HCVpp) displaying El E2 from diverse genotypes (Table 12) See Wakita et ai, Nat Med. 11 : 791-96 (2005); Bartosch ef α/., J Exp. Med. 197: 633-42 (2003); Hsu et al, Proc Natl. Acad. Sci. USA 100: 7271-7276 (2003)).
Table 11: Binding properties of E2-specifϊc IgGs
Figure imgf000109_0001
" Antibody concentration (nM) to inhibit 50% of E1-E2 (isolate H77) binding to immobilized recombinant large extracellular loop of CD81 (CD81-LEL)
1VaCCmIa-CXPrCSSCd E1-E2
Ε1-E2 produced by transfected 293T cells dApparent affinity is defined as the antibody concentration required to achieve half-maximal binding in an ELISA Data shown are the means of at least two independent experiments All mAbs bind natively folded, but not reduced and denatured, E2 GTIa indicates genotype Ia, GT2a indicates genotype 2a and dashes indicate that no significant inhibition or binding was observed with the highest mAb concentration tested
Figure imgf000109_0002
Figure imgf000110_0001
mAbs at 50, 25, 10, 5 or 1 μg/mL were tested for virus neutralization, and the lowest antibody concentrations that reduced >50% of virus infectivity are shown Dashes indicate no or <50% virus neutralization with 50 μg/mL mAb. Data shown are the means of at least two experiments. b Neutralization of HC Vpp was determined by the reduction in luciferase activity in Huh-7 cells infected with HCVpp displaying Env from different HCV isolates. The panel of HCVpps shown includes HCV Env proteins that produce a signal at least tenfold higher than the background signal induced by the control pseudotype virus generated without HCV Env cDNA Many HCV Env proteins, including CH35 (genotype Ib), UKN3A1.28c (genotype 3a), UKN6.5.8 (genotype 6) and 13 different
KP Env clones (genotype Ia, see FIG. 6), did not produce a consistent signal tenfold higher than background and were excluded from this analysis
The above results indicate that all recombinant mAbs bound the E1-E2 complex from HCV genotype 1 a with approximately similar apparent affinities, in the range of 0.4-6 nM, but only antigenic region 3 (AR3)-specific mAbs reacted with genotype 2a HCV, suggesting that epitopes in AR3 are highly conserved. Monoclonal antibodies ARIA and AR3A-D inhibited the binding of E1-E2 to the virus co-receptor CD81 (Piled et al., Science 282, 938-41 (1998); Cocquerel et al., J. Virol. 77, 10677-83 (2003)) at nanomolar concentrations, suggesting that these antibodies can block HCV interaction with CD81 and thereby inhibit infection. In addition, these experiments indicate the following. First, antibodies that bind E2 in an ELISA did not necessaπly neutralize the corresponding virus. The ARl -specific antibodies bound recombinant E1-E2 from genotype Ia HCV isolate H77 with a similar or higher affinity than AR3-specific antibodies, but they did not neutralize the virus, suggesting that the ARl epitopes are available on isolated envelope proteins but not on infectious virions. Of note, the Fab fragments of antibodies ARIA and ARlB (that is, B2 and Dl, Table 11) did neutralize HCVpp-H77 (FIG. 2), indicating that steπc hindrance, possibly by El (FIG. IA), prevents virus neutralization by whole ARl -specific antibodies. Second, the ability of the antibodies to inhibit E1-E2 binding to CD81 in the
'neutralization of binding' assay (Rosa et al., Proc. Natl. Acad. Sci C/&4 93: 1759- 63 (1996)) did not fully predict virus neutralization Third, and most notably, the AR3 -specific antibodies bound E1-E2 from both genotypes Ia and 2a at nanomolar affinities and cross-neutralized many HCVpps tested. These results show that AR3 is a relatively conserved neutralizing site on HCV E2.
The specificity, affinity and neutralizing activities of the E2-specific human monoclonal antibodies were evaluated by mapping the antigenic regions using competition ELISA and alanine-mutagenesis scanning. Results are shown in the following tables.
Table 13 - Antibody Competition
544- 640- 197- 207
|I
I f
Figure imgf000112_0001
Numbers indicate percentage of residual binding signals of biotinylated human mAbs in the presence of blocking mAbs. Origin: h, human; m, mouse; r, rat.
Table 14 - Alanine-scanning Mutagenesis
69 74 34 0379 16 20 0 0257 83151 156
177228259
64155134
Figure imgf000112_0002
The panel of variants (top row) includes substitutions at conserved residues in the putative CD81 -binding regions of E2. Substitutions important for CD81 binding are shaded and include L413A, W420A, H421A, I422A, N423A, S424A, G523A, T526A, Y527A, W529A, G530A, D535A, V538A, N540A and F550A. (Owsianka, A.M. et al. J. Virol 80, 8695-8704 (2006)).
The antibody competition study shows that mAbs AP33 and 3/11 (*) recognize epitopes partially dependent on proper protein folding (Tarr, A.W. et al, Hepatology 43, 592-601 (2006)). The results confirm the broad designation of the antigenic regions and suggest that the discontinuous epitopes in AR3 are formed by at least three segments between amino acids 396-424, 436—447 and 523-540; the first and third segments also contribute to the CD81 -binding domain of E2 (Owsianka, A.M. et al, J. Virol. 80, 8695-8704 (2006)), and the conserved residues Ser424, Gly523, Pro525, Gly530, Asp535, Val538 and Asn540 (Owsianka, A.M. et al., J. Virol. 80, 8695-8704 (2006)) are probably involved in the binding of the AR3- specific antibodies (FIG. 3).
A key question is whether broadly neutralizing AR3-specific antibodies can protect against infection by heterologous HCV quasispecies. As a first step to evaluate the mAbs and establish the essential parameters for passive antibody protection, the human liver-chimeric Alb-uPA/SCID mouse model was used (Kneteman, N.M. et al, Hepatology 43, 1346-1353 (2006); Lindenbach, B.D. et al, Proc. Natl. Acad. Sd. USA 103, 3805-3809 (2006)). Although this animal model is not suitable for studying virus pathogenesis, owing to its lack of a functional adaptive immune system, the question of whether antibodies can protect against HCV challenge is appropriate. Previous passive antibody studies in animal models have reported relatively high antibody concentrations are needed for protection. For instance, to achieve sterilizing immunity by single mAB treatment against HIV in hu-PBL/SCID mice (Gauduin et al. Nat. Med. 3, 1389-1393 (1997)) and against chimeric simian/human immunodeficiency virus (SHIV) in macaques (Parren et al. J. Virol. 75, 8340-8347 (2001)), serum concentrations in the animals of the order of 100-fold in vitro 90% neutralization titers (ICg0) have been required. The IC90 titers (against HCVpp-H77) for MAb AR3A and AR3B are 11 and 20 g/mL, respectively, suggesting that relatively large doses of antibody may be required for protection. The kinetics and tolerability were first established in the animal model for the antibodies AR3 A, AR3B and a human isotype control IgGl to HIV-I , b6. Transplanted Alb-uPA/SCID mice with a low level of human liver chimerism were injected intraperitoneally with 100, 150 or 200 mg/kg MAb, and blood samples were collected by tail bleed. Human antibody in the murine sera was measured by a quantitative sandwich ELISA using conjugated and unconjugated goat anti-human F(ab)'2 antibody. The antibodies did not show adverse effects in control mice. No specific weight loss or signs of illness associated with the administration of the MAbs were noted in the mice during the experiment. One mouse (N457) was euthanized due to unrelated morbidity at Day 7. A dose of 200 mg/kg given through intraperitoneal injection was required to achieve mean serum titers approximately 100 x higher than in vitro neutralization titers. Such titers have previously been found to be necessary to achieve sterilizing immunity in other viral disease models. The neutralizing activity in mouse sera collected ten days after injection was determined by HCVpp-H77 neutralization assay. Mouse sera containing anti-HCV MAbs AR3A and AR3B neutralized 50% of HCVpp infectivity (IC5o) in the range of 1:200 to 1:1000. Control mouse sera from mice injected with an isotype MAb DEN3 or PBS neutralized non-specifically -50% virus infectivity at 10-fold dilution. Non- specific neutralization was not observed when the control sera were diluted 100-fold. The conservation of virus neutralizing activity of anti-HCV MAbs was determined by normalizing to the level of human IgG in the mouse sera quantified in the kinetics and tolerability study. In this experiment, MAbs AR3A and AR3B were titrated alongside with the mouse sera to construct the standard curves and the IC50 titers of MAbs AR3A and AR3B were 0.4 and 1 μg/mL, respectively. The IC50 titers of mouse sera containing anti-HCV MAbs AR3A and AR3B were in the range of 0.4-1.1 (mean 0.8 ± s.d. 0.3) and 0.5-3 (mean 1.2 ± s.d. 0.9) μg/mL, respectively. Isotype control MAbs b6 & DEN3 did not neutralize HCVpp. The observed half-lives of mAbs AR3 A, AR3B and b6 were 6.0 ± 2.2 d, 9.0 ± 1.3 d and 7.3 ± 1.8 d (mean ± s.d.), respectively, and their specific neutralizing activities (that is, neutralizing activity relative to serum mAb concentration) were stable for at least 10 days in the mice.
The mAbs were administered intraperitoneally in passive transfer experiments to mice with high levels of human liver chimerism (see Example 1), and the mean serum titers of mAbs AR3A, AR3B and the control mAb b6, at 24 hours after injection were -2.5 ± 0.3 mg/mL, 3.1 ± 0.5 mg/mL and 2.6 ± 0.3 mg/mL, respectively (FIG. 4). To simulate a natural human exposure to virus, we inoculated genotype Ia HCV-infected human serum intravenously into the mice. The partial amino acid sequences (residues 384-622) of forty HCVs found in the viral quasispecies population in the HCV genotype la-infected human serum are shown below.
Table 15: Cloned Variant Sequences of E2 Amino Acid Residues 384-622
Name Sequence
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP S9 CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 701)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLAGCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN Rl 4 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 702)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP S6 CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPSYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 703)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI
KP AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN S18 WFGCTWMDSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 704)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN S16 WFGCTWMNSTGFTKVCGALPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 705)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP R8 CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCVIGGVGSNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 706)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHVNRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVHCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN S20 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 707)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI VGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPI SYVNVSGPGERPYCWHYPPRP
KP S4 CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 708)
ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFDSSGCPERLASCRRLDDFAQGWGPISYVNVSGPGERPYCWHYPPRP
KP R3 CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 709) ETHVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFDSSGCPERLASCRRLDDFAQGWGPISYVNVSGPGERPYCWHYPPRP
KP S3 CGIVPARDVCGPVYCFTPSPWVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCDIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 710)
ETHVTGGATAHGASVLASLLTPGAKQHVQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFDSSGCPERLASCRRLDDFAQGWGPISYVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN S12 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 711)
ETHVTGGATAHGASVLASLLTPGAKQNIQLINTNGSWHINRTALNCNDSLHTGFV AGLFYYNKFDSSGCPERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN S15 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 712)
ETHVTGGATAHGASVLASLLTPGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFDSSGCLERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP
KP S5 CGIVPARDVCGPVYCFTPSPVWGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCAIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 713)
ETHVTGGATAHGASVLASLLTPGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP R7 CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCAIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 714)
ETHVTGGATAHGASVLASLLTPGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN RIl WFGCTWMNTTGFTKVCGAPSCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 715)
ETHVTGGATAHGASVLASLLTPGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP Rl CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNTTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 716)
ETHVTGGATAHGASVLASLLTPGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN R12 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 717)
ETHVTGGATAHGASVLASLLTPGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP S7 CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPCI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 718)
ETHVTGGATAHGASVLTSLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI
KP AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP CGIVPARDVCGPVYCFTPSPVVVGTTDRAGAPTYNWGANETDIFVLNNTRPPLGN R15 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 719) ETYVTGGATAHGASVLASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN R18 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 720)
ETHVTGGATAHGASVFASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFNSSGCPERLASCRRLDDFAQGWGPI SHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPSPWVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN SIl WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 721)
ETHVTGGATAHGASVFASLLTTGAKQNIQLINTNGSWHINRTALNCNDSLHTGFI AGLFYYNKFDSSGCPERLASCRRLDDFAQGWGPISHVNVSGPGERPYCWHYPPRP
KP CGIVPARDVCGPVYCFTPS PVVVGTTDRAGAPTYNWGANETDVFVLNNTRPPLGN R20 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLRCPTDCFRKHPDATYSRCGSGPWI TPRCLVDYPYRLWHYPCTI (SEQ ID NO: 722)
ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWL AGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRP CGIVPAKSVCGPVYCFTPS PVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGN
H77 WFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWI TPRCMVDYPYRLWHYPCTI (SEQ ID NO: 723)
RTRTTGGSAAQTTYGLTSLFRSGPSQKIQLVNTNGSWHINRTALNCNDSLNTGFL AALFYVRNFNSSGCPERMASCRPIDTFDQGWGPITYTEPHSLDQRPYCWHYAPQP
UKNIb CGIVPAAQVCGPVYCFTPS PVVVGTTDRFGAPTYTWGENETDVLILNNTRPPQGN 12.16 WFGCTWMNGTGFTKTCGGPPCNIGGAGNNTL^CPTDCFRKHPEATYTRCGSGPWL TPRCMVDYPYRLWHYPCTV (SEQ ID NO: 724)
The alignment of these sequences are shown in FIG. 6. Infection was monitored by assessing serum viral load up to 6 weeks after inoculation (FIG. 5). Protection in this mouse model is defined as the absence of serum HCV RNA as detected by quantitative PCR at or after 6 days post virus challenge. All mice in the control group (« = 4) were infected, and serum viral load was maintained at >10,000 RNA copies/mL until the completion of the study. In mice that received mAb AR3A {n = 5) or AR3B (n = 4), HCV was detected the day after challenge in five of nine mice, but was cleared 6 days after virus challenge. High levels of HCV RNA were detected in four mice between weeks 2 and 4, indicating virus replication concurrent with the decay of antibody in these mice. By week 6, when the mAbs would have decayed to <10% of the initial serum level, two of five mice receiving mAb AR3A and three of four mice receiving mAb AR3B were still protected. The protection was highly significant compared to the isotype control antibody group (two-tailed log-rank test: AR3A, P = 0.0298; AR3B, P = 0.0171). The experiments ended at week 6 because two mice became morbid and were killed on day 41 and day 45, respectively, but the remaining mice were monitored to week 8, and a signal below the sensitivity of the quantitative PCR assay (6.0 x 102 international units/mL) was noted in one additional mouse in each neutralizing antibody-treated group (mice N681 and N697). In summary, (i) it is possible to use mAbs against AR3 to protect against challenge with a heterologous HCV quasispecies swarm, consistent with the notion that AR3 is the principal conserved neutralizing antibody determinant of HCV; (ii) high concentrations of the mAbs were required for protection, suggesting that more potent antibody preparations will likely be required in immunotherapy, but that the mAbs described will be useful for comparative in vitro studies with newly identified mAbs and combinations of mAbs; and (iii) considering that one- third of the 115 phage-Fab clones isolated in this study are AR3 specific and are diverse in their heavy-chain sequences (FIGs. 1, 2 and Table 10), and similar mAbs were isolated from different HCV-infected donors elsewhere (Table 13)(Keck, Z.Y. et al., J. Virol. 78, 9224-9232 (2004)), AR3 seems to be relatively immunogenic in humans and thus a favorable target for vaccine design. So, despite the enormous diversity of HCV, the prospects for developing a vaccine against this virus, that can target both conserved B and T cell epitopes (Elmowalid, G. A. et al. Proc. Natl. Acad. Sd. USA 104, 8427- 8432 (2007); Folgori, A. et al, Nat. Med. 12, 190-197 (2006)), seem favorable. Example 3.
Generation and Characterization of HCV E2 Mutants The HCV E2 glycoprotein is a major target for virus neutralizing antibodies and an important component in a HCV vaccine. E2 has encoded several features to evade antibodies. First, E2 encodes regions that are highly mutable. Rapid changes in viral sequence facilitate virus escape. Second, E2 is highly glycosylated and the associated glycans help shield the neutralizing epitopes from antibodies. Despite these escape features, we have identified the antigenic region 3 (AR3) on E2 as a relatively conserved target for antibody neutralization in vitro and antibody protection in vivo. The amino acid residues important for the binding of AR3-specific antibodies is described above. The following show how these residues organize together to form the AR3 conformational epitopes. To identify a form of E2 that displays AR3 properly while silencing some of the variable sequences that are usually immunogenic but are not targets of broadly neutralizing antibodies, a panel of E2 truncation mutants were constructed. To identify the minimal E2 fragment that displays the CD81 -binding sites and the broadly neutralizing epitopes correctly, the binding of these E2 mutants with CDE81- LEL or various mAb were studied.
Construction of expression DNA plasmids ofE2 mutants The E2 mutants were constructed by deletion of highly variable regions, specific N-glycosylation signals, or every other cysteine residues from C- or N- terminus of wildtype (WT) E2. The panel of E2 mutants in fusion with the Flag tags at their C-termini are illustrated in FIG. 7, and their sequences are shown in Table 16. Table 16— Hepatitis C virus E2 Glycoprotein Mutants
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
E2384-746 ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTG WLAGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHY PPRPCGIVPAKSVCGPVYCFTSPVWGTTDRSGAPTYSWGANDTDVFVLNNTR PPLGNWFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLLCPTDCFRKHPEATYSR CGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHLEAACNWTRGE RCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQNIVDVQYLY GVGSSIASWAIKWEYVVLLFLLLADARVCSCLWMMLLISQAEA (SEQ ID NO: 743)
The cDNA encoding these mutants were generated by polymerase chain reaction (PCR) or by splicing by overlap extension polymerase chain reaction (SOE- PCR) as described in Horton et al., Biotechniques 8:528-535 (1990). In the reaction, the plasmid pCV-H77c (Genbank accession# AFOl 1751) encoding wildtype E2 gene of the isolate H77 was used as a template. The primers used in the reactions are enlisted below.
Table 17. Primers for cloning 17 mutants Primer SEQ ID NO: Sequence (from 5'- to -3')
E2wtF 744 AATAACGCGTGAAACCCACGTCACCGG
E2flF 745 AATAACGCGTCAACTGATCAACACCAACG
E2f2F 746 AATAACGCGTTTGGCCAGCTGCCGACGC
E2f3F 747 AATAACGCGTAGACCTTGTGGCATTGTGC
E2f4F 748 AATAACGCGTGTATATTGCTTCACTCCCAG
E2f5F 749 AATAACGCGTACTGGATTCACCAAAGTGTG
E2wtR 750 TATTCTCGAGCTCCCACTTAATGGCCCAG
E2rl 751 TATTCTCGAGCTCGGACCTGTCCCTGTC
E2r2 752 TATTCTCGAGCCGCGTCCAGTTGCAGGC
E2r2a 753 TATTCTCGAGGTTGCAGGCCGCTTCCAGC
E2r3 754 TATTCTCGAGGTAGTCGACCATGCACCTG
E2r4 755 TATTCTCGAGATGTTTGCGGAAGCAATCAG
E2r5 756 TATTCTCGAGCACCCCTCCGATGACACAAG
E2r6 757 TATTCTCGAGTGAGTTCATCCAGGTACAAC
E2r7 758 TATTCTCGAGCGGGCCACACACGCTCTTTG delHVR2F 759 TGCGGCTCTAGCGGATGCTGGCACTACCCTCCAAG delHVR2R 760 CAGCATCCGCTAGAGCCGCAGCTGGCCAACCTCTC delHVR3F 761 TGTGGAAGCTCTGGCTGCCCCACTGATTGCTTCC delHVR3R 762 GCAGCCAGAGCTTCCACAAGGGGGCGCTCCGCAC
The experimental conditions for generating the E2 mutant genes are shown below.
Table 18. Generation of E2 mutants by PCR and SOE-PCR
PCR Template Forward Reverse Product primer primer
1 pCV-H77c E2wtF E2wtR E2ΔTM
2 pCV-H77c E2flF E2rl E2(412-661)
3 pCV-H77c E2flF E2r2 E2(412-647)
4 pCV-H77c E2flF E2r2a E2(412-645)
5 pCV-H77c E2flF E2r3 E2(412-611)
6 pCV-H77c E2flF E2r4 E2(412-589)
7 pCV-H77c E2flF E2r5 E2(412-574)
8 pCV-H77c E2flF E2r6 E2(412-557)
9 pCV-H77c E2flF E2r7 E2(412-505)
10 pCV-H77c E2f2F E2r2a E2(456-645)
11 pCV-H77c E2f3F E2r2a E2(492-645)
12 pCV-H77c E2f4F E2r2a E2(506-645)
13 pCV-H77c E2f5F E2r2a E2(558-645)
14 pCV-H77c E2flF delHVR2R product#14
15 pCV-H77c delHVR2F E2r2a product#15 product#14 and 16 product# 15 E2flF E2r2a E2ΔN5
17 pCV-H77c E2flF delHVR3R product#17
18 pCV-H77c delHVR3F E2r2a product#18 product#17 and 19 product#18 E2flF E2r2a E2ΔN9
20 E2ΔN5 E2flF delHVR3R product#20 product#17 and
21 product#20 E2flF E2r2a E2ΔN5N9
PCR conditions: 94°C, 3 min; 25 cycles of (94°C, 30 s; 55°C, 30 s; 700C, 90 s); & 700C, 10 min; PCR system: Platinum Pfx DNA polymerase (Invitrogen); PCR instrument: GeneAmp PCR System 9700 (Applied Biosystems).
The PCR products generated in Table 18 were resolved by agarose gel electroporesis and the DNA bands of correct size were excised and purified. The products were either used as templates in a second PCR, or were digested with MIu I and Xho I restriction enzymes. The digested products were gel-purified and inserted between the BssH II and Xho I sites of the plasmid pCMV-Tag4A-tpaJR-FLgpl20 (Pantophlet et al., J Virol 77:642-658 (2003); Law et ai, J Virol 81 :4272-4285 (2007)). The inserted products replaced the HIV genes in the plasmid and are in frame with a 5 '-signal peptide and a 3'-FLAG tag to facilitate protein secretion and for detection. The nucleotide sequences of the E2 mutants were verified by DNA sequencing.
Expression of E2 mutants The E2 mutants were expressed by transient transfection of 293T cells. Cell monolayers were co-transfected with the expression plasmids encoding the different E2 mutants and pAdVAntage plasmid (Promega) at 1:1 ratio by polyethylenimine (Boxxssif et al., Proc Natl Acad Sd USA 92:7297-7301 (1995)). Cell supernatants were collected 3 days post-transfection and were clarified by centrifugation.
To identify E2 mutants correctly presenting the different conformation- dependent epitopes, a panel of monoclonal antibodies (MAbs) or the HCV co-receptor CD81 were used to capture the mutants in a capture ELISA. MAb ARIA, ARlB, AR2A, AR3A, AR3B, AR3C, AR3D or maltose binding protein tused-large extracellular loop of CD81 (CD81 -LEL) (Chan-Fook et a!., Virology 273 :60-66
(2000)) at 5 μg/mL were coated onto ELISA microwells overnight at 40C. After the microwells were blocked with 4% non-fat milk (Bio-Rad) and 0.05% Tween 20 in PBS, serially diluted transfected cell supernatants from above were added to the microwells for 1 hour at room temperature. Mutants with correctly folded antibody epitopes or CD81 -binding sites were captured by the corresponding reagents and the captured mutants were detected with a mouse anti-FLAG tag MAb (Sigma), followed by a secondary antibody (Peroxidase- conjugated AffiniPure Goat Anti-mouse IgG from Jackson ImmunoResearch Laboratories) and the colorimetic peroxidase substrate TMB (Pierce). The peroxidase reaction was stopped by adding sulfuric acid.
Specific binding of the E2 mutants to the capturing reagents were detected by measuring the absorbances of the samples at 450 nm using a microplate reader (Molecular Devices). The results are summarized in FIG.8. The CD81 -binding sites and AR3 are presented well on the E2 mutants E2ΔTM, E2flrl, E2flr2, E2flr2a, E2ΔN5 and E2ΔN9. The mutant E2ΔN5N9 was captured by MAbs AR3A or AR3C at a comparable level to the above mutants but at a much reduced level by CD81-LEL, MAbs AR3B or AR3D. In contrast, the mutants E2flr3, E2flr4, E2flr5, E2f2r2a and E2f3r2a were captured by the non-neutralizing MAbs ARIA and ARlB but not CD81-LEL or AR3-specific MAbs, suggesting that the CD81 -binding sites and the broadly neutralizing epitopes in AR3 are not present or folded correctly in these mutants. The fact that fragments E2flrl and E2flr2abind to the conformation- dependent, broadly neutralizing MAb AR3A and CD81-LEL indicates that the E2 residues 412-645 and cysteines 1-16 are important for correct folding of AR3 (within this region, residues 460-485 and 570-580 are not required). Of note, E2ΔTM binds all Abs recognizing ARl , 2 and 3, but weakly to CD81-LEL.
Example 4.
Purification of £2 Mutants
Generally, the HCV envelope El and E2 glycoproteins are technically challenging to produce as El does not fold properly in the absence of E2 (Michalak et al, J Gen Virol 78:2299-2306 (1997) and Patel et al, Virology 279:58-68 (2001)) and efficient production of E2 is influenced by El (Cocquerel et al., J Virol 77:10677- 10683 (2003), Brazzoli et al, Virology 332:438-453 (2005)). A truncated version of E2 (known as E2661) can be expressed independently and retain its function in binding to the co-receptor CD81 (Michalak et al., J Gen Virol 78:2299-2306 (1997); Flint et al., J Virol 73 :6235-6244 (1999); Flint et al, J Virol 74:702-709 (2000)). This truncated E2 has not been shown to be produced in a highly purified form suitable for biochemical analysis and crystallization attempts (Flint et al., J Virol 74:702-709 (2000)).
To purify E2 displaying correctly folded AR3 epitopes, a protein production and purification method was developed. The plasmids encoding the E2 mutants pE2ΔTM and pE2flr2a were co-transfected with pAdVAntage plasmid (Promega) at 1:1 ratio into FreeStyle 293 cells (Invitrogen) using 293fectin Transfection Reagent (Invitrogen). Cell supernatants were collected twice at 3-day and 5-day post- transfection. If necessary, kifunensine (at 7.5 μM, Cayman Chemical) (Elbein et al., J Biol Chem 265:15599-15605 (1990); Chang et al., Structure 15:267-273 (2007)) was added to cell culture media to improve glycan homogeneity on E2. The E2 mutants were purified by antibody affinity chromatography. To purify correctly folded E2 • mutants, the MAb AR3A, which can distinguish folded from misfolded protein, was used. The MAb AR3A recognizes a conformation-dependent epitope on E2, neutralizes HCV in vitro and offers protection against HCV infection in vivo as shown above. It also binds natively folded E2 at high affinity but not denatured and reduced E2. To prepare conjugated MAb AR3A-affinity matrix, MAb AR3A was first captured by Protein A-Sepharose (GE Healthcare) at a ratio of 10 mg MAb per mL Sepharose beads. After overnight incubation, the beads were washed 3 times with 0.2 M sodium borate buffer (pH 9). MAb AR3A was then crosslinked chemically to the Protein A-beads using dimethyl pimelimidate (Thermo Scientific). The reaction was stopped after lhour incubation at room temperature by pelleting the beads and washing the beads 3 times with 0.2 M ethanolamine (pH 8). The MAb-conjugated beads were packed into an Econo-Column (Bio-Rad) and the beads were rinsed once with 0.2 M glycine (pH 2.2) followed by PBS to equilibrate the column for affinity purification of the E2 mutants. Cell supernatants containing the E2 mutants were clarified by low-speed centrifugation and filtration through a 0.22-μm filter before loading onto the affinity columns by gravity flow. The flow-through solutions were collected and the columns were washed with PBS. Bound proteins were released from the affinity columns using different elution conditions and the antigenicity of the eluted proteins were investigated (see below). The eluants were concentrated and monomers of the E2 mutants were purified by size-exclusion chromatography using a Superdex 75 column (Amersham Biosciences).
Three batches of E2flr2a were produced by transient transfection of 293T cells (~5 x 108 cells per batch) with the corresponding expression plasmid. Batch 3 was produced in the presence of 10 μM kifunesine (BIOMOL), a potent inhibitor of the glycoprotein processing α-mannosidase I and is used to improve glycan homogeneity in the glycoproteins. Cell supernatants were loaded onto an antibody- affinity column (MAb AR3A, 5 mL) by gravity flow and bound proteins were eluted with a low pH buffer (0.2 M glycine, pH 2.7). Batches 2 and 3 was purified twice to monitor purification efficiency. Note that the majority of E2flr2a was isolated in the first round for these batches. The eluents were collected into tubes with 0.1 volume of neutralizing buffer (2 M Tris-HCl, pH 9). The eluants were analyzed by 4-15 % gradient SDS-PAGE (BIO-RAD). For Batches 1 and 2, monomelic E2flr2a was purified to greater than 90 % purity and has a similar apparent size (55-65 kDa) under both reducing and non-reducing conditions. For Batch 3, additional higher molecular weight impurities were found, which can probably be removed by a second chromatographic step. In the presence of kifunensine, the E2flr2a protein bands appear less diffused and more distinct, indicating that N-glycans on the Batch 3 recombinant proteins (glycoforms) are more homogeneous than that of Batches 1 and 2. The yields of E2flr2a in the three batches were approximately 1 mg.
E2flr2a was further purified and analyzed by size-exclusion chromatography. E2flr2a purified by MAb AR3A affinity column was concentrated to 0.5 mL using an ultra-centrifugal filter device with a 30 kDa nominal molecular weight limit (Millipore). The concentrated proteins were loaded onto a Sephadex 75 size- exclusion column (GE Healthcare) using a AKTA Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare). The proteins were separated in Tris buffer (0.1 M Tris-HCl pH 7.4 and 150 mM NaCl) and elution fractions of 0.5 mL were collected by an automatic fractionator. The chromatogram of E2flr2a was compared to the chromatogram of protein standards, including: (A) blue dextran 2000, (B) bovine serum albumin 67 kDa, (C) ovalbumin 43 kDa, and (D) chymotrypsinogen 25 kDa (GE Healthcare). Fractions 14-22 were analyzed by non-reducing SDS- PAGE (4-15% gradient, BIO-RAD). The results showed that the high molecular weight impurities eluted from the MAb AR3A-afflnity column were separated from the glycoforms of E2flr2a, which appear to be monomers of size between 43-67 kDa in gel filtration.
As an alternative to the procedure described above for purification of E2flr2a by MAb AR3A affinity chromatography using low pH elution buffer, additional experiments were performed wherein neutral and high pH elution buffers were utilized and the results were analyzed by SDS-PAGE. Using a neutral pH elution, E2flr2a was produced in the presence of kifunensine, loaded onto a MAb AR3A- affinity column and eluted with an increasing step-gradient of the chaotropic salt sodium thiocyanate (NaSCN). Reaction conditions included (1) E2flr2a eluted with low pH buffer as a control; (2) E2flr2a eluted with 0.5 M NaSCN; (3) 1 M NaSCN; and (4) 2 M NaSCN. The purified proteins were analyzed by non-reducing SDS- PAGE (4-15% gradient, BIO-RAD). The results show that neutral pH elution conditions can be used to purify the E2flr2a glycoforms, with the exception of elution with 0.5 M NaSCN, which led to high molecular weight impurities. Using high pH elution, E2flr2a was produced in the presence of kifunensine, loaded onto a MAb AR3A-affinity column and eluted with a step-gradient of buffers with increasing pH. The eluents were collected into tubes with 0.1 volume of neutralizing buffer (2 M Tπs-HCl, pH 7.4). Reaction conditions included (1) E2flr2a eluted with 2 M NaSCN as a control; (2) E2flr2a eluted with 0.2 M glycine pH 9.5; (3) pH 10.5; (4) pH 11.5; (5) pH 12.5; and (6) pH 11.5 sample filtered through an ultra-centrifugal filter device with a 100 kDa nominal molecular weight limit (Milhpore). The results show that both 2 M NaSCN and 0 2 M glycine pH 11.5 elution conditions led to pure E2flr2a protein.
E2ΔTM was purified using a MAb AR3A-conjugated affinity column with a 2M NaSCN, pH 7.4 elution and analyzed by SDS-PAGE. High molecular weight impurities were removed by filtering through an ultracentrifugal filter device with a 100 kDa nominal molecular weight limit (Milhpore). The purified proteins were analyzed by 4-15% gradient non-reducing SDS-PAGE (BIO-RAD). The results show that ultracentrifugal filtration removed high molecular weight impurities in samples produced in both the absence and presence of kifunensine. Protein concentration was quantified by the Bradford method (Bradford et al.,
AnalBiochem 72:248-254 (1976)) (Quick Start Bradford Dye Reagent, BioRad) or optical density reading at 280 nm based on calculated extinction coefficients listed in Table 19.
Table 19. Biochemical properties of E2 mutants
Molar 1 absorbance
Length Molecular extinction (280nm) corrected
E2 mutants (residues) weight (Da) Pi coefficient to (mg/mL)
1 E2ΔTM 344 38020 679 95330 04
2 E2(412661) 260 29124 663 75580 0 39
3 E2(412-647) 247 27563 791 75460 0 37
4 E2(412-645) 244 27119 756 69770 0 39
5 E2(412-611) 210 23036 644 58720 0 39
6 E2(412-589) 188 20565 644 50230 041
7 E2(412-574) 173 18864 585 49990 0 38
8 E2(412-557) 156 17276 547 49750 0 35
9 E2(412-505) 104 11594 583 29880 0 39
10 E2(456-645) 200 22213 757 56870 0 39
11 E2(492645) 164 18146 76 41410 0 44
12 E2(506-645) 151 16891 63 41170 041
13 E2(558-645) 99 11210 696 21300 0 53 14 E2ΔN5 222 24499 7.56 61520 0.4
15 E2ΔN9 237 26369 7.56 69770 0.38
16 E2ΔN5N9 215 23749 7.56 61520 0.39
Note: The properties were calculated using VectorNTI software (version 10, Invitrogen). Signal peptides and post-translational modifications of the mutants were excluded in the calculation
Three protein elution conditions, 0.2M glycine pH 2.2, 2M sodium thiocyanate (pH adjusted to pH 7.4 with 5OmM Tris-HCl) and 0.2M glycine pH 11.5, were examined, and the purified proteins were found to be essentially the same under the different conditions (FIGs. 9-10).
The recombinant E2 fragment E2flr2a can be purified to greater than 90 % by a single affinity chromatography step. In addition, the purification method is applicable to E2flr2a produced in the presence of the plant alkaloid kifunensine, a potent inhibitor of the glycoprotein processing α-mannosidase I. N-glycans on recombinant proteins produced in the presence of kifunensine are almost exclusively high-mannose type oligosaccharides, which can be readily trimmed by endoglycosidase H digestion to improve protein homogeneity.
Overall, the results show that the E2 mutants E2ΔTM and E2flr2a can be purified as monomers. The recombinant E2 fragments purified by the above method adopt a native fold as found on viral surface. The purified E2 mutants will be extremely useful in research and discovery of anti-viral drugs and HCV vaccines.
Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims

CLAIMS:
1. A mutant hepatitis C viral E2 polypeptide, the amino acid sequence of which comprises, from the amino terminus to the carboxy terminus: (1) a first segment that corresponds to amino acid residues 412 to 459 of the E2 polypeptide of a select hepatitis C virus (HCV), (2) a second segment that corresponds to amino acid residues 486 to 569 of the E2 polypeptide of the select hepatitis C virus, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the E2 polypeptide of the select hepatitis C virus, wherein: the segments are linked directly or via a polypeptide linker, with the proviso that the mutant polypeptide does not include a contiguous sequence of amino acids corresponding to all of amino acid residues 411 to 662 of the E2 polypeptide of the select hepatitis C virus; the mutant polypeptide has deletions of amino acid residues corresponding to amino acids 384 to 411 and amino acids 718 to 746 of the full-length E2 polypeptide of the select hepatitis C virus; and the mutant polypeptide can specifically bind an antibody that binds to a conformational epitope on the E2 polypeptide that contains at least amino acids 411 to 462.
2. A mutant E2 polypeptide of claim 1 that consists of (1) a first segment that corresponds to amino acid residues 412 to 459 of the E2 polypeptide of a select hepatitis C virus, (2) a second segment that corresponds to amino acid residues 486 to 569 of the E2 polypeptide of the select hepatitis C virus, and (3) a third segment that corresponds to amino acid residues 581 to 645 of the E2 polypeptide of the select hepatitis C virus, wherein: the segments are linked directly or via a polypeptide linker, with the proviso that the mutant polypeptide does not include a contiguous sequence of amino acids corresponding to all of amino acid residues 411 to 662 of the E2 polypeptide of the select hepatitis C virus; the mutant polypeptide has deletions of amino acid residues corresponding to amino acids 384 to 411 and amino acids 718 to 746 relative to the full-length E2 polypeptide of the select hepatitis C virus.
3. The mutant E2 polypeptide of claim 1 or 2, wherein the conformational epitope contains amino acids corresponding to amino acids 412 to 424, 436 to 447 and 523 to 540 of the select hepatitis C virus.
4. The mutant E2 polypeptide of any of claims 1 -3 that does not contain the contiguous amino acid residues that correspond to amino acid residues 460 to 485 and/or 570 to 580 of the E2 polypeptide of the select hepatitis C virus.
5. The mutant E2 polypeptide of any of claims 1 -4, wherein: the first and second segments are linked directly; or the second and third segments are linked directly to each other; or all three segments are linked directly.
6. The mutant E2 polypeptide of claim 5, wherein first and second segments are linked by a linker.
7. The mutant E2 polypeptide of any of claims 1 -4 or 6, wherein the linker located between the first and second segments contains 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues.
8. The mutant E2 polypeptide of claim 5, wherein the second and third segments are linked by a linker.
9. The mutant E2 polypeptide of any of claims 1-4 or 8, wherein the linker located between the second and third segments contains 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues.
10. The mutant E2 polypeptide of any of claims 1 -9, wherein the first and second segments are linked via a linker that is about 26 amino acid residues in length and the second and third segments are linked via a linker that is about 12 amino acid residues.
11. The mutant E2 polypeptide of any of claims 1-10, wherein the first and second segments are linked via a linker that comprises amino acids corresponding to amino acids 460 to 485 of the full-length E2 polypeptide of a select hepatitis C virus.
12. The mutant E2 polypeptide of any of claims 1-10, wherein the second and third segments are linked via a linker that comprises amino acids corresponding to amino acids 570 to 580 of the full-length E2 polypeptide of a select hepatitis C virus.
13. The mutant E2 polypeptide of any of claims 1-12, wherein the select hepatitis C virus is selected from among subtypes Ia, Ib, Ic, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p or 6t.
14. The mutant E2 polypeptide of any of claims 1-13, wherein the select 5 hepatitis C virus is selected from among subtypes Ia, Ib, or Ic.
15. The mutant E2 polypeptide of any of claims 1-14, wherein the select hepatitis C virus is selected from among H77, HCV-L2, or HCV-G9.
16. The mutant E2 polypeptide of any of claims 1-15, wherein the first segment is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %,
10 97 %, 98 %, or 99 % identical to amino acids 412 to 459 of hepatitis C virus H77.
17. The mutant E2 polypeptide of any of claims 1-16, wherein the second segment is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 486 to 569 of hepatitis C virus H77.
18. The mutant E2 polypeptide of any of claims 1-17, wherein the third
15 segment is 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identical to amino acids 581 to 645 of hepatitis C virus H77.
19. The mutant E2 polypeptide of any of claims 1-18, wherein the first amino acid segment has the sequence of any one of SEQ ID NOS: 888-912 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 %
20 sequence identity therewith.
20. The mutant E2 polypeptide of any of claims 1-19, wherein the second amino acid segment has the sequence of any one of SEQ ID NOS: 913-937 or has 65 %, 70 %, 75 %, 80 % 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
25 21. The mutant E2 polypeptide of any of claims 1 -20, wherein the third amino acid segment has the sequence of any one of SEQ ID NOS: 938-962 or has 65 %, 70 %, 75 %, 80 %> 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
22. The mutant E2 polypeptide any of claims 1 -21 that has the sequence of
30 any one of SEQ ID NOS: 727-730 and 740-742 or has 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % sequence identity therewith.
23. The mutant E2 polypeptide of any of claims 1 -22, further comprising a polypeptide tag.
24. The mutant E2 polypeptide of claim 23, wherein the tag is linked directly or indirectly to the amino terminus or carboxy terminus of the polypeptide.
25. The mutant E2 polypeptide of claim 24, wherein the polypeptide tag comprises a poly-histidine peptide, a FLAG (DYKDDDDK) peptide, an HA peptide, a myc peptide, a V5 peptide, a chitin binding protein peptide, a maltose binding protein peptide or a glutathione-S-transferase peptide.
26. An isolated nucleic acid molecule that encodes the mutant E2 polypeptide of any of claims 1-25.
27. The nucleic acid molecule of claim 26 operably linked to an expression control sequence.
28. The nucleic acid molecule of claim 27, wherein the expression control sequence is a promoter.
29. The nucleic acid molecule of claim 28, wherein the promoter is a viral promoter, a bacterial promoter, or a mammalian promoter.
30. The nucleic acid molecule of claim 29, wherein the promoter is a SV40 promoter, a Rous Sarcoma Virus promoter, or a cytomegalovirus immediate early promoter.
31. A vector, comprising the nucleic acid molecule of any of claims 26-30.
32. The vector of claim 31 that is a viral vector.
33. A cell, comprising the mutant E2 polypeptide of any of claims 1 -25.
34. A cell, comprising the nucleic acid of any of claims 26-30 or the vector of claim 31.
35. The cell of claim 33 or 34 that is a bacterial cell.
36. The cell of claim 33 or 34 that is a mammalian cell.
37. The cell of claim 36 that is a Chinese hamster ovary cell or a human embryonic kidney 293 cell.
38. A pharmaceutical composition, comprising a mutant E2 polypeptide of any of claims 1-25 and a pharmaceutically acceptable carrier.
39. A pharmaceutical composition, comprising the nucleic acid of any of , claims 26-30 or the vector of claim 31 and a pharmaceutically acceptable carrier.
40. A pharmaceutical composition, comprising the cell of claim 34 and a pharmaceutically acceptable carrier.
41. A vaccine or immunogenic composition, comprising a mutant E2 polypeptide of any of claims 1-25, the nucleic acid of any of claims 26-30, or the vector of claim 31.
42. Use of a mutant E2 polypeptide of any of claims 1-25 or the nucleic acid of any of claims 26-30 for the treatment or prevention of HCV infection in a subject.
43. Use of a mutant E2 polypeptide of any of claims 1 -25 or the nucleic acid of any of claims 26-30 for the preparation of a pharmaceutical composition for the treatment or prevention of HCV infection in a subject.
44. Use of a mutant E2 polypeptide of any of claims 1 -25 for the preparation of a vaccine for the prevention of HCV infection in a subject.
45. A method of eliciting an immune response in a subject, comprising administering to the subject the mutant E2 polypeptide of any of claims 1-25 or the pharmaceutical composition of claim 38.
46. The method of claim 45, wherein the mutant E2 polypeptide is in a pharmaceutical composition that comprises a pharmaceutically acceptable carrier.
47. The method of claim 45 or 46, wherein the subject is a mammal.
48. The method of claim 45 or 47, wherein the mammal is a mouse, sheep, goat, horse, rabbit, hamster, rat or human.
49. The method of claim 48, wherein the mammal is a human.
50. The method of any one of claims 45-49, further comprising obtaining a blood sample from the subject.
51. The method of any of claims 45-49, wherein the mutant E2 polypeptide is in an amount effective to prevent or treat a hepatitis C viral infection in the mammal.
52. The method of any of claims 45-51, further comprising administering to the mammal a second dose of the mutant E2 polypeptide at a selected time after the first administration.
53. The method of any one of claims 45-52, further comprising isolating an antibody or antibody-producing cell from the mammal, wherein the isolated antibody binds to a conformational epitope on an E2 polypeptide.
54. The method of claim 53, wherein the conformational epitope contains amino acids corresponding to 412-424, 436-447 and 523-540 of the select hepatitis C virus.
55. The method of claim 53 or 54, wherein the antibody is a neutralizing antibody.
56. The method of claim 55, wherein the antibody is a cross-neutralizing antibody.
57. The method of claim 53, further comprising fusing the antibody- producing cell from the mammal with a myeloma cell to obtain an antibody-producing hybridoma.
58. A method of eliciting an immune response in a mammal, comprising administering to the mammal the nucleic acid of any of claims 26-30, or the vector of claim 31.
59. The method of claim 58, wherein the nucleic acid or vector comprises a nucleic acid encoding a mutant E2 polypeptide of any of SEQ ID NOS: 866, 867, 868, 869 or 870.
60. The method of claim 58 or 59, wherein the nucleic acid has a sequence of nucleotides that comprises any one of SEQ ID NOS: 874, 875, 876, 877, 878, 879, 880 or 881.
61. A purified preparation of the mutant E2 polypeptide of any one of claims
1-25, wherein at least 80 % of the polypeptides are in a conformation capable of binding to a conformation-dependent or cross-neutralizing antibody.
62. A method for purifying a mutant E2 polypeptide of any one of claims 1 - 25, comprising: (1) contacting a sample that contains a mutant E2 polypeptide with a conformation-dependent antibody that binds specifically with the mutant E2 polypeptide of any one of claims 1-25 or with a conformational epitope on E2 under conditions effective for formation of a polypeptide-antibody complex; and
(2) separating the polypeptide-antibody complex from unrelated polypeptides in the sample.
63. The method of claim 62, wherein the conformational epitope contains amino acids corresponding to 412-424, 436-447 and 523-540 of the select hepatitis C virus.
64. The method of claim 62, further comprising separating the mutant E2 polypeptide from the antibody to obtain a preparation that comprises at least 50 % mutant E2 polypeptides by weight.
65. The method of claim 64, wherein the mutant E2 polypeptide is separated 5 from the antibody by elution with 0.2 M glycine at pH 2.2; 2M sodium thiocyanate at pH 7.4; or 0.2 M glycine at pH 11.5.
66. The method of any of claims 62-65 , further comprising purifying the mutant E2 polypeptide using size-exclusion chromatography.
67. The method of any of claims 62-66, wherein the purified mutant E2 10 polypeptide is a monomer.
68. A preparation obtained by the method of any one of claims 62-67, wherein at least 50 % of the polypeptides are in a conformation capable of binding to a conformation dependent antibody.
69. The preparation of claim 67, wherein at least 60 %, 65 %, 70 %, 75 %, 15 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96%, 97 %, 98 %, 99 % or more of the polypeptides in the preparation are in a conformation that can bind to a conformation dependent antibody.
70. The preparation of claim 68 or 69, wherein at least 60 %, 65 %, 70 %, 75 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96%, 97 %, 98 %, 99 % or more of
20 the polypeptides in the preparation are monomelic polypeptides.
71. The preparation of any one of claims 68-70, wherein the conformation- dependent antibody binds specifically to a conformational epitope that contains amino acids corresponding to 412-424, 436-447 and 523-540 of the select hepatitis C virus.
72. The preparation of claim 71 , wherein the conformational epitope 25 contains: (1) amino acids having the sequence
TAGLVGLLTPGAKQNIQLINTNGSWHINS (SEQ ID NO: 694); (2) amino acids having the sequence GWLAGLFYQHKF (SEQ ID NO: 695); and (3) amino acids having the sequence GAPTYSWGANDTDVFVLN (SEQ ID NO: 696).
73. The preparation of claim 71 , wherein the conformational epitope
30 contains: (1) amino acids having the sequence QLINTNGS WHINS (SEQ ID NO: 688); (2) amino acids having the sequence GWLAGLFYQHKF (SEQ ID NO: 695), and (3) amino acids having the sequence GAPTYSWGANDTD VFVLN (SEQ ID NO: 696).
74. A method for determining whether a mammal has been infected with a hepatitis C virus, comprising: contacting a blood sample from the mammal with the mutant E2 polypeptide of any one of claims 1-25; and determining whether the polypeptide binds specifically to an antibody from the blood of the mammal to form a polypeptide-antibody complex, wherein the presence of the complex indicates that the mammal has been infected with a hepatitis C virus and the absence of the complex indicates that the mammal has not been infected with the virus.
75. A method for identifying an anti-hepatitis C viral agent, comprising: contacting a candidate agent with the mutant E2 polypeptide of any one of claims
1-25; and if the candidate agent binds to the E2 polypeptide and prevents its binding with a cell receptor or prevents its ability to inhibit viral replication, identifying the candidate agent as an anti-hepatitis C viral agent.
76. The method of claim 75, wherein the cell receptor is CD81.
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