WO2014018904A1 - Fused antigen vaccines and compositions against streptococcus pneumoniae - Google Patents

Fused antigen vaccines and compositions against streptococcus pneumoniae Download PDF

Info

Publication number
WO2014018904A1
WO2014018904A1 PCT/US2013/052352 US2013052352W WO2014018904A1 WO 2014018904 A1 WO2014018904 A1 WO 2014018904A1 US 2013052352 W US2013052352 W US 2013052352W WO 2014018904 A1 WO2014018904 A1 WO 2014018904A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
pneumoniae
polypeptide
vaccine formulation
fusion protein
Prior art date
Application number
PCT/US2013/052352
Other languages
French (fr)
Inventor
Jean-Francois Lucien Maisonneuve
David E. Briles
Jon Scott Munzer
Original Assignee
Genocea Biosciences, Inc.
Path Vaccine Solutions
The Uab Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genocea Biosciences, Inc., Path Vaccine Solutions, The Uab Research Foundation filed Critical Genocea Biosciences, Inc.
Publication of WO2014018904A1 publication Critical patent/WO2014018904A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • Pneumococcal disease continues to be a leading cause of sickness and death in the United States and throughout the world. Each year, millions of cases of pneumonia, meningitis, bacteremia, and otitis media are attributed to infection with the pathogen
  • Streptococcus pneumoniae is a Gram-positive encapsulated coccus that colonizes the nasopharynx in about 5-10% of healthy adults and 20-40% of healthy children. Normal colonization becomes pathogenic when 5 * . pneumoniae is carried into the Eustachian tubes, nasal sinuses, lungs, bloodstream, meninges, joint spaces, bones, or peritoneal cavity.
  • S. pneumoniae has several virulence factors that enable the organism to evade the immune system. Examples include a polysaccharide capsule that prevents phagocytosis by host immune cells, proteases that inhibit complement-mediated opsonization, and proteins that cause lysis of host cells. In the polysaccharide capsule, the presence of complex
  • polysaccharides forms the basis for classifying pneumococci into different serotypes. To date, 93 serotypes of S. pneumoniae have been identified.
  • CPs capsular polysaccharides
  • anticapsular antibodies The antibodies raised against the S. pneumoniae capsular polysaccharides, termed anticapsular antibodies, are recognized as protective in adult and immunocompetent individuals. However, children under 2 years of age and
  • S. pneumoniae vaccine includes bacterial polysaccharides from 7 S. pneumoniae serovars conjugated to the diphtheria toxoid protein. This vaccine induces both B and T cell responses. However, because it only protects against 7 pneumococcal serotypes, serotype replacement can render Prevnar ineffective against non-vaccine serovars. Serotype replacement has already been demonstrated in several clinical trials and epidemiologic studies, necessitating development of different formulations of these vaccines.
  • polysaccharides has broader coverage, but does not provide protection to children under the age of 2 years, a population which is at the highest risk for pneumococcal disease.
  • Streptococcus pneumoniae is a major health concern, especially in very young, elderly, or immunocompromised patients. While DNA and protein sequence information for S. pneumoniae have been known for some time, and researchers have long attempted to produce vaccines against S. pneumoniae, a major challenge was how to elicit an immune response that is both long-lived and effective. Fusion proteins described herein exhibit surprising and unexpected properties, including eliciting immune responses that are long- lived and/or effective. Certain of the S. pneumoniae antigens included in fusion proteins described herein were initially identified by screening immune cells from mice either immunized with unencapsulated killed whole S. pneumoniae or infected with S.
  • the present disclosure provides, inter alia, certain highly effective vaccines against S. pneumoniae.
  • the vaccines may be used therapeutically and/or prophylactically.
  • the present disclosure also provides fusion proteins comprising specific antigens and methods for using such fusion proteins to elicit an immune response against 5 * . pneumoniae.
  • the present disclosure provides fusion proteins comprising or consisting of (i) one or more immunogenic polypeptides having or comprising the amino acid sequence of one of SEQ ID NOS: 1-8 or an immunogenic fragment or variant thereof; and (ii) one or more S. pneumoniae antigens that are predominantly an antibody target.
  • the immunogenic polypeptide is N-terminal to the S. pneumoniae antigen.
  • the immunogenic polypeptide is lipidated.
  • the immunogenic polypeptide has or comprises the amino acid sequence of SEQ ID NO: 2.
  • the immunogenic polypeptide has or comprises the amino acid sequence of SEQ ID NO: 6.
  • an immunogenic polypeptide of a fusion protein described herein is directly bonded to a S. pneumoniae antigen.
  • a fusion protein described herein further comprises a linker polypeptide.
  • a linker polypeptide is positioned between the immunogenic polypeptide and the S.
  • the linker polypeptide is rigid. In certain embodiments, the linker polypeptide comprises SEQ ID NO: 93. In some embodiments, the linker polypeptide is semi-rigid. In certain embodiments, the linker polypeptide comprises SEQ ID NO: 94.
  • the S. pneumoniae antigen of a fusion protein described herein is Pneumococcal surface protein A (PspA) or a derivative thereof.
  • the PspA derivative is or comprises all or a fragment of the proline-rich region of PspA.
  • the PspA derivative is or comprises a non-proline block.
  • the PspA derivative is or comprises SEQ ID NO: 29.
  • the PspA derivative is or comprises SEQ ID NO: 33.
  • the S. pneumoniae antigen of a fusion protein described herein is or comprises pneumolysin, a pneumolysoid, or a derivative thereof.
  • the present disclosure provides vaccine formulations comprising one or more fusion protein described herein and a pharmaceutically acceptable carrier.
  • a vaccine formulation comprises a fusion protein described herein and a second immunogenic polypeptide having or comprising an amino acid sequence of one of SEQ ID NOS: 1-11 or an immunogenic fragment thereof.
  • a vaccine formulation comprises a first fusion protein described herein and a second fusion protein comprising two immunogenic polypeptides, each having or comprising an amino acid sequence of one of SEQ ID NOS: 1-1 1 or an immunogenic fragment thereof.
  • a vaccine formulation described herein further comprises an adjuvant.
  • the adjuvant is an agonist of toll-like receptors (TLRs).
  • TLRs toll-like receptors
  • the adjuvant is alum.
  • a vaccine formulation described herein comprises 1 -
  • each fusion protein and/ or polypeptide 1000 ⁇ g of each fusion protein and/ or polypeptide and 25-500 ⁇ g of the adjuvant.
  • compositions and methods described herein are used for prophylaxis and/or treatment of any pneumococcal disease, disorder, and/or condition due to a pneumococcal infection.
  • a v ac c i n e composition described herein reduces risk of infection by, and/or treats, alleviates, ameliorates, relieves, delays onset of, inhibits progression of, reduces severity of, and/or reduces incidence of one or more symptoms or features of a pneumococcal disease, disorder, and/or condition.
  • prophylaxis and/or treatment of pneumococcal infection comprises administering a therapeutically effective amount of a v ac c in e composition described herein to a subject in need thereof, e.g., in such amounts and/or for such time as is necessary to achieve the desired result.
  • a "therapeutically effective amount" of a vaccine composition described herein is an amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of pneumococcal infection.
  • prophylactic, prognostic and/or therapeutic methods involve administering a therapeutically effective amount of one or more vacc ine compositions described herein to a subject such that an immune response is stimulated in one or both of T cells and B cells.
  • a vaccine formulation described herein is characterized in that, upon contacting a TH17 cell, the vaccine formulation induces a TH17 cell response at least 1.5-fold greater than that induced by a control (e.g., an unrelated antigen) after contacting TH17 cells.
  • a vaccine formulation described herein inhibits infection by S. pneumoniae in an uninfected subject (e.g., the vaccine formulation is characterized in that, upon administration to an uninfected subject, the vaccine formulation inhibits infection by S. pneumoniae in the uninfected subject).
  • a vaccine formulation described herein inhibits S.
  • a vaccine formulation described herein inhibits S. pneumoniae symptoms (e.g., the vaccine formulation is characterized in that, upon administration to a subject, the vaccine formulation inhibits S. pneumoniae symptoms in the subject).
  • a vaccine formulation described herein inhibits S. pneumoniae-m ' Jerusalem invasive disease (e.g., the vaccine formulation is characterized in that, upon administration to a subject, the vaccine formulation inhibits S. pneumoniae-m ' Jerusalem invasive disease in the subject).
  • the vaccine formulation inhibits S. pneumoniae-m ' Jerusalem sepsis (e.g., the vaccine formulation is characterized in that, upon administration to a subject, the vaccine formulation inhibits S. pneumoniae-m ' Jerusalem sepsis in the subject).
  • the present disclosure provides methods for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering to the subject an effective amount of a vaccine formulation described herein.
  • the subject is an uninfected subject, and the method inhibits infection by S. pneumoniae in the uninfected subject. In certain embodiments, the method inhibits S. pneumoniae colonization in a subject. In some embodiments, the method inhibits S. pneumoniae symptoms. In some embodiments, the method inhibits S. pneumonia- induced invasive disease. In certain embodiments, the method inhibits S. pneumoniae-m ' Jerusalem sepsis.
  • the method treats a subject with one dose. In certain embodiments, the method treats a subject within three doses. In some embodiments, the subject is a human.
  • FIG. 1 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then stimulated with killed, unencapsulated whole cell S. pneumoniae, as described in Example 1.
  • the left panel shows the data in scatter format, and the right panel shows the average and standard deviation for each sample.
  • Immunization group "All 3" represents animals immunized with a combination of SP2108, SP0148, and SP1634.
  • Example 2 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then stimulated with a combination of three proteins (SP2108, SP0148, and SP 1634), as described in Example 1.
  • FIG. 3 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then challenged with intranasal administration of S. pneumoniae, as described in Example 1.
  • 003 represents a control unrelated antigen.
  • FIG. 4 shows the number of S. pneumoniae colonies obtained from a nasal wash in BALB/c mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then challenged by intranasal administration of S. pneumoniae, as described in Example 2.
  • FIG. 5 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated proteins and cholera toxin adjuvant, then stimulated with the protein of immunization (A) or killed, unencapsulated whole cell S. pneumoniae (B), as described in Example 3.
  • FIG. 6 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and cholera toxin adjuvant, then challenged by intranasal administration of S. pneumoniae, as described in Example 4.
  • FIG. 7 shows survival of mice that were immunized with the indicated proteins and the adjuvant alum, then underwent aspiration challenge with 5 * . pneumoniae as described in Example 5.
  • FIG. 8 shows survival of mice that were immunized with the indicated proteins and the adjuvant alum, then underwent aspiration challenge with 5 * . pneumoniae as described in Example 6.
  • FIG. 9 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and cholera toxin adjuvant, then challenged by intranasal administration of S. pneumoniae, as described in Example 7.
  • FIG. 10 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated proteins and alum, then stimulated with the proteins indicated at upper left, as described in Example 8.
  • FIG. 1 1 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and alum or with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), then challenged by intranasal administration of S. pneumoniae, as described in Example 9.
  • FIG. 12 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and alum or with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), then challenged by intranasal administration of S. pneumoniae, in two pooled studies as described in Example 10.
  • FIG. 13 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and alum or with killed, unencapsulated whole cell S. pneumoniae plus alum (WCB), then challenged by intranasal administration of S. pneumoniae, as described in Example 1 1.
  • FIG. 14 shows survival of mice that were injected with antibodies or sera specific to the indicated proteins, then underwent aspiration challenge with S. pneumoniae, as described in Example 12.
  • FIG. 15 shows the percent of animals protected from sepsis in six separate aspiration challenge studies, two of which are described in more detail in Examples 6 and 12.
  • FIG. 16A, 16B, and 16C show the mean IL-17A response generated by blood samples from mice that were immunized with the indicated proteins and alum, then stimulated with the proteins indicated at upper right, as described in Example 13.
  • FIG. 17 shows the combined number of S. pneumoniae colonies, obtained in two separate studies, from nasal washes of mice that were immunized with the indicated fusion proteins adsorbed to alum, then challenged by intranasal administration of S.
  • FIG. 18 shows hours to moribund for mice that were immunized with the indicated fusion proteins adsorbed to alum, then challenged by intravenous administration of S pneumoniae, as described in Example 17.
  • FIG. 19A and 19B shows the concentration of IL- 17A generated by blood samples from mice that were immunized with the indicated fusion proteins adsorbed to alum, following stimulation with killed, unencapsulated whole cell S. pneumoniae, as described in Example 18.
  • FIG. 20 shows the total IgG concentration against CD2 or H70 in blood samples from mice immunized with the indicated fusion proteins adsorbed to alum, then challenged by intravenous administration of S pneumoniae, as described in Example 19.
  • This application describes vaccine formulations that include one or more S. pneumoniae fusion proteins, e.g., fusion proteins that include one or more S. pneumoniae polypeptides or fragments thereof.
  • the fusion proteins can include a first polypeptide that elicits primarily a T cell response and a second polypeptide that elicits primarily a B cell (e.g., antibody) response.
  • the first polypeptide elicits an immune response that is primarily a T cell response.
  • the second polypeptide elicits an immune response that is primarily a B cell response.
  • a suitable immunogenicity assay e.g., an assay described herein
  • more than 50%, 60%, 70%, 80%, 90%, or more, of the immune response elicited by the second polypeptide is a B cell response.
  • a fusion protein described herein includes one, two or more of the polypeptides listed (or polypeptides encoded by the genes listed) in Table 1 and/or 2.
  • a fusion protein includes a polypeptide listed (or encoded by a gene listed) in Table 1 fused to a polypeptide listed (or encoded by a gene listed) in Table 2.
  • a fusion protein comprises an immunogenic peptide having or comprising an amino acid sequence of one of SEQ ID NOS: 1-8, shown in Table 1, or an immunogenic fragment thereof; and a 5 * . pneumoniae antigen that is or comprises predominantly an antibody target.
  • the immunogenic peptide is SP2108 (SEQ ID NO: 6).
  • the immunogenic peptide is SP0148 (SEQ ID NO: 2).
  • a fusion protein includes a S. pneumoniae antigen that is or comprises predominantly an antibody target, e.g., an antigen shown in Table 2, e.g., PspA (SEQ ID NO: 27) or fragments or variants thereof.
  • fragments or variants of PspA comprise proline-rich segments with the non-proline block (PR+NPB), for example the CD2 sequence SEQ ID NO: 29.
  • fragments or variants of PspA comprise proline-rich segments with the non-proline block and 10, 20 30, 40 or more additional amino acids of PspA sequence, for example the H70 sequence (SEQ ID NO: 33).
  • the S. pneumoniae antigen that is predominantly an antibody target comprises pneumolysin or a pneumolysoid.
  • Fusion proteins described herein can include polypeptides homologous to the polypeptides of Tables 1 (for example, SP 1912, SP1912L, SP0148 with or without a signal sequence, SP2108 with or without a signal sequence) and Table 2 (for example, PspA and fragments and variants thereof).
  • Tables 1 for example, SP 1912, SP1912L, SP0148 with or without a signal sequence, SP2108 with or without a signal sequence
  • Table 2 for example, PspA and fragments and variants thereof.
  • Individual strains of S. pneumoniae contain numerous mutations relative to each other, and some of these result in different protein sequences between the different strains.
  • One of skill in the art may readily substitute an amino acid sequence, or a portion thereof, with the homologous amino acid sequence from a different S. pneumoniae strain.
  • a fusion protein described herein includes one or more polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to the polypeptides of Tables 1 and 2 or an immunogenic fragment thereof. Serotypic variation may be used to design such variants of the polypeptides of Tables 1 and 2.
  • a fusion protein described herein includes a fragment of a polypeptide of Table 1 (for example, fragments of SP 1912, SP1912L, SP0148 with or without a signal sequence, SP2108 with or without a signal sequence), and/or a fragment of a polypeptide of Table 2 (for example, PspA and variants thereof).
  • a fusion protein herein comprises one or more truncation mutants that are close in size to the polypeptides of Tables 1 and 2. For example, they may lack at most one, two three, four, five, ten, or twenty amino acids from one or both termini (referring to component polypeptides in a fusion protein).
  • the fragment is a truncated fragment of any of SEQ ID NOS: 1-8 or 27-36, wherein from 1-5, 1-10, or 1-20 amino acid residues are removed from the N-terminus, C-terminus, or both.
  • the fragment is a truncated fragment of any of SEQ ID NOS: 1-8 or 27-36, wherein from 1-10 amino acid residues are removed from the N-terminus, C-terminus, or both. For instance, 10 amino acid residues may be removed from each of the N-terminus and C-terminus resulting in a protein with 20 amino acid residues removed. Internal deletions, e.g., of 1-10, 1 1-20, 21- 30, or 31-40 amino acids, are also contemplated.
  • SP2108 including signal sequence 6 NC 003028.31:2020750- 2022021
  • the fusion protein comprises an N-terminal peptide and a C-terminal peptide.
  • the N-terminal peptide comprises the immunogenic polypeptide, for example, a polypeptide having an amino acid sequence of one of SEQ ID NOS: 1-8 or immunogenic fragments or variants thereof, and the C-terminal peptide comprises a 5 * . pneumoniae antigen that is predominantly an antibody target as described herein.
  • the N-terminal peptide and the C-terminal peptide are directly bound to each other.
  • the N-terminal peptide and the C- terminal peptide are linked via a linker peptide.
  • the length and/or amino acids of a linker when present, can be adjusted to obtain a more flexible or rigid linker.
  • Exemplary peptide linkers are shown as SEQ ID NOS: 92-94.
  • a linker can generally be from 1-40, such as 10- 30 and specifically 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.
  • the fusion protein comprises an immunogenic fragment of the fusion proteins shown in Table 3.
  • the fragment is a truncated fusion protein of any of SEQ ID NOS: 47-66.
  • they may lack at most one, two three, four, five, ten, or twenty amino acids from the N-terminus, C-terminus, or both.
  • the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus.
  • the N- terminal polypeptide is not truncated so as not to prevent lipidation.
  • this application provides fusion proteins with at least 90%
  • a vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 47-66, or an immunogenic fragment thereof.
  • vaccines or pharmaceutical compositions described herein include one or more fusion proteins containing at least one lipidated polypeptide.
  • the fusion protein is lipidated.
  • the fusion protein is lipidated on the N-terminal peptide. Conjugation to the lipid moiety may be direct or indirect (e.g., via a linker). The lipid moiety may be synthetic or naturally produced.
  • a polypeptide from Table 1, 2 or 3 may be chemically conjugated to a lipid moiety.
  • a construct may comprise a gene or polypeptide from Table 1, 2 or 3, or an immunogenic fragment or variant thereof, and a lipidation sequence including a lipobox motif.
  • a canonical lipobox motif is shown as SEQ ID NO: 89.
  • a lipidation sequence may be N-terminal or C-terminal to the protein, and may be embedded in a signal or other sequence, or in a fusion protein.
  • Exemplary lipidation sequences include the signal sequence of SP2108 (SEQ ID NO: 90) and the signal sequence of the E. coli gene RlpB (SEQ ID NO: 91).
  • a signal sequence may be, for example, an E. coli or S. pneumoniae signal sequence.
  • Exemplary E. coli signal sequences include the mlpA signal sequence (Lin, J.J. et al, "An Escherichia coli mutant with an amino acid alteration within the signal sequence of outer membrane prolipoprotein" Proc Natl Acad Sci U S A. 1978
  • Lpp is an exemplary E. coli signal sequence that directs lipidation (Cullen, P.A. et al.
  • E. coli signal sequences that direct lipidation are also described in Legrain, M. et al. ("Production of lipidated meningococcal transferrin binding protein 2 in Escherichia coli” Protein Expr Purif. 1995 Oct;6(5):570-8), e.g. the signal sequence of the gene RlpB (SEQ ID NO: 91) Numerous S. pneumoniae signal sequences are known in the art. One such signal sequence is SEQ ID NO: 90.
  • vaccines or pharmaceutical compositions described herein include a fusion protein comprising a polypeptide encoded by a polynucleotide from Table 1, 2, or 3, a polypeptide from Table 1, 2 or 3, or an immunogenic fragment or variant thereof, and a tag.
  • a tag may be N-terminal or C-terminal.
  • tags may be added to the nucleic acid or polypeptide to facilitate purification, detection, solubility, or confer other desirable characteristics on the protein or nucleic acid.
  • a purification tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification.
  • the polypeptide is free of tags such as protein purification tags, and is purified by a method not relying on affinity for a purification tag.
  • the fused portion is short. This, in some instances, the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids on one or both termini of the polypeptide of Table 1, 2 or 3.
  • this application provides nucleic acids encoding one or more of the fusion proteins described herein, such as DNA (e.g., cDNA), RNA, or an analog thereof.
  • DNA e.g., cDNA
  • RNA e.g., RNA
  • the underlying DNA sequences for the polypeptides described above may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention.
  • the DNA sequence may be codon-optimized to improve expression in a host such as E. coli, an insect cell line (e.g., using the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
  • this application provides polynucleotides (such as
  • the polynucleotide is 600-2000, 800-1800, 1000- 1600, 1200-1400 nucleotides in length. In some embodiments, the polynucleotide is 600- 1600, 800-1800, 1000-2000, 2000-3000, or 3000-4000 nucleotides in length.
  • the nucleic acids may be used, for example, for recombinant production of the polypeptides of Table 1 or 2, or the fusion proteins of Table 3, or immunogenic fragments thereof.
  • Polynucleotides encoding peptides of Tables 1, 2 or 3 or fragments thereof can be cloned into any of a variety of expression vectors, under the control of a variety of regulatory elements, and fusions can be created with other peptides of Tables 1, 2, or 3 or with other sequences of interest.
  • Methods of cloning nucleic acids are routine and conventional in the art.
  • isolating, cloning, modifying, labeling, manipulating, sequencing and otherwise treating or analyzing nucleic acids and/or proteins see, e.g., Sambrook, J. et al. (1989).
  • the fusion proteins include one, two or more of the polypeptides (or polypeptides encoded by the genes) listed in Table 1 and/or 2, or variants or fragments thereof as described below.
  • the fusion proteins contain one or more polypeptides from Table 1 and one or more polypeptides from Table 2.
  • the DNA and protein sequence of each gene and polypeptide may be found by searching for the Locus Tag in the publicly available database, Entrez Gene (on the NCBI NIH web site on the World Wide Web, at
  • SP0148 SEQ ID NO: 2 and variants thereof
  • the protein SP0148 is named "ABC transporter, substrate-binding protein”.
  • SP0148 is typically extracellular proteins that interact transiently with a transmembrane protein complex. Such complexes use energy generated by ATP hydrolysis to translocate specific substrates across a cell membrane.
  • SP0148 is a 276 or 277 (depending on the isolate) amino acid protein that contains a conserved PBPb (periplasmic binding protein) domain, spanning amino acids 40-246, which is typical of membrane-bound transport complexes.
  • PBPb peripheral binding protein domain
  • SP0148 has a bacterial extracellular solute-binding proteins family 3 domain which is largely co-extensive with the PBPb domain and extends from amino acid 40 to 244.
  • a vaccine or other composition comprises a truncation mutant of SP0148 comprising or lacking one or more of said domains and motifs.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a fusion protein containing a polypeptide containing at least 20 consecutive amino acid residues selected from SP0148.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 275, 250, 225, 200, 175, 150, 125, or 100 consecutive amino acids from SP0148.
  • Endogenous SP0148 comprises a signal sequence that directs its secretion and potential lipidation.
  • the signal sequence of the polypeptide of SEQ ID NO: 2 is partially or fully processed by an expression host, e.g. E. coli.
  • an expression host e.g. E. coli.
  • a variant of SP0148 that lacks the signal sequence (SEQ ID NO: 1) is used.
  • the polypeptide of SEQ ID NO: 1 is encoded by the nucleic acid of SEQ ID NO: 37, although other nucleic acid sequences (including codon-optimized sequences) may be used.
  • SEQ ID NO: 38 encodes the full length sequence of SP0148 used in the screens herein.
  • the vaccine formulation comprises a fusion protein that includes a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 3-4, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 1 or 2).
  • a fusion protein comprises or consists of an
  • immunogenic fragment of SP0148 include ALGLVAAGV (SEQ ID NO: 97), ELTGYEIEV (SEQ ID NO: 98), AVNNLSYTK (SEQ ID NO: 99), TYLPAEADI (SEQ ID NO: 100), RYNMAVNNL (SEQ ID NO: 101),
  • DFQQIMVRL (SEQ ID NO: 102), EHTDNPTIL (SEQ ID NO: 103), APIAQNPNV (SEQ ID NO: 104), LPSDQQPYV (SEQ ID NO: 105), YVYPLLAQG (SEQ ID NO: 106), QGLDNLKVI (SEQ ID NO: 107), KYLYAAPI (SEQ ID NO: 108), GELTGYEI (SEQ ID NO: 109), NPNVLVVKK (SEQ ID NO: 1 10), KLSKQFFGD (SEQ ID NO: 1 11),
  • GSPRPFIYE (SEQ ID NO: 1 12), AVNNLSYTK (SEQ ID NO: 1 13), KIFDKIGVE (SEQ ID NO: 1 14), MVRLSDGQF (SEQ ID NO: 1 15), YVYPLLAQG (SEQ ID NO: 116),
  • WQATTSAK (SEQ ID NO: 117), TLEKLSKQF (SEQ ID NO: 1 18), VAAGVLAAC (SEQ ID NO: 119), LDNLKVIEL (SEQ ID NO: 120), and NMAVNNLSY (SEQ ID NO: 121).
  • SP2108 (SEQ ID NO: 6) and variants thereof
  • the polypeptide SP2108 is 423 amino acids in length and is alternatively known as MalX, maltose/maltodextrin ABC transporter, or maltose/maltodextrin-binding protein. Much of the protein (amino acids 3-423) is classified as a MalE (Maltose-binding periplasmic) domain.
  • SP2108 contains a signal sequence that directs its secretion and potential lipidation.
  • the signal sequence of the polypeptide of SEQ ID NO: 6 is partially or fully processed by an expression host, e.g. E. coli.
  • a vaccine or other composition comprises a truncation mutant of SP2108 comprising one or more of said domains and motifs.
  • compositions and methods herein call for the use of an SP2108 variant that lacks the signal sequence.
  • This variant is represented by polypeptide sequence SEQ ID NO: 5 and may be encoded by, for example, a nucleic acid according to SEQ ID NO: 39, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) may be used.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a fusion protein containing a polypeptide containing at least 20 consecutive amino acid residues selected from SP2108.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids from SP2108.
  • the vaccine formulation comprises a fusion protein containing a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 7-8, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 5 or 6).
  • a fusion protein comprises or consists of an
  • immunogenic fragment of SP2108 include AIIDGPWKA (SEQ ID NO: 122), VMMAPYDRV (SEQ ID NO: 123), SIAGINYAK (SEQ ID NO: 124), VWDPAKNML (SEQ ID NO: 125), QPLPNISQM (SEQ ID NO: 126), APYDRVGSL (SEQ ID NO: 127), APAVIESLV (SEQ ID NO: 128), FYYTYGLLA (SEQ ID NO: 129), SKYAFAGE (SEQ ID NO: 130), TEGAGNLI (SEQ ID NO: 131),
  • LADWTNFYY (SEQ ID NO: 132), SLVMYYNKD (SEQ ID NO: 133), KEAGVKVTL (SEQ ID NO: 134), KSTAVLGTV (SEQ ID NO: 135), GAKTDDTTK (SEQ ID NO: 136), SQKFVDFLV (SEQ ID NO: 137), QAFKDAKVN (SEQ ID NO: 138), AVIESLVMY (SEQ ID NO: 139), DAKTAANDA (SEQ ID NO: 140), YGVATIPTL (SEQ ID NO: 141), KTAAIIDGP (SEQ ID NO: 142), KAYEKEAGV (SEQ ID NO: 143), AGNGAYVFG (SEQ ID NO: 144), and AWVIPQAVK (SEQ ID NO: 145).
  • SP 1912 is a hypothetical protein of 99 amino acids. While the protein function is not definitively known, sequence analysis suggests it is a putative thioredoxin.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP1912.
  • the polypeptide may also be a variant of the at least 20 residue fragment.
  • the polypeptide includes no more than 90, 75, 60, 45 or 30 consecutive amino acids from SP1912.
  • compositions and methods herein call for the use of an SP 1912 variant that comprises an exogenous lipidation sequence.
  • a signal sequence directs lipidation.
  • the lipidation signal may be, e.g., the signal sequence of SP2108 (SEQ ID NO: 90) or SP0148, or an E. coli signal sequence.
  • the exemplary variant SP1912L, comprising the signal sequence of the E. coli gene RlpB (SEQ ID NO: 91) is represented by polypeptide sequence SEQ ID NO: 10.
  • SP 1912 (SEQ ID NO: 9) and SP1912L (SEQ ID NO: 10) may be encoded, respectively, by nucleic acids according to SEQ ID NO: 40 and 41, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) may be used.
  • the lipidation sequence is provided by fusing a full-length polypeptide such as SP2108 (SEQ ID NO: 6) or SP0148 (SEQ ID NO: 2) to SP1912.
  • Exemplary fusions are SEQ ID NOS: 49-54.
  • the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, SEQ ID NO: 11, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising SEQ ID NO: 9).
  • a fusion protein comprises or consists of an
  • immunogenic fragment of SP1912 include KMWMAGLALLGIGSL (SEQ ID NO: 146), LLGIGSLALATKKVA (SEQ ID NO: 147), MAGLALLGIGSLALA (SEQ ID NO: 148), WMAGLALLGIGSLAL (SEQ ID NO: 149), GLALLGIG SL ALATK (SEQ ID NO: 150), LALLGIGSLALATKK (SEQ ID NO: 151), FSDMGEIATLYVQVY (SEQ ID NO: 152), KAKKMWMAGLALLGI (SEQ ID NO: 153), ALLGIGSLALATKKVAK (SEQ ID NO: 154), SDMGEIATLYVQVYE (SEQ ID NO: 155), DMGEIATLYVQVYES (SEQ ID NO: 156), AGLALLGIGSLALAT (SEQ ID NO: 157), MGEIATLYVQVYESS (SEQ ID NO: 158), KKMWMAG
  • the vaccine formulations provided herein comprise or further comprise one or more, or two or more, known S. pneumoniae antigens.
  • vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a fusion protein containing a known S. pneumoniae antigen.
  • the known 5 * . pneumoniae antigens are predominantly antibody targets.
  • the known S. pneumoniae antigens protect from S pneumoniae colonization, or from S pneumoniae-m ' Jerusalem sepsis.
  • PspA Pneumococcal surface protein A
  • derivatives of PspA is Pneumococcal surface protein A (PspA) (SEQ ID NO: 27) and derivatives of PspA.
  • H70 is one exemplary sequence which includes the proline-rich region and non-proline-block encompassing amino acids 290-410 of PspA.
  • An example of the non- proline-block has the exemplary sequence EKSADQQAEEDYARRSEEEYNRLTQQQ (SEQ ID NO: 34), which generally has no proline residues in an otherwise proline-rich area of the non-coiled region of PspA.
  • Other embodiments of non-proline block (NPB) sequences include SEQ ID NOS: 35 and 36 and PspA and its derivatives can include genes expressing similar proline-rich structures (i.e. PKP, PKEPEQ and PEKP), with or without the NPB.
  • amino acids at either end of the NPB mark the boundaries of the proline-rich region.
  • the amino-terminal boundary to the PR-region is DLKKAVNE (SEQ ID NO: 95)
  • the carboxy-terminal boundary is (K/G)TGW(K/G)QENGMW (SEQ ID NO: 96).
  • immunogenic PspA polypeptide derivatives containing the coiled-coil structure include SEQ ID NOS: 28 and 31. Particular embodiments of the immunogenic PspA polypeptide derivatives lacking the coiled-coil structure have the amino acid sequences shown as SEQ ID NOS: 29, 30 and 32.
  • Immunogenic PspA polypeptides SEQ ID NO: 28-30 include both PR and NPB sequences (PR+NPB). Immunogenic PspA polypeptides of SEQ ID NOS: 31 and 32 include only a PR sequence (PR only) and lack the NPB.
  • Pneumolysoids have homology to the S. pneumoniae protein pneumolysin (PLY), but have reduced toxicity compared to pneumolysin. Pneumolysoids can be naturally occurring or engineered derivatives of pneumolysin. In some embodiments, a pneumolysoid has at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to pneumolysin.
  • the pneumolysoid demonstrates less than 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500, or 1/1000 the toxicity of pneumolysin in an assay for one or both of hemolytic activity towards erythrocytes and inhibition of polymorphonuclear leukocytes. Both assays are described in Saunders F.K. et al. ("Pneumolysin, the thiol-activated toxin of
  • Streptococcus pneumoniae does not require a thiol group for in vitro activity" Infect Immun. 1989 Aug;57(8):2547-52).
  • Exemplary pneumolysoids include PdT (a triple mutant further described in Berry, A.M. et al. (1995) Infection and Immunity 63: 1969-74); Pd-A and Pd-B (Paton J.C. et al. "Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide" Infect Immun. 1991 Jul;59(7):2297-304); rPd2 and rPd3 (Ferreira et al.
  • DNA vaccines based on genetically detoxified derivatives of pneumolysin fail to protect mice against challenge with Streptococcus pneumoniae
  • FEMS Immunol Med Microbiol (2006) 46: 291-297 Ply8, A6PLY, L460D (see, e.g., US 2009/0285846 and L. Mitchell, Protective Immune Responses to Streptococcus pneumoniae Pneumolysoids, ASM2011 conference abstract, 201 1), or a variant thereof.
  • the pneumolysin has a mutation in the catalytic center, such as at amino acid 428 or 433 or the vicinity.
  • S. pneumoniae antigens include Choline-binding protein A
  • CbpA and derivatives thereof (AD Ogunniyi et ah, "Protection against Streptococcus pneumoniae elicited by immunization with pneumolysin and CbpA," Infect Immun. 2001 Oct;69(10):5997-6003); Pneumococcal surface adhesin A (PsaA); caseinolytic protease; sortase A (SrtA); pilus 1 RrgA adhesin; PpmA; PrtA; PavA; LytA; Stk-PR; PcsB; RrgB and derivatives thereof.
  • CpbA derivatives include fusion proteins described in WO 2012/134975.
  • Such fusion proteins may comprise one or more copies of the R2 domain, R2i and/or R3 ⁇ 4 subdomains of CpbA, or active variants and fragments thereof, or any combination thereof.
  • Such fusion proteins may further comprise a pneumolysoid.
  • the construct YLN for example, comprises CpbA polypeptides YPT and NEEK, and pneumolysoid L460D.
  • the antigen is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the corresponding wild-type S. pneumoniae protein.
  • Sequences of the above-mentioned polypeptides, and nucleic acids that encode them, are known; see, for example, the S. pneumoniae ATCC 700669 complete genome sequence under GenBank accession number FM21 1187.1 and linked polypeptide sequences therein.
  • fusion proteins described herein include polypeptides, and fragments and variants thereof, that are immunogenic. These polypeptides may be immunogenic in mammals, for example mice, guinea pigs, or humans.
  • An immunogenic polypeptide is typically one capable of raising a significant immune response in an assay or in a subject.
  • the immune response may be innate, adaptive, humoral, cell-mediated, or mucosal (combining elements of innate, adaptive, humoral and cell-mediated immunity).
  • an immunogenic polypeptide may increase the amount of IL-17 produced by T cells.
  • an immunogenic polypeptide may (i) induce production of antibodies, e.g., neutralizing antibodies, that bind to the polypeptide and/or the whole bacteria, (ii) induce T H 17 immunity, (iii) activate the CD4 + T cell response, for example by increasing CD4 + T cells and/or increasing localization of CD4 + T cells to the site of infection or reinfection, (iv) activate the CD8 + CTL response, for example by increasing CD8 + T cells and/or increasing localization of CD8 + T cells to the site of infection or reinfection, (v) induce THI immunity, and/or (vi) activate innate immunity.
  • an immunogenic polypeptide causes the production of a detectable amount of antibody specific to that antigen.
  • polypeptides have less than 20%, 30%, 40%, 50%,
  • human autoantigens and/or gut commensal bacteria e.g., certain Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus , Bifidobacterium, Escherichia and Lactobacillus species.
  • human autoantigens include insulin, proliferating cell nuclear antigen, cytochrome P450, and myelin basic protein.
  • a polypeptide may comprise one or more immunogenic portions and one or more non- immunogenic portions.
  • the immunogenic portions may be identified by various methods, including protein microarrays, ELISPOT/ELISA techniques, and/or specific assays on different deletion mutants (e.g., fragments) of the polypeptide in question.
  • Immunogenic portions may also be identified by computer algorithms. Some such algorithms, like
  • EpiMatrix produced by EpiVax
  • Other computational tools for identifying antigenic epitopes include PEPVAC (Promiscuous EPitope-based VACcine, hosted by Dana Farber Cancer Institute on the world wide web at
  • MHCPred which uses a partial least squares approach and is hosted by The Jenner Institute on the world wide web at www.jenner.ac.uk/MHCPred
  • Immune Epitope Database algorithms on the world wide web at www.jenner.ac.uk/MHCPred
  • An immunogenic fragment of a polypeptide described herein comprises at least one immunogenic portion, as measured experimentally or identified by algorithm.
  • this application provides a fusion protein that includes one or more immunogenic fragments of an antigen described herein.
  • the fragments in some instances, are close in size to the full-length polypeptide or the polypeptide of Table 1, 2 or 3. For example, they may lack at most one, two, three, four, five, ten, twenty, or thirty amino acids from one or both termini.
  • the polypeptide is 100-500 amino acids in length, or 150-450, or 200-400, or 250-250 amino acids in length.
  • the polypeptide is 100-200, 150-250, 200-300, 250-350, 300-400, 350-450, or 400-500 amino acids in length.
  • the fragments result from
  • an expression host e.g. E. coli, an insect cell line (e.g., the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
  • the fragments described above or sub-fragments thereof e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues
  • a mammalian cell line e.g., human or Chinese Hamster Ovary
  • the fragments described above or sub-fragments thereof e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues
  • a fragment may be used as the polypeptide in the vaccines described herein or may be fused to another protein, protein fragment or a polypeptide.
  • the fragment is a truncated fragment of any of SEQ ID NO: 1
  • NOS: 1-36 or 47-66 having from 1-5, 1-10, or 1-20 amino acid residues removed from the N-terminus, C-terminus, or both.
  • the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus.
  • this application provides fusion proteins that include one or more immunogenic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to a polypeptide of Table 1, 2 or 3.
  • the fusion protein comprises at least two different polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 1-36 or 47-66, or an immunogenic fragment thereof.
  • this application provides nucleic acids encoding one or more of the polypeptides described above, such as cDNA, DNA, RNA, or an analog thereof.
  • the underlying DNA sequences for the polypeptides described above may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention.
  • the DNA sequence may be codon-optimized to improve expression in a host such as E. coli, an insect cell line (e.g., using the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
  • this application provides nucleic acids (such as cDNA, DNA, RNA, or an analog thereof) that are at least 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a gene in Table 1, 2 or 3, or a variant or portion of said gene.
  • the nucleic acid is 600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In some embodiments, the nucleic acid is 600-1600, 800-1800, or 1000-2000 nucleotides in length.
  • the nucleic acids may be used, for example, for recombinant production of the polypeptides of Tables 1, 2 and 3, or immunogenic fragments or fusion proteins thereof.
  • the present disclosure also provides pharmaceutical compositions containing immunogenic polypeptides or polynucleotides encoding these immunogenic polypeptides, including fusion proteins, together with a pharmaceutical carrier.
  • the S. pneumoniae antigens used were identified by screening immune cells from mice either immunized with unencapsulated killed whole S. pneumoniae or infected with S. pneumoniae, or from healthy human donors, that elicit a T cell specific immune response. The human donors had almost certainly been exposed to S. pneumoniae at some point during their lifetimes, because S. pneumoniae is a very common disease and colonizing pathogen.
  • these antigens are fused to known S. pneumoniae capsular antigens, known to stimulate a B- cell specific immunity.
  • compositions of e.g., fusion proteins including) 5 * . pneumoniae antigens that elicit an immune response in all age groups that is strong and long-lived in immunized or infected mice or humans, for counteracting infection by S. pneumoniae.
  • a vaccine composition described herein induces a
  • TH17 cell response at least 1.5-fold greater than that induced by a control unrelated antigen (such as the HSV-2 protein ICP47 with the gene name US 12) after contacting TH17 cells.
  • a vaccine formulation described herein inhibits infection by S.
  • a vaccine formulation reduces occurrence or duration of S. pneumoniae nasopharyngeal colonization in an individual infected by S. pneumoniae. In some embodiments, a vaccine formulation inhibits development of sepsis in an individual infected by S. pneumoniae. In some embodiments, a vaccine formulation inhibits development of invasive diseases such as pneumonia, meningitis, otitis media, sinusitis or infection of other sites or organs with S. pneumoniae.
  • An immunogenic composition may also comprise portions of said
  • Streptococcus polypeptides including fusion proteins, for example deletion mutants, truncation mutants, oligonucleotides, and peptide fragments.
  • the portions of said polypeptides are immunogenic.
  • the immunogenicity of a portion of a protein is readily determined using the same assays that are used to determine the immunogenicity of the full-length protein.
  • the portion of the polypeptide has substantially the same immunogenicity as the full-length proteins.
  • the immunogenicity is no more than 10%, 20%, 30%, 40%, or 50% less than that of the full-length protein (e.g., polypeptides of Tables 1, 2 and 3).
  • the peptide fragments may be, for example, linear, circular, or branched.
  • a vaccine formulation contains one or more fusion proteins described herein in combination with one or more, e.g., two, three, four, or more polypeptides from Table 1 or 2 or immunogenic fragments or variants thereof in a mixture.
  • the mixture contains both full-length polypeptides and fragments resulting from processing, or partial processing, of signal sequences by an expression host, e.g. E. coli, an insect cell line (e.g., the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
  • a vaccine formulation contains one or more fusion proteins of any of SEQ ID NOS: 55-66 in the absence of any other antigens. In some embodiments, a vaccine formulation contains one or more fusion proteins of any of SEQ ID NOS: 55-66 in combination with one or more additional proteins of any of SEQ ID NOS: 1-26 and 47-54 for eliciting a T cell response, in the absence of other antigens.
  • polypeptides described in Tables 1 and 2 may be used without modification in conjunction with the fusion proteins described herein.
  • modification may be useful in eliciting the desired immune response.
  • the smaller polypeptides can be conjugated to an appropriate immunogenic carrier such as tetanus toxoid, keyhole limpet hemocyanin or the like.
  • polypeptides described herein may be conjugated to S. pneumoniae polysaccharides. The conjugated polysaccharides may be, for example, as described in US Patent 5,623,057, US Patent 5,371, 197, or PCT/US2011/023526.
  • compositions described herein include an immunogenic polypeptide, including a fusion protein (e.g., a polypeptide of Table 1, 2 or 3) that contains a membrane translocating sequence (MTS), to facilitate introduction of the polypeptide into the mammalian cell and subsequent stimulation of the cell-mediated immune response.
  • a fusion protein e.g., a polypeptide of Table 1, 2 or 3
  • MTS membrane translocating sequence
  • Exemplary membrane translocating sequences include hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of a-synuclein, ⁇ -synuclein, or ⁇ -synuclein, the third helix of the Antennapedia homeodomain, SN50, integrin ⁇ 3 h-region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and those MTSs described in US Patents 6,248,558, 6,432,680 and 6,248,558.
  • an antigen e.g., a polypeptide of Table 1, 2 or 3
  • an antigen is covalently bound to another molecule. This may, for example, increase the half-life, solubility, bioabailability, affinity, or immunogenicity of the antigen, or raise an immune reaction against more than one pathogen.
  • Molecules that may be covalently bound to the antigen include a carbohydrate, biotin, avidin or avidin derivatives, poly(ethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA.
  • PEG polypeptide described herein
  • a polypeptide described herein is covalently bound to a moeity that stimulates the immune system.
  • a moeity is a lipid moeity.
  • lipid moieties are recognized by a Toll-like receptor (TLR) such as TLR-2 or TLR-4, and activate the innate immune system.
  • TLR Toll-like receptor
  • a vaccine or immunogenic composition described herein comprises one or more fusion proteins described herein and one or more of the following: an adjuvant, vaccine scaffold, stabilizer, buffer, surfactant, controlled release component, salt, preservative, and/or an antibody specific to a polypeptide included in such composition.
  • the vaccine formulations and immunogenic compositions described herein may include an adjuvant.
  • Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g., Singh et ah, Curr. HIV Res. 1 :309-20, 2003).
  • the adjuvant provides a signal to the immune system so that it generates a response to the antigen, and the antigen is required for driving the specificity of the response to the pathogen.
  • Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune-stimulating complexes (ISCOMs), nanoparticles, which may be, for example, particles and/or matrices, and liposomes.
  • immunostimulatory adjuvants are sometimes derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system.
  • PAMP pathogen associated molecular patterns
  • LPS lipopolysaccharides
  • MPL monophosphoryl lipid
  • CpG-containing DNA which activate cells of the innate immune system.
  • adjuvants may be classified as organic and inorganic.
  • Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum
  • Organic adjuvants comprise organic molecules including macromolecules.
  • An example of an organic adjuvant is cholera toxin.
  • Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the activation of 3 ⁇ 41 cells or TH2 cells. In other embodiments, the adjuvant induces the activation of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.
  • the adjuvant induces the activation of T H 17 cells. It may promote the CD4 + or CD8 + T cells to secrete IL-17.
  • an adjuvant that induces the activation of TH17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL-17 levels secreted by two populations of cells: (1) cells from animals immunized with the adjuvant and a polypeptide known to induce TH17 activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide.
  • An adjuvant that induces the activation of TH17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2).
  • IL-17 may be measured, for example, by ELISA or ELISPOT.
  • the adjuvant is a toxin. Cholera toxin was successfully used in the mouse model to induce protective immunity in conjunction with certain polypeptides from Table 1 (see Examples 1-2). One form of labile toxin is produced by Intercell.
  • Mutant derivates of labile toxin that are active as adjuvants but significantly less toxic can be used as well.
  • Exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP-ribosyltransferase activity.
  • Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et ah, "Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants" PNAS Vol. 92, pp.
  • LTK63 Wang et al. "Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease” The Journal of Immunology, 2004, 173: 7435-7443), LT- G192 (Douce et al. "Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants" Infect Immun.
  • LTR72 Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. J Exp Med. 1998 Apr 6; 187(7): 1123-32).
  • the adjuvant comprises a VLP (virus-like particle).
  • Alphavirus replicons induces the activation of T H 17 cells using alphavirus and is produced by Alphavax.
  • alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL-17 or a cytokine that stimulates IL-17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Patent Nos. 5,643,576 and 6,783,939.
  • a vaccine formulation is administered to a patient in combination with a nucleic acid encoding a cytokine.
  • TLRs toll-like receptors
  • MPL Monophosphoryl Lipid A
  • MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from Gram negative bacteria, such as S. Minnesota.
  • LPS lipopolysaccharide
  • sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS.
  • TLR-4 agonists synthetic TLR agonists
  • TLR-4 ligands MPL adjuvant and Ribi.529 activate the innate immune system via TLR.
  • TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka ST et al. "E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant” Expert Rev. Vaccines 2007 Oct;
  • TLR agonists include TLR-4 agonists
  • TLR-4 agonists have been produced and/or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich.
  • Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and
  • TLR-activating adjuvant is R848 (resiquimod).
  • the adjuvant is or comprises a saponin.
  • the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree.
  • a saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity.
  • Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C.
  • a particular form of saponins that may be used in vaccine formulations described herein is immunostimulating complexes (ISCOMs).
  • ISCOMs are an art-recognized class of adjuvants, that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline).
  • an ISCOM is assembled together with a polypeptide or nucleic acid of interest.
  • different saponin fractions may be used in different ratios.
  • the different saponin fractions may either exist together in the same particles or have substantially only one fraction per particle (such that the indicated ratio of fractions A and C are generated by mixing together particles with the different fractions).
  • substantially refers to less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or even 1%.
  • Such adjuvants may comprise fraction A and fraction C mixed into a ratio of 70-95 A: 30-5 C, such as 70 A : 30 C to 75 A : 5 C, 75 A : 5 C to 80 A : 20 C, 80 A : 20 C to 85 A : 15 C, 85 A : 15 C to 90 A : 10 C, 90 A : 10 C to 95 A : 5 C, or 95 A : 5 C to 99 A : 1 C.
  • combinations of adjuvants are used.
  • Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.
  • Adjuvants may be covalently bound to antigens (e.g., to one or more polypeptides included in a fusion protein described herein).
  • the adjuvant may comprise a protein which induces inflammatory responses through activation of antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (see Wu et al, Cancer Res 2005; 65(1 1), pp 4947-4954).
  • a polypeptide, including a fusion protein, described herein is presented in the context of the trivalent conjugate system, comprising a fusion protein of S. pneumoniae Pneumococcal surface adhesin A (PsaA) with the pneumolysoid PdT and a cell wall polysaccharide (PsaA:PdT-CPs), described in Lu et al. ("Protection against Pneumococcal colonization and fatal pneumonia by a trivalent conjugate of a fusion protein with the cell wall polysaccharide.” Infect Immun. 2009 May;77(5):2076-83).
  • PdT carries three amino acid substitutions (W433F, D385N, and C428G) which render the molecule nontoxic but do not interfere with its TLR-4-mediated inflammatory properties. Conjugation of a
  • polysaccharide to the fusion of a polypeptide to the TLR-4-agonist PdT enhances
  • polypeptides described herein are used in place of PsaA in the trivalent conjugate.
  • the trivalent conjugate system typically includes alum and is usually administered parenterally.
  • Other exemplary adjuvants that may be covalently bound to antigens comprise polysaccharides, pneumolysin, synthetic peptides, lipopeptides, and nucleic acids.
  • the same adjuvant or mixture of adjuvants is present in each dose of a vaccine.
  • an adjuvant may be administered with the first dose of vaccine and not with subsequent doses (i.e., booster shots).
  • a strong adjuvant may be administered with the first dose of vaccine and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses.
  • the adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human patients, non-human animals, or both.
  • a vaccine formulation or immunogenic composition may include one or more additional components.
  • the vaccine formulation or immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L- glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
  • stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or mono
  • the vaccine formulation or immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid.
  • the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.
  • PBS phosphate buffered saline
  • the vaccine formulation or immunogenic composition includes one or more surfactants such as polysorbate 80 (Tween 80), Triton X-100,
  • Polyethylene glycol tert-octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-( 1, 1,3,3- Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100); Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN 20); and 4-(l, 1,3,3- Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL).
  • a surfactant can be ionic or nonionic.
  • the vaccine formulation or immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.
  • a preservative is included in the vaccine or immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.
  • the vaccine formulation or immunogenic composition is a controlled release formulation.
  • S. pneumoniae vaccines described herein may be used for prophylactic and/or therapeutic treatment of S. pneumoniae.
  • vaccine compositions may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce risk of infection by, and reduce severity of, and/or reduce incidence of one or more symptoms or features of a pneumococcal disease, disorder, and/or condition.
  • vaccine compositions may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of pneumococcal infection (e.g., but not limited to sepsis, pneumonia, acute otitis media, bacteremia, and bacterial meningitis).
  • pneumococcal infection e.g., but not limited to sepsis, pneumonia, acute otitis media, bacteremia, and bacterial meningitis.
  • vaccine compositions may be used to reduce the incidence of pneumococcal infection in subjects administered the composition. In some embodiments, vaccine compositions may be used to reduce the incidence of nasopharyngeal colonization by a pneumococcal organism. In some embodiments, vaccine compositions may be used to reduce the likelihood of chronic infection by a pneumococcal organism. In some embodiments, vaccine compositions may be used to reduce the likelihood of suffering from pelvic inflammatory disease or infertility subsequent to a pneumococcal infection.
  • prophylactic and/or therapeutic protocols involve administering a therapeutically effective amount of one or more vacc ine compositions de s c rib e d h e re in to a healthy subject (i.e., a subject who does not display any symptoms of, and/or who has not been diagnosed with, pneumococcal infection).
  • healthy individuals may be vaccinated using inventive vaccine compositions prior to pneumococcal colonization, development of pneumococcal infection and/or onset of symptoms of pneumococcal infection; at risk individuals (e.g., patients exposed to individuals suffering from pneumococcal infection, patients with HIV or a compromised immune system, or a subject who is susceptible is to pneumococcal infection such as a subject with Job's syndrome (subject lacking T H 17 cell-mediated response) or a subject with aggamaglobunemic (a subject lacking antibody-mediated response) can be treated substantially contemporaneously with (e.g., within 48 hours, within 24 hours, or within 12 hours of) the onset of symptoms of and/or exposure to pneumococcal infection.
  • individuals known to have pneumococcus infection or sepsis may receive treatment at any time.
  • prophylactic and/or therapeutic protocols involve administering a therapeutically effective amount of one or more inventive vaccine compositions to a subject such that an immune response is stimulated in both T cells and B cells.
  • immune responses e.g. T cell and/or B cell responses
  • T cell and/or B cell responses can be tailored to preferentially elicit the most desirable type of immune response for a given indication, e.g., humoral response, mucosal response, T H l-biased T cell response, T H 17-biased T cell response, IFN- ⁇ secretion by antigen-specific T cells, IL- 17A secretion by antigen-specific T cells, cytotoxic T cell response, antibody response, B cell response, ad aptiv e immune re sp o n s e , innate immune response, or a combination of these responses .
  • this application provides a method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering an effective amount of any of the vaccine formulations described herein.
  • the method inhibits S. pneumoniae colonization in an individual.
  • the method inhibits S. pneumoniae symptoms, invasive disease or sequelae, such as sepsis, pneumonia, meningitis, otitis media, sinusitis or infection of other sites or organs with S. pneumoniae.
  • the subject receiving the vaccination may be a male or a female, and may be a child or adult.
  • the subject being treated is a human.
  • the subject is a non-human animal. 1. Prophylactic use
  • the vaccine is administered to a subject to induce an immune response that can help protect against the establishment of S. pneumoniae, for example by protecting against colonization, the first and necessary step in disease.
  • the method inhibits infection by S. pneumoniae in a non-colonized or uninfected subject.
  • the method may reduce the duration of colonization in an individual who is already colonized.
  • the vaccine compositions described herein confer protective immunity, allowing a vaccinated individual to exhibit delayed onset of symptoms or sequelae, or reduced severity of symptoms or sequelae, as the result of his or her exposure to the vaccine.
  • the reduction in severity of symptoms or sequelae is at least 25%, 40%, 50%, 60%, 70%, 80% or even 90%.
  • vaccinated individuals may display no symptoms or sequelae upon contact with S.
  • Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts.
  • the sIGA antibodies are generated after a series of events, mediated by antigen-processing cells, B and T lymphocytes, that results in sIGA production by B lymphocytes on mucosa- lined tissues of the body.
  • Humoral immunity is typically the result of IgG antibodies and IgM antibodies in serum.
  • Cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies.
  • cellular immunity may be mediated by THI or TH17 cells.
  • a vaccine formulation as described herein is administered to patients that are immunocompromised.
  • An immunocompromising condition e.g., arising from a medical treatment, can expose the individual in question to a higher risk of infection with 5 * . pneumoniae. It is possible to treat an infection prophylactically in an individual having an
  • an antigenic composition e.g., a fusion protein described herein
  • an antigenic composition e.g., a fusion protein described herein
  • antibodies reactive to two or more antigens from Table 1 or 2, or to an antigen from Table 3, before or during a treatment known to compromise immune function it is possible to prevent a subsequent S. pneumoniae infection or to reduce the risk of the individual contracting an infection due to the immunocompromised condition.
  • an antigenic composition e.g., a fusion protein described herein
  • antibodies reactive to two or more antigens from Table 1 or 2 or to an antigen from Table 3, before or during a treatment known to compromise immune function it is possible to prevent a subsequent S. pneumoniae infection or to reduce the risk of the individual contracting an infection due to the immunocompromised condition.
  • the individual contract an 5 * . pneumoniae infection e.g., following a treatment leading to an immunocompromised condition it is also possible to treat the infection by administering to the individual an antigen composition.
  • the following groups are at increased risk of pneumococcal disease or its complications, and therefore it is advantageous for subjects falling into one or more of these groups to receive a vaccine formulation described herein: children, especially those from 1 month to 5 years old or 2 months to 2 years old; children who are at least 2 years of age with asplenia, splenic dysfunction or sickle-cell disease; children who are at least 2 years of age with nephrotic syndrome, chronic cerebrospinal fluid leak, HIV infection or other conditions associated with immunosuppression.
  • At least one dose of a vaccine composition described helrein is given to adults in the following groups at increased risk of pneumococcal disease or its complications: all persons 65 years of age; adults with asplenia, splenic dysfunction or sickle-cell disease; adults with the following conditions: chronic cardiorespiratory disease, cirrhosis, alcoholism, chronic renal disease, nephrotic syndrome, diabetes mellitus, chronic cerebrospinal fluid leak, HIV infection, AIDS and other conditions associated with immunosuppression (Hodgkin's disease, lymphoma, multiple myeloma, immunosuppression for organ transplantation), individuals with cochlear implants; individuals with long-term health problems such as heart disease and lung disease, as well as individuals who are taking any drug or treatment that lowers the body's resistance to infection, such as long-term steroids, certain cancer drugs, radiation therapy; Alaskan natives and certain Native
  • a vaccine formulation described herein may be administered to a patient suffering from 5 * . pneumoniae infection, in an amount sufficient to treat the patient.
  • Treating the patient refers to reducing S. pneumoniae symptoms and/or bacterial load and/or sequelae in an infected individual.
  • treating the patient refers to reducing the duration of symptoms or sequelae, or reducing the intensity of symptoms or sequelae.
  • treating the patient refers to eliminating pneumococcal organisms in the nasopharynx or reducing the duration of nasopharyngeal colonization.
  • the vaccine reduces transmissibility of S. pneumoniae from the vaccinated patient.
  • the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or even 90%.
  • a vaccine is administered to an individual postinfection.
  • the vaccine may be administered shortly after infection, e.g. before symptoms or sequelae manifest, or may be administered during or after manifestation of symptoms or sequelae.
  • a vaccine formulation described herein can reduce the intensity and/or duration of the various symptoms or sequelae of S. pneumoniae infection.
  • Symptoms or sequelae of S. pneumoniae infection can take many forms.
  • an infected patient develops pneumonia, acute sinusitis, otitis media (ear infection), meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brain abscess.
  • Sepsis is a rare but life-threatening complication of S. pneumoniae infection, where the bacterium invades the bloodstream and systemic inflammation results. Typically, fever is observed and white blood cell count increases.
  • a further description of sepsis is found in Goldstein, B. et al. "International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics.” Pediatr Crit Care Med. Jan 2005;6 (l):2-8. 3. Assaying vaccination efficacy
  • the efficacy of vaccination with vaccines disclosed herein may be determined in a number of ways, in addition to the clinical outcomes described above.
  • IL-17 levels particularly IL-17A
  • the IL-17 levels may be compared to IL-17 levels in the same subject before vaccination.
  • Increased IL-17 (e.g., IL-17 A) levels such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine.
  • IL-17 A e.g., IL-17 A
  • neutrophils in the presence of T cells or antibodies from the patient for pneumococcal killing.
  • Increased pneumococcal killing such as a 1.5 fold, 2-fold, 5-fold, 10- fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine.
  • One may also measure levels of an antibody specific to the vaccine where increased levels of the specific antibody, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, are correlated with increased vaccine efficacy. In certain embodiments, two or more of these assays are used.
  • additional cytokines and transcription factors in the IL-17 lineage e.g., cytokines IL-22, IL-23, 11-6 and TGF-B, and transcription factors STAT3, RORyt and RORa.
  • epidemiological markers such as incidence of, severity of, or duration of pneumococcal infection in vaccinated individuals compared to unvaccinated individuals.
  • Vaccine efficacy may also be assayed in various model systems such as the mouse model. For instance, BALB/c or C57BL/6 strains of mice may be used. After administering a vaccine formulation described herein to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of S. pneumoniae. In some cases, a challenge dose administered intranasally is sufficient to cause S. pneumoniae colonization (especially nasal colonization) in an unvaccinated animal, and in some cases a challenge dose administered via aspiration is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. One can then measure the reduction in colonization or the reduction in lethality in vaccinated animals.
  • Examples 1-2 and 4 show the efficacy of polypeptides of Table 1 in inhibiting 5 * . pneumoniae nasal colonization following intranasal challenge in the mouse model.
  • Examples 5 and 6 show the efficacy of polypeptides of Table 1 in protecting against sepsis and death following infection with 5 * . pneumoniae via aspiration in the mouse model.
  • compositions described herein e.g., ones comprising one or more fusion proteins described herein
  • compositions described herein may stimulate an antibody response or a cell-mediated immune response, or both, in the mammal to which it is administered.
  • the composition stimulates a 3 ⁇ 41 -biased CD4 + T cell response, a T H 17-biased CD4 + T cell response and/or a CD8 + T cell response.
  • the composition elicits IFN- ⁇ secretion by antigen-specific T cells.
  • the composition elicits IL-17A secretion by antigen-specific T cells.
  • the composition stimulates an antibody response (e.g., an IgG, IgM and/or IgA response).
  • the composition stimulates a B cell response.
  • the composition stimulates a cytotoxic T cell (CTL) response.
  • CTL cytotoxic T cell
  • the composition stimulates an adaptive immune response.
  • the composition stimulates an innate immune response.
  • the composition stimulates both a T cell response and a B cell response.
  • the composition stimulates a T H l-biased CD4 + T cell response, T H 17-biased CD4 + T cell response and/or a CD8 + T cell response, and an antibody and/or B cell response.
  • the composition (e.g., one comprising one or more fusion proteins described herein) includes a cytokine or nucleotide coding region encoding a cytokine such as IL- 17, to provide additional stimulation to the immune system of the mammal.
  • the composition comprises a cytokine such as IL-17.
  • a T H 17 cell response is desirable in mounting an immune response to the compositions disclosed herein.
  • an active TH17 response is beneficial in clearing a pneumococcal infection. For instance, mice lacking the IL-17A receptor show decreased whole cell vaccine-based protection from a pneumococcal challenge (Lu et ah, "Interleukin-17A mediates acquired immunity to pneumococcal colonization.” PLoS Pathog. 2008 Sep 19;4(9)).
  • the present disclosure includes methods of increasing IL-17 production by administering one or more compositions described herein (e.g., ones comprising one or more fusion proteins described herein) to a subject.
  • one or more compositions described herein e.g., ones comprising one or more fusion proteins described herein
  • the present disclosure includes methods of activating T H 17 cells by
  • IL-17A levels result in increased pneumococcal killing by neutrophils or neutrophil-like cells, for instance by inducing recruitment and activation of neutrophils of neutrophil-like cells.
  • this pneumococcal killing is independent of antibodies and
  • Immunogenic compositions containing one or more fusion proteins described herein (or nucleic acids encoding a fusion protein described herein) together with a pharmaceutical carrier are also provided.
  • the immunogenic composition comprises one or more nucleic acids (e.g., cDNA molecules) encoding one or more polypeptides of SEQ ID NOS: 1- 66, such as one or more polypeptides selected from SEQ ID NOS 55-66.
  • these nucleic acids are expressed in the immunized individual, producing the encoded S. pneumoniae antigens, and the S. pneumoniae antigens so produced can produce an immunostimulatory effect in the immunized individual.
  • Such a nucleic acid-containing immunostimulatory composition may comprise, for example, an origin of replication, and a promoter that drives expression of one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 55-66.
  • Such a composition may also comprise a bacterial plasmid vector into which is inserted a promoter (sometimes a strong viral promoter), one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 55-66, and a polyadenylation/transcriptional termination sequence.
  • the nucleic acid is DNA.
  • the amount of a fusion protein described herein in each vaccine or immunogenic composition dose is selected as an effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses.
  • the dose is without significant adverse side effects in typical vaccinees.
  • Such amount will vary depending upon which specific antigen is employed.
  • a dose will comprise 1-1000 ⁇ g of each protein, in some instances 2-100 ⁇ g, for instance 4-40 ⁇ g.
  • the vaccine formulation comprises 1-1000 ⁇ g of the polypeptide and 1-350 ⁇ g of the adjuvant.
  • the appropriate amount of antigen to be delivered will depend on the age, weight, and health (e.g. immune-compromised status) of a subject.
  • typically an adjuvant will be present in amounts from 1 ⁇ g - 350 ⁇ g per dose, for example 50-150 ⁇ g, 75-125 ⁇ g or 100 ⁇ &
  • only one dose of the vaccine is administered to achieve the results described above.
  • subjects receive one or more boost vaccinations, for a total of two, three, four or five vaccinations.
  • the number is three or fewer.
  • a boost vaccination may be administered, for example, about 1 month, 2 months, 4 months, 6 months, or 12 months after the initial vaccination, such that one vaccination regimen involves administration at 0, 0.5-2 and 4-8 months. It may be advantageous to administer split doses of vaccines which may be administered by the same or different routes.
  • the vaccines and immunogenic compositions described herein may take on a variety of dosage forms.
  • the composition is provided in solid or powdered (e.g., lyophilized) form; it also may be provided in solution form.
  • a dosage form is provided as a dose of lyophilized composition and at least one separate sterile container of diluent.
  • the composition will be administered in a dose escalation manner, such that successive administrations of the composition contain a higher concentration of composition than previous administrations. In some embodiments, the composition will be administered in a manner such that successive administrations of the composition contain a lower concentration of composition than previous administrations.
  • compositions are administered to a patient suffering from a disease in an amount sufficient to treat the patient.
  • Therapeutic applications of a composition described herein include reducing transmissibility, slowing disease progression, reducing bacterial viability or replication, or inhibiting the expression of proteins required for toxicity, such as by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the levels at which they would occur in individuals who are not treated with the composition.
  • compositions are administered to a human or other mammal to induce an immune response that can inhibit the establishment of an infectious disease or other condition.
  • a composition may partially block the bacterium from establishing an infection.
  • a therapeutically effective amount may be determined on a population basis, and is not required to be an amount that naturally induces a protective response in a particular subject.
  • the compositions are administered in combination with antibiotics.
  • antibiotics are used to treat pneumococcal infections, including penicillin, amoxicillin, amoxicillin/clavulanate, cefuroxime, cefotaxime, ceftriaxone, and vancomycin.
  • the appropriate antibiotic may be selected based on the type and severity of the infection, as well as any known antibiotic resistance of the infection (Jacobs MR "Drug- resistant Streptococcus pneumoniae: rational antibiotic choices" Am J Med. 1999 May 3; 106(5A): 19S-25S).
  • compositions are administered in combination with one or more additional vaccines, such as Prevnar-13 (Wyeth Pharmaceuticals, Inc., Philadelphia, PA) or Pneumovax (Merck & Co., Inc., Whitehouse Station, NJ ) .
  • additional vaccines such as Prevnar-13 (Wyeth Pharmaceuticals, Inc., Philadelphia, PA) or Pneumovax (Merck & Co., Inc., Whitehouse Station, NJ ) .
  • This coadministration can be used, for example, to treat a patient who has been exposed to, colonized or infected by S. pneumoniae serotypes that are not represented in said existing vaccine.
  • the vaccine formulations and pharmaceutical compositions herein can be delivered by administration to an individual, typically by systemic administration (e.g., by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, or buccal routes; by inhalation; or intratracheal or bronchial instillation) or they can be administered by topical application.
  • the route of administration is intramuscular.
  • the route of administration is subcutaneous.
  • the route of administration is mucosal.
  • the route of administration is transdermal or intradermal.
  • transdermal administration is one suitable route of administration for 5 * . pneumoniae vaccines comprising toxins (e.g. cholera toxin or labile toxin); in other embodiments, the toxins (e.g. cholera toxin or labile toxin); in other embodiments, the toxins (e.g. cholera toxin or labile toxin); in other embodiments, the toxins (e.g. cholera toxin or labile toxin); in other embodiments, the
  • Vaccines formulated with Alphavirus replicons may be administered, for example, by the intramuscular or the subcutaneous route.
  • Vaccines comprising aluminum hydroxide or aluminum phosphate (alum), Monophosphory Lipid A (MPL), Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA) are suitable (inter alia) for intramuscular and subcutaneous administration.
  • a vaccine comprising resiquimod may be administered topically or subcutaneously, for example.
  • the vaccine formulation or immunogenic composition may be suitable for administration to a human patient, and vaccine or immunogenic composition preparation may conform to USFDA guidelines.
  • the vaccine formulation or immunogenic composition is suitable for administration to a non-human animal.
  • the vaccine or immunogenic composition is substantially free of either endotoxins or exotoxins. Endotoxins may include pyrogens, such as lipopolysaccharide (LPS) molecules.
  • LPS lipopolysaccharide
  • the vaccine or immunogenic composition may also be substantially free of inactive protein fragments which may cause a fever or other side effects.
  • the composition contains less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/or inactive protein fragments.
  • the vaccine or immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water. Other vaccine or
  • immunogenic composition components may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation.
  • the pyrogens may be inactivated or destroyed prior to administration to a patient.
  • Raw materials for vaccines such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in the vaccine may be sterile, and each lot of the vaccine may be tested for sterility.
  • the endotoxin levels in the vaccine fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU kg of product for a non- intrathecal injectable composition, and 0.25-0.5 EU/mL for sterile water.
  • EU endotoxin
  • the preparation comprises less than 50%, 20%, 10%, or 5% (by dry weight) contaminating protein.
  • a fusion protein described herein is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture).
  • the vaccine or immunogenic composition comprising purified subunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of protein from host cells in which the subunit proteins were expressed, relative to the amount of purified subunit.
  • the desired polypeptides are substantially free of nucleic acids and/or carbohydrates.
  • the vaccine or immunogenic composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNA and/or RNA.
  • At least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of biological macromolecules of the same type are present in the preparation (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • the vaccine or immunogenic composition has low or no toxicity, within a reasonable risk-benefit ratio.
  • the vaccine or immunogenic composition comprises ingredients at concentrations that are less than LD5 0 measurements for the animal being vaccinated. LD50 measurements may be obtained in mice or other experimental model systems, and extrapolated to humans and other animals.
  • a vaccine formulation or immunogenic composition might have an LD5 0 value in rats of greater than 100 g/kg, greater than 50g/kg, greater than 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2 g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200 mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20 mg/kg, or greater than 10 mg/kg.
  • a vaccine formulation or immunogenic composition that comprises a toxin such as botulinum toxin (which can be used as an adjuvant) should contain significantly less than the LD5 0 of botulinum toxin.
  • formulations suitable for introduction of the vaccine formulations or pharmaceutical composition vary according to route of administration.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the cells can be administered intravenously or parenterally.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of a fusion protein described herein or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount of a fusion protein described herein or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400
  • diluents such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • the pharmaceutical compositions can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.
  • a fusion protein described herein, alone or in combination with other suitable components, can be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
  • pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
  • Suitable formulations for vaginal or rectal administration include, for example, suppositories, which consist of the polypeptides or packaged nucleic acids with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the polypeptides or packaged nucleic acids with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • the S. pneumoniae vaccines and immunogenic compositions described herein may be produced using a variety of techniques.
  • a polypeptide e.g., a fusion protein
  • a suitable host cell may be bacterial, yeast, mammalian, or other type of cell.
  • the host cell may be modified to express an exogenous copy of one of the relevant polypeptide genes.
  • the gene is operably linked to appropriate regulatory sequences such as a strong promoter and a polyadenylation sequence.
  • the promoter is inducible or repressible.
  • the gene may be present on an extrachromosomal plasmid, or may be integrated into the host genome.
  • One of skill in the art will recognize that it is not necessary to use a nucleic acid 100% identical to the naturally-occurring sequence. Rather, some alterations to these sequences are tolerated and may be desirable.
  • the nucleic acid may be altered to take advantage of the degeneracy of the genetic code such that the encoded polypeptide remains the same.
  • the gene is codon-optimized to improve expression in a particular host.
  • the nucleic acid may be produced, for example, by PCR or by chemical synthesis.
  • a polypeptide e.g., fusion protein
  • the isolation may be accomplished, for example, by affinity purification techniques or by physical separation techniques (e.g., a size column).
  • a method of manufacture comprising mixing one or more fusion proteins described herein with a carrier and/or an adjuvant.
  • fusion proteins for inclusion the vaccine formulations and immunogenic compositions may be produced in cell culture.
  • One method comprises providing one or more expression vectors and cloning nucleotides encoding one or more fusion proteins described herein, then expressing and isolating the fusion proteins.
  • the immunogenic fusion proteins described herein, and nucleic acid compositions that express the fusion proteins can be packaged in packs, dispenser devices, and kits for administering nucleic acid compositions to a mammal.
  • packs or dispenser devices that contain one or more unit dosage forms are provided.
  • instructions for administration of the composition will be provided with the packaging, along with a suitable indication on the label that the composition is suitable for treatment of an indicated condition, such as those disclosed herein.
  • SP2108 polypeptide SEQ ID NO: 6
  • SP0148 polypeptide SEQ ID NO: 6
  • results of the IL-17 immunogenicity assay are shown in FIGS. 1 and 2, where the left panels show data in scatter format, and the right panels show data as averages with standard deviations.
  • the subjects were allowed to rest an additional 2 weeks, at which time they were challenged with intranasal administration of live, encapsulated S. pneumoniae.
  • the subjects were sacrificed a week later, and the number of colony-forming units (CFU) was counted from nasal washes. Results of the colonization assay are shown in FIG. 3.
  • Vaccine compositions comprised the polypeptides SP0148 (SEQ ID NO: 2), SP0314 (SEQ ID NO: 26), SP0882 (SEQ ID NO: 17) or SP2108 (SEQ ID NO: 6), and cholera toxin adjuvant (CT).
  • CT cholera toxin adjuvant
  • SP1912, SP2108 and SP0148 polypeptides IL-17A immunogenicity assay
  • polypeptides SP1912 SEQ ID NO: 9
  • SP2108 SEQ ID NO: 6
  • SP0148 (SEQ ID NO: 2) were formulated as vaccine compositions with cholera toxin adjuvant (CT).
  • CT cholera toxin adjuvant
  • the vaccine compositions were administered to mice two times, one week apart.
  • the positive control was killed, unencapsulated whole cell S. pneumoniae + CT (WCB), and the negative controls were CT alone or recombinant proteins without CT (with the exception of SP1912).
  • peripheral blood was collected from the retroorbital sinus and evaluated in a whole blood assay. Briefly, the heparizined whole blood was diluted in media and then cultured in duplicate with A) the protein of immunization, or B) the whole cell vaccine for six days. The supernatants were harvested and IL-17A levels measured by ELISA. Results of the IL-17A immunogenicity assay are shown in FIG. 5. Each symbol in the graph represents responses from individual mice, and the line indicates the median response of the group.
  • SP1912, SP2108 and SP0148 polypeptides colonization assay
  • SP1912 polypeptide aspiration challenge (sepsis assay)
  • Polypeptide SP1912 was evaluated for its ability to protect mice from sepsis.
  • mice were subcutaneously immunized three times, two weeks apart with vaccine compositions comprising either the SP1912 polypeptide (SEQ ID NO: 9) or pneumolysoid (PdT) adsorbed to alum.
  • the positive control was killed, unencapsulated whole cell S. pneumoniae + alum (WCB), and the negative control was alum alone.
  • WB unencapsulated whole cell S. pneumoniae + alum
  • WCB unencapsulated whole cell S. pneumoniae + alum
  • the negative control was alum alone.
  • blood was collected for evaluation of IL-17A response and antibody levels, and then one week later, the mice underwent aspiration challenge with 10 7 live strain 0603 (type 6B) 5 * . pneumoniae. Animals were monitored for survival for eight days. Results of the aspiration challenge are shown in FIG. 7 as survival curves for each immunized group.
  • Pneumolysoid PdT, SP0148 and SP0641N polypeptides aspiration challenge (sepsis assay)
  • Polypeptide SP0148 was evaluated for its ability to protect mice from sepsis when immunized singly or in combination with SP0641N and/or pneumolysoid (PdT).
  • mice Groups of ten mice were subcutaneously immunized three times, two weeks apart with vaccine compositions comprising polypeptide SP0148 (SEQ ID NO: 2), singly or in combination with polypeptide SP0641N (SEQ ID NO: 13) and/or PdT, adsorbed to alum.
  • the positive control was killed, unencapsulated whole cell S. pneumoniae + alum (WCB), and the negative control was alum alone.
  • blood was collected for evaluation of IL- 17 and antibody, and then one week later, the mice underwent aspiration challenge with 10 7 live strain 0603 (type 6B) S. pneumoniae. Animals were monitored for survival for eight days. The data are shown in FIG. 8 as survival curves for each immunized group.
  • SP1912, SP2108 and SP0148 polypeptides colonization assay
  • Example 4 Additional studies were performed essentially as described in Example 4, for a total of four separate studies. Briefly, animals were immunized with vaccine formulations comprising the polypeptides SP 1912 (SEQ ID NO: 9), SP2108 (SEQ ID NO: 6), SP0148 (SEQ ID NO: 2), or additionally SP2108 plus SP0148, and cholera toxin adjuvant (CT) as described in Example 3. Control animals were immunized with killed, unencapsulated whole cell S. pneumoniae plus CT (WCV), or CT alone. Immunized animals were challenged intranasally with 10 7 live type 6B 5 * . pneumoniae four weeks after the last immunization.
  • vaccine formulations comprising the polypeptides SP 1912 (SEQ ID NO: 9), SP2108 (SEQ ID NO: 6), SP0148 (SEQ ID NO: 2), or additionally SP2108 plus SP0148, and cholera toxin adjuvant (CT) as described in Example 3. Control animals were immunized with killed, unencapsulated whole
  • SP1912 and SP0148 polypeptides IL-17A immunogenicity assay
  • mice Groups of ten mice were subcutaneously immunized twice, two weeks apart with vaccine compositions comprising either SP 1912 polypeptide (SEQ ID NO: 9), SP0148 polypeptide (SEQ ID NO: 2), or both adsorbed to alum. Control animals were immunized with alum alone.
  • heparinized blood was collected by cardiac puncture and evaluated for IL-17A levels in a whole blood assay. Briefly, the heparizined whole blood was diluted in media and then cultured for six days with the protein(s) of immunization. The supernatants were harvested and IL-17A levels measured by ELISA. Results of the IL-17A immunogenicity assay are shown in FIG. 10. Each symbol in the graph represents responses from individual mice, and the line indicates the median response of the group.
  • Example 10 1 1 as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. The number of animals protected from colonization out of the number of animals in the group is indicated at the top of the figure.
  • CFU colony forming units of bacteria
  • SP1912, SP0148, and SP2108 polypeptides colonization assay
  • mice were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 2) and SP0148 plus SP 1912 (SEQ ID NO: 9), or additionally with SP2108 (SEQ ID NO: 6), SP2108 plus SP0148, and SP2108 plus SP1912, adsorbed to alum.
  • Control animals were immunized with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), or alum alone. Immunized animals were challenged intranasally with 10 7 live type 6B 5 * . pneumoniae four weeks after the last immunization.
  • Pneumolysoid L460D PspA derivative CD2, SP1912, SP0148, and SP2108 polypeptides: colonization assay
  • the chart below shows the absolute number and corresponding percentage of animals protected from colonization in the four studies described in Examples 10 and 1 1.
  • PspA, SP0148 and SP2108 passive antibody transfer and aspiration challenge (sepsis assay)
  • mice were injected with monoclonal antibodies specific for
  • FIG. 15 shows the percent of animals protected from sepsis in six separate aspiration challenge studies, two of which aredescribed in more detail in Examples 6 and 12.
  • Example 13 shows the percent of animals protected from sepsis in six separate aspiration challenge studies, two of which aredescribed in more detail in Examples 6 and 12.
  • SP0148, SP2108, and SP1912 fusion proteins IL-17A immunogenicity assay
  • mice were subcutaneously immunized three times, two weeks apart with vaccine compositions comprising the fusion proteins SP2108/0148/1912 (SEQ ID NO: 53), SP0148/2108 (SEQ ID NO: 48), SP0148/1912 (SEQ ID NO: 50) or the comparable combinations of the single antigens SP2108 (SED ID NO: 6), SP0148 (SEQ ID NO: 2) and SP1912 (SEQ ID NO: 9), adsorbed to alum. Control animals were immunized with alum alone. Two weeks after the last immunization, peripheral blood was collected from the retroorbital sinus and evaluated for IL-17A levels in a whole blood assay.
  • vaccine compositions comprising the fusion proteins SP2108/0148/1912 (SEQ ID NO: 53), SP0148/2108 (SEQ ID NO: 48), SP0148/1912 (SEQ ID NO: 50) or the comparable combinations of the single antigens SP2108 (SED ID NO: 6), SP0148 (SEQ ID NO: 2) and SP
  • Control animals are immunized with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), or alum alone.
  • Immunized animals are challenged intranasally with 10 7 live type 6B 5 * . pneumoniae four weeks after the last immunization. Seven days after challenge, animals are euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Results are shown as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. The absolute number and corresponding percentage of animals protected from colonization are also indicated.
  • CFU colony forming units of bacteria
  • SP0148, SP2108, and SP1912 fusion proteins aspiration challenge (sepsis assay)
  • mice Groups of ten mice are subcutaneously immunized three times, two weeks apart with vaccine compositions comprising the fusion proteins SP2108/0148 (SEQ ID NO: 47), SP0148/2108 (SEQ ID NO: 48), SP2108/1912 (SEQ ID NO: 49), SP0148/1912 (SEQ ID NO: 50), SP2108/1912/0148 (SEQ ID NO: 51), SP0148/1912/2108 (SEQ ID NO: 52), SP2108/0148/1912 (SEQ ID NO: 53), or SP0148/2108/1912 (SEQ ID NO: 54), adsorbed to alum.
  • Control animals are immunized with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), or alum alone.
  • mice were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the fusion proteins SP0148/1912 (SEQ ID NO: 50), SP0148/1912 (SEQ ID NO: 50) plus SP2108 (SEQ ID NO: 6), SP2108/1912 (SEQ ID NO: 49), SP2108/1912 (SEQ ID NO: 49) plus SP0148 (SEQ ID NO: 2), SP2108/CD2 (SEQ ID NO: 57) plus SP0148/1912 (SEQ ID NO: 50), SP0148/CD2 (SEQ ID NO: 57) plus SP2108/1912 (SEQ ID NO: 49), SP2108/H70 (SEQ ID NO: 58) plus SP0148/1912 (SEQ ID NO: 50), and SP0148/H70 (SEQ ID NO: 56) plus SP2108/1912 (SEQ ID NO: 49), adsorbed to alum.
  • vaccine formulations comprising the fusion proteins SP0148/1912 (SEQ ID NO: 50), SP0148/1912
  • Control animals were immunized with SP2108 (SEQ ID NO: 6) plus SP0148 (SEQ ID NO: 2) plus SP 1912 (SEQ ID NO: 9) adsorbed to alum, or alum alone. Immunized animals were challenged intranasally with 10 7 live type 6B 5 * . pneumoniae four weeks after the last immunization. Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S.
  • CFU colony forming units of bacteria
  • SP0148, SP2108, SP1912, CD2 and H70 fusion proteins intravenous challenge (sepsis assay)
  • mice were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the fusion proteins SP2108/H70 (SEQ ID NO: 58), SP2108/CD2 (SEQ ID NO: 57), SP0148/H70 (SEQ ID NO: 56), SP0148/CD2 (SEQ ID NO: 55), SP2108/H70 (SEQ ID NO: 58) plus SP0148/1912 (SEQ ID NO: 50), and SP0148/H70 (SEQ ID NO: 56) plus SP2108/1912 (SEQ ID NO: 49), adsorbed to alum.
  • vaccine formulations comprising the fusion proteins SP2108/H70 (SEQ ID NO: 58), SP2108/CD2 (SEQ ID NO: 57), SP0148/H70 (SEQ ID NO: 56), SP0148/CD2 (SEQ ID NO: 55), SP2108/H70 (SEQ ID NO: 58) plus SP0148/1912 (SEQ ID NO: 50), and SP0148/H70
  • Control animals were immunized with H70 (SEQ ID NO: 33) or CD2 (SEQ ID NO: 29) adsorbed to alum, or alum alone. Immunized animals were challenged intravenously with 368 CFUs of strain A66.1 S. pneumoniae three weeks after the last immunization. Mice were observed daily for fourteen days. Results are shown in FIG. 18 as hours to moribund. [NB: In this figure, CD2 is labeled PRN.] Each symbol represents the survival hours of an individual mouse, and the horizontal line represents the median hours to moribund of the group. The table below the figure indicates the mean and median hours to moribund and the standard error.
  • peripheral blood was collected from the retroorbital sinus and evaluated for IL-17A levels in a whole blood assay. Briefly, the heparizined whole blood was diluted in media and then cultured for six days with killed, unencapsulated whole cell S. pneumoniae (WCA). The supernatants were harvested and IL-17A levels measured by ELISA. Results of the IL-17A immunogenicity assay for each study are shown in FIG. 19, Panels A and B. Individual symbols represent the cytokine secretion in whole blood from each mouse. Each horizontal line represents the mean IL-17A response from each group of animals in response to the indicated antigen.
  • FIG. 20 shows the mean ( ⁇ SD) IgG titer for the group expressed as the log 10 ⁇ ⁇ .
  • N In this figure, CD2 is labeled PRN.
  • SP0148 including signal sequence (277 amino acids with N-terminal E) MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKE
  • Immunogenic PspA/PspC polypeptides including the coiled-coil structure (PR + NPB)
  • Immunogenic PspA/PspC polypeptides including the coiled-coil structure (only)
  • NPB Non-proline Block
  • NPB Non-proline Block
  • NPB Non-proline Block
  • SP0148 including signal sequence (nucleotides)
  • SP2108/SP0148 fusion protein (675 amino acids) MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAV

Abstract

Streptococcus pneumoniae is a major health concern, especially in very young, elderly, or immunocompromised patients. The present disclosure provides, inter alia, certain highly effective vaccines and pharmaceutical compositions for Streptococcus pneumoniae that contain fusion proteins. The vaccines and pharmaceutical compositions may be used therapeutically and/or prophylactically.

Description

FUSED ANTIGEN VACCINES AND COMPOSITIONS AGAINST
STREPTOCOCCUS PNEUMONIAE
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S. provisional application
Serial No. 61/676,276, filed July 26, 2012, the contents of which are herein incorporated by reference in their entirety.
BACKGROUND
[0002] Pneumococcal disease continues to be a leading cause of sickness and death in the United States and throughout the world. Each year, millions of cases of pneumonia, meningitis, bacteremia, and otitis media are attributed to infection with the pathogen
Streptococcus pneumoniae. S. pneumoniae is a Gram-positive encapsulated coccus that colonizes the nasopharynx in about 5-10% of healthy adults and 20-40% of healthy children. Normal colonization becomes pathogenic when 5*. pneumoniae is carried into the Eustachian tubes, nasal sinuses, lungs, bloodstream, meninges, joint spaces, bones, or peritoneal cavity. S. pneumoniae has several virulence factors that enable the organism to evade the immune system. Examples include a polysaccharide capsule that prevents phagocytosis by host immune cells, proteases that inhibit complement-mediated opsonization, and proteins that cause lysis of host cells. In the polysaccharide capsule, the presence of complex
polysaccharides forms the basis for classifying pneumococci into different serotypes. To date, 93 serotypes of S. pneumoniae have been identified.
[0003] Various pharmaceutical compositions have been used to harness an immune response against infection by S. pneumoniae. A polyvalent pneumococcal vaccine, PPV-23, was developed for preventing pneumonia and other invasive diseases due to S. pneumoniae in the adult and aging populations. The vaccine contains capsular polysaccharides (CPs) from 23 serotypes of S. pneumoniae. As T-independent antigens, these CPs induce only shortlived antibody responses, necessitating repeated doses, which increases the risk of immunological tolerance. The antibodies raised against the S. pneumoniae capsular polysaccharides, termed anticapsular antibodies, are recognized as protective in adult and immunocompetent individuals. However, children under 2 years of age and
immunocompromised individuals, including the elderly, do not respond well to T- independent antigens and, therefore, are not afforded optimal protection by PPV-23. Another S. pneumoniae vaccine, Prevnar, includes bacterial polysaccharides from 7 S. pneumoniae serovars conjugated to the diphtheria toxoid protein. This vaccine induces both B and T cell responses. However, because it only protects against 7 pneumococcal serotypes, serotype replacement can render Prevnar ineffective against non-vaccine serovars. Serotype replacement has already been demonstrated in several clinical trials and epidemiologic studies, necessitating development of different formulations of these vaccines. An example is the recently introduced Prevnar 13, directed to 13 pneumococcal serotypes. Furthermore, the two Prevnar formulations are expensive to manufacture, greatly limiting their availability in the developing world. PPV-23, which consists of 23 purified but unconjugated
polysaccharides, has broader coverage, but does not provide protection to children under the age of 2 years, a population which is at the highest risk for pneumococcal disease.
[0004] Thus, there remains a need to design more effective pharmaceutical compositions than the current strategies offer. In particular, there remains a need for such compositions incorporating novel or specific antigens that elicit an immune response against S. pneumoniae.
SUMMARY OF THE INVENTION
[0005] Streptococcus pneumoniae is a major health concern, especially in very young, elderly, or immunocompromised patients. While DNA and protein sequence information for S. pneumoniae have been known for some time, and researchers have long attempted to produce vaccines against S. pneumoniae, a major challenge was how to elicit an immune response that is both long-lived and effective. Fusion proteins described herein exhibit surprising and unexpected properties, including eliciting immune responses that are long- lived and/or effective. Certain of the S. pneumoniae antigens included in fusion proteins described herein were initially identified by screening immune cells from mice either immunized with unencapsulated killed whole S. pneumoniae or infected with S.
pneumoniae, or from healthy human donors, for T-cell specific immune responses. In certain embodiments, such antigens are fused to S. pneumoniae capsular antigens, e.g., antigens that stimulate B cell specific immunity. Accordingly, the present disclosure provides, inter alia, certain highly effective vaccines against S. pneumoniae. The vaccines may be used therapeutically and/or prophylactically. The present disclosure also provides fusion proteins comprising specific antigens and methods for using such fusion proteins to elicit an immune response against 5*. pneumoniae. [0006] In certain aspects, the present disclosure provides fusion proteins comprising or consisting of (i) one or more immunogenic polypeptides having or comprising the amino acid sequence of one of SEQ ID NOS: 1-8 or an immunogenic fragment or variant thereof; and (ii) one or more S. pneumoniae antigens that are predominantly an antibody target. In some embodiments, the immunogenic polypeptide is N-terminal to the S. pneumoniae antigen. In certain embodiments, the immunogenic polypeptide is lipidated. In some embodiments, the immunogenic polypeptide has or comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the immunogenic polypeptide has or comprises the amino acid sequence of SEQ ID NO: 6.
[0007] In certain embodiments, an immunogenic polypeptide of a fusion protein described herein is directly bonded to a S. pneumoniae antigen. In some embodiments, a fusion protein described herein further comprisesa linker polypeptide. In some embodiments, a linker polypeptide is positioned between the immunogenic polypeptide and the S.
pneumoniae antigen. In some embodiments, the linker polypeptide is rigid. In certain embodiments, the linker polypeptide comprises SEQ ID NO: 93. In some embodiments, the linker polypeptide is semi-rigid. In certain embodiments, the linker polypeptide comprises SEQ ID NO: 94.
[0008] In some embodiments, the S. pneumoniae antigen of a fusion protein described herein is Pneumococcal surface protein A (PspA) or a derivative thereof. In some embodiments, the PspA derivative is or comprises all or a fragment of the proline-rich region of PspA. In certain embodiments, the PspA derivative is or comprises a non-proline block. In some embodiments, the PspA derivative is or comprises SEQ ID NO: 29. In certain embodiments, the PspA derivative is or comprises SEQ ID NO: 33.
[0009] In some embodiments, the S. pneumoniae antigen of a fusion protein described herein is or comprises pneumolysin, a pneumolysoid, or a derivative thereof.
[0010] In additional aspects, the present disclosure provides vaccine formulations comprising one or more fusion protein described herein and a pharmaceutically acceptable carrier.
[0011] In some embodiments, a vaccine formulation comprises a fusion protein described herein and a second immunogenic polypeptide having or comprising an amino acid sequence of one of SEQ ID NOS: 1-11 or an immunogenic fragment thereof. In some embodiments, a vaccine formulation comprises a first fusion protein described herein and a second fusion protein comprising two immunogenic polypeptides, each having or comprising an amino acid sequence of one of SEQ ID NOS: 1-1 1 or an immunogenic fragment thereof. [0012] In some embodiments, a vaccine formulation described herein further comprises an adjuvant. In some embodiments, the adjuvant is an agonist of toll-like receptors (TLRs). In some embodiments, the adjuvant is alum.
[0013] In certain embodiments, a vaccine formulation described herein comprises 1 -
1000 μg of each fusion protein and/ or polypeptide and 25-500 μg of the adjuvant.
[0014] In some aspects, compositions and methods described herein are used for prophylaxis and/or treatment of any pneumococcal disease, disorder, and/or condition due to a pneumococcal infection. In some embodiments, a v ac c i n e composition described herein reduces risk of infection by, and/or treats, alleviates, ameliorates, relieves, delays onset of, inhibits progression of, reduces severity of, and/or reduces incidence of one or more symptoms or features of a pneumococcal disease, disorder, and/or condition. In some embodiments, prophylaxis and/or treatment of pneumococcal infection comprises administering a therapeutically effective amount of a v ac c in e composition described herein to a subject in need thereof, e.g., in such amounts and/or for such time as is necessary to achieve the desired result. In certain embodiments, a "therapeutically effective amount" of a vaccine composition described herein is an amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of pneumococcal infection.
[0015] In some embodiments, prophylactic, prognostic and/or therapeutic methods involve administering a therapeutically effective amount of one or more vacc ine compositions described herein to a subject such that an immune response is stimulated in one or both of T cells and B cells.
[0016] In some embodiments, a vaccine formulation described herein is characterized in that, upon contacting a TH17 cell, the vaccine formulation induces a TH17 cell response at least 1.5-fold greater than that induced by a control (e.g., an unrelated antigen) after contacting TH17 cells. In some embodiments, a vaccine formulation described herein inhibits infection by S. pneumoniae in an uninfected subject (e.g., the vaccine formulation is characterized in that, upon administration to an uninfected subject, the vaccine formulation inhibits infection by S. pneumoniae in the uninfected subject). In certain embodiments, a vaccine formulation described herein inhibits S. pneumoniae colonization in a subject (e.g., the vaccine formulation is characterized in that, upon administration to a subject, the vaccine formulation inhibits S. pneumoniae colonization in the subject). In some embodiments, a vaccine formulation described herein inhibits S. pneumoniae symptoms (e.g., the vaccine formulation is characterized in that, upon administration to a subject, the vaccine formulation inhibits S. pneumoniae symptoms in the subject). In certain embodiments, a vaccine formulation described herein inhibits S. pneumoniae-m' duced invasive disease (e.g., the vaccine formulation is characterized in that, upon administration to a subject, the vaccine formulation inhibits S. pneumoniae-m' duced invasive disease in the subject). In certain embodiments, the vaccine formulation inhibits S. pneumoniae-m' duced sepsis (e.g., the vaccine formulation is characterized in that, upon administration to a subject, the vaccine formulation inhibits S. pneumoniae-m' duced sepsis in the subject).
[0017] In further aspects, the present disclosure provides methods for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering to the subject an effective amount of a vaccine formulation described herein.
[0018] In some embodiments, the subject is an uninfected subject, and the method inhibits infection by S. pneumoniae in the uninfected subject. In certain embodiments, the method inhibits S. pneumoniae colonization in a subject. In some embodiments, the method inhibits S. pneumoniae symptoms. In some embodiments, the method inhibits S. pneumonia- induced invasive disease. In certain embodiments, the method inhibits S. pneumoniae-m ' duced sepsis.
[0019] In some embodiments, the method treats a subject with one dose. In certain embodiments, the method treats a subject within three doses. In some embodiments, the subject is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.
[0021] FIG. 1 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then stimulated with killed, unencapsulated whole cell S. pneumoniae, as described in Example 1. The left panel shows the data in scatter format, and the right panel shows the average and standard deviation for each sample. Immunization group "All 3" represents animals immunized with a combination of SP2108, SP0148, and SP1634. [0022] FIG. 2 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then stimulated with a combination of three proteins (SP2108, SP0148, and SP 1634), as described in Example 1.
[0023] FIG. 3 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then challenged with intranasal administration of S. pneumoniae, as described in Example 1. 003 represents a control unrelated antigen.
[0024] FIG. 4 shows the number of S. pneumoniae colonies obtained from a nasal wash in BALB/c mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then challenged by intranasal administration of S. pneumoniae, as described in Example 2.
[0025] FIG. 5 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated proteins and cholera toxin adjuvant, then stimulated with the protein of immunization (A) or killed, unencapsulated whole cell S. pneumoniae (B), as described in Example 3.
[0026] FIG. 6 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and cholera toxin adjuvant, then challenged by intranasal administration of S. pneumoniae, as described in Example 4.
[0027] FIG. 7 shows survival of mice that were immunized with the indicated proteins and the adjuvant alum, then underwent aspiration challenge with 5*. pneumoniae as described in Example 5.
[0028] FIG. 8 shows survival of mice that were immunized with the indicated proteins and the adjuvant alum, then underwent aspiration challenge with 5*. pneumoniae as described in Example 6.
[0029] FIG. 9 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and cholera toxin adjuvant, then challenged by intranasal administration of S. pneumoniae, as described in Example 7.
[0030] FIG. 10 shows the concentration of IL-17A generated by blood samples from mice that were immunized with the indicated proteins and alum, then stimulated with the proteins indicated at upper left, as described in Example 8.
[0031] FIG. 1 1 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and alum or with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), then challenged by intranasal administration of S. pneumoniae, as described in Example 9.
[0032] FIG. 12 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and alum or with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), then challenged by intranasal administration of S. pneumoniae, in two pooled studies as described in Example 10.
[0033] FIG. 13 shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the indicated proteins and alum or with killed, unencapsulated whole cell S. pneumoniae plus alum (WCB), then challenged by intranasal administration of S. pneumoniae, as described in Example 1 1.
[0034] FIG. 14 shows survival of mice that were injected with antibodies or sera specific to the indicated proteins, then underwent aspiration challenge with S. pneumoniae, as described in Example 12.
[0035] FIG. 15 shows the percent of animals protected from sepsis in six separate aspiration challenge studies, two of which are described in more detail in Examples 6 and 12.
[0036] FIG. 16A, 16B, and 16C show the mean IL-17A response generated by blood samples from mice that were immunized with the indicated proteins and alum, then stimulated with the proteins indicated at upper right, as described in Example 13.
[0037] FIG. 17 shows the combined number of S. pneumoniae colonies, obtained in two separate studies, from nasal washes of mice that were immunized with the indicated fusion proteins adsorbed to alum, then challenged by intranasal administration of S.
pneumoniae, as described in Example 16.
[0038] FIG. 18 shows hours to moribund for mice that were immunized with the indicated fusion proteins adsorbed to alum, then challenged by intravenous administration of S pneumoniae, as described in Example 17.
[0039] FIG. 19A and 19B shows the concentration of IL- 17A generated by blood samples from mice that were immunized with the indicated fusion proteins adsorbed to alum, following stimulation with killed, unencapsulated whole cell S. pneumoniae, as described in Example 18.
[0040] FIG. 20 shows the total IgG concentration against CD2 or H70 in blood samples from mice immunized with the indicated fusion proteins adsorbed to alum, then challenged by intravenous administration of S pneumoniae, as described in Example 19. DETAILED DESCRIPTION
A. Fusion Proteins
[0041] This application describes vaccine formulations that include one or more S. pneumoniae fusion proteins, e.g., fusion proteins that include one or more S. pneumoniae polypeptides or fragments thereof. The fusion proteins can include a first polypeptide that elicits primarily a T cell response and a second polypeptide that elicits primarily a B cell (e.g., antibody) response. In some embodiments, the first polypeptide elicits an immune response that is primarily a T cell response. For example, when assayed using a suitable immunogenicity assay (e.g., an assay described herein), more than 50%, 60%, 70%, 80%, 90%, or more, of the immune response elicited by the first polypeptide is a T cell response. In some embodiments, the second polypeptide elicits an immune response that is primarily a B cell response. For example, when assayed using a suitable immunogenicity assay (e.g., an assay described herein), more than 50%, 60%, 70%, 80%, 90%, or more, of the immune response elicited by the second polypeptide is a B cell response.
[0042] In certain embodiments, a fusion protein described herein includes one, two or more of the polypeptides listed (or polypeptides encoded by the genes listed) in Table 1 and/or 2. In certain embodiments, a fusion protein includes a polypeptide listed (or encoded by a gene listed) in Table 1 fused to a polypeptide listed (or encoded by a gene listed) in Table 2.
[0043] In some embodiments, a fusion protein comprises an immunogenic peptide having or comprising an amino acid sequence of one of SEQ ID NOS: 1-8, shown in Table 1, or an immunogenic fragment thereof; and a 5*. pneumoniae antigen that is or comprises predominantly an antibody target. In some embodiments, the immunogenic peptide is SP2108 (SEQ ID NO: 6). In some embodiments, the immunogenic peptide is SP0148 (SEQ ID NO: 2). In some embodiments, a fusion protein includes a S. pneumoniae antigen that is or comprises predominantly an antibody target, e.g., an antigen shown in Table 2, e.g., PspA (SEQ ID NO: 27) or fragments or variants thereof. In some embodiments, fragments or variants of PspA comprise proline-rich segments with the non-proline block (PR+NPB), for example the CD2 sequence SEQ ID NO: 29. In some embodiments, fragments or variants of PspA comprise proline-rich segments with the non-proline block and 10, 20 30, 40 or more additional amino acids of PspA sequence, for example the H70 sequence (SEQ ID NO: 33). In some embodiments, the S. pneumoniae antigen that is predominantly an antibody target comprises pneumolysin or a pneumolysoid. [0044] Fusion proteins described herein can include polypeptides homologous to the polypeptides of Tables 1 (for example, SP 1912, SP1912L, SP0148 with or without a signal sequence, SP2108 with or without a signal sequence) and Table 2 (for example, PspA and fragments and variants thereof). Individual strains of S. pneumoniae contain numerous mutations relative to each other, and some of these result in different protein sequences between the different strains. One of skill in the art may readily substitute an amino acid sequence, or a portion thereof, with the homologous amino acid sequence from a different S. pneumoniae strain. In certain aspects, a fusion protein described herein includes one or more polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to the polypeptides of Tables 1 and 2 or an immunogenic fragment thereof. Serotypic variation may be used to design such variants of the polypeptides of Tables 1 and 2.
[0045] In some embodiments, a fusion protein described herein includes a fragment of a polypeptide of Table 1 (for example, fragments of SP 1912, SP1912L, SP0148 with or without a signal sequence, SP2108 with or without a signal sequence), and/or a fragment of a polypeptide of Table 2 (for example, PspA and variants thereof). In some embodiments, a fusion protein herein comprises one or more truncation mutants that are close in size to the polypeptides of Tables 1 and 2. For example, they may lack at most one, two three, four, five, ten, or twenty amino acids from one or both termini (referring to component polypeptides in a fusion protein). In certain embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: 1-8 or 27-36, wherein from 1-5, 1-10, or 1-20 amino acid residues are removed from the N-terminus, C-terminus, or both. In certain embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: 1-8 or 27-36, wherein from 1-10 amino acid residues are removed from the N-terminus, C-terminus, or both. For instance, 10 amino acid residues may be removed from each of the N-terminus and C-terminus resulting in a protein with 20 amino acid residues removed. Internal deletions, e.g., of 1-10, 1 1-20, 21- 30, or 31-40 amino acids, are also contemplated.
Table 1. Immunogenic polypeptides for vaccine formulations
Figure imgf000010_0001
146,343*
SP0148 consensus lacking signal 3 - - sequence
SP0148 consensus including signal 4 - - sequence
SP2108 lacking signal sequence 5 39 -
SP2108 including signal sequence 6 NC 003028.31:2020750- 2022021
SP2108 consensus lacking signal 7 - - sequence
SP2108 consensus including signal 8 - - sequence
SP1912 9 40 NC 003028.31: 1824672- 1824971
SP1912L 10 41 -
SP1912 consensus 11 - -
SP0641 12
SP0641N 13 42
SP0641M 14 -
SP0641N consensus 15 - -
SP0641M consensus 16 - -
SP0882 17 NC 003028.31:831804-
832628
SP0882N 18 43 -
SP0882 with exogenous signal 19 44 - sequence
SP0882N with exogenous signal 20 45 - sequence
SP0882 consensus 21 - -
SP0882N consensus 22 - -
SP0882 consensus with exogenous 23
leader
SP0882N consensus with exogenous 24 - - leader
SP1634 25 NC 003028.31: 1534348- 1535421
SP0314 26 NC_003028.3 |:287483- 290683
Table 2. Immunogenic polypeptides for vaccine formulations
Figure imgf000012_0001
*NB: The database sequence incorrectly lists TTG (encoding Leu) at nucleotide positions 541-543. The correct sequence, as shown in SEQ ID NO: 38, has TTC at that codon and encodes Phe. The database sequence further does not include a C-terminal Glu found in certain isolates.
[0046] Particular examples of fusion proteins are provided in Table 3.
Table 3. Immunogenic fusion proteins for vaccine formulations
Figure imgf000012_0002
SP0148/SP 1912/SP2108 52 72
SP2108/SP0148/SP1912 53 73
SP0148/SP2108/SP1912 54 74
SP0148/CD2 55 75
SP0148/H70 56 76
SP2108/CD2 57 77
SP2108/ H70 58 78
SP0148/LC/CD2 59 79
SP0148/ LC/H70 60 80
SP2108/ LC/CD2 61 81
SP2108/LC/H70 62 82
SP0148/LR/CD2 63 83
SP0148/LR/H70 64 84
SP2108/LR/CD2 65 85
SP2108/LR/H70 66 86
[0047] In some embodiments, the fusion protein comprises an N-terminal peptide and a C-terminal peptide. In some embodiments, the N-terminal peptide comprises the immunogenic polypeptide, for example, a polypeptide having an amino acid sequence of one of SEQ ID NOS: 1-8 or immunogenic fragments or variants thereof, and the C-terminal peptide comprises a 5*. pneumoniae antigen that is predominantly an antibody target as described herein.
[0048] In some embodiments, the N-terminal peptide and the C-terminal peptide are directly bound to each other. In some embodiments, the N-terminal peptide and the C- terminal peptide are linked via a linker peptide. The length and/or amino acids of a linker, when present, can be adjusted to obtain a more flexible or rigid linker. Exemplary peptide linkers are shown as SEQ ID NOS: 92-94. A linker can generally be from 1-40, such as 10- 30 and specifically 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.
[0049] In some embodiments, the fusion protein comprises an immunogenic fragment of the fusion proteins shown in Table 3. In some embodiments, the fragment is a truncated fusion protein of any of SEQ ID NOS: 47-66. For example, they may lack at most one, two three, four, five, ten, or twenty amino acids from the N-terminus, C-terminus, or both. In some such embodiments, the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus. Advantageously, in some embodiments the N- terminal polypeptide is not truncated so as not to prevent lipidation.
[0050] In certain aspects, this application provides fusion proteins with at least 90%,
95%, 97%, 98%, 99%, or 99.5% identity to a fusion protein of Table 3. In certain embodiments, a vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 47-66, or an immunogenic fragment thereof.
[0051] In certain embodiments, vaccines or pharmaceutical compositions described herein include one or more fusion proteins containing at least one lipidated polypeptide. In some embodiments, the fusion protein is lipidated. In certain embodiments, the fusion protein is lipidated on the N-terminal peptide. Conjugation to the lipid moiety may be direct or indirect (e.g., via a linker). The lipid moiety may be synthetic or naturally produced. In certain embodiments, a polypeptide from Table 1, 2 or 3 may be chemically conjugated to a lipid moiety. In certain embodiments, a construct may comprise a gene or polypeptide from Table 1, 2 or 3, or an immunogenic fragment or variant thereof, and a lipidation sequence including a lipobox motif. A canonical lipobox motif is shown as SEQ ID NO: 89. A lipidation sequence may be N-terminal or C-terminal to the protein, and may be embedded in a signal or other sequence, or in a fusion protein. Exemplary lipidation sequences include the signal sequence of SP2108 (SEQ ID NO: 90) and the signal sequence of the E. coli gene RlpB (SEQ ID NO: 91). A signal sequence may be, for example, an E. coli or S. pneumoniae signal sequence. Exemplary E. coli signal sequences include the mlpA signal sequence (Lin, J.J. et al, "An Escherichia coli mutant with an amino acid alteration within the signal sequence of outer membrane prolipoprotein" Proc Natl Acad Sci U S A. 1978
Oct;75(10):4891-5 ), the lamB signal sequence (Emr, S.D. et al. "Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein", Proc Natl Acad Sci U S A. 1978 Dec;75(12):5802-6), or the MBP signal sequence (Bassford, P. J., "Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm" J Bacteriol. 1979 Jul; 139(1): 19-31). Lpp is an exemplary E. coli signal sequence that directs lipidation (Cullen, P.A. et al. "Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coi Plasmid. 2003 Jan;49(l): 18-29.) E. coli signal sequences that direct lipidation are also described in Legrain, M. et al. ("Production of lipidated meningococcal transferrin binding protein 2 in Escherichia coli" Protein Expr Purif. 1995 Oct;6(5):570-8), e.g. the signal sequence of the gene RlpB (SEQ ID NO: 91) Numerous S. pneumoniae signal sequences are known in the art. One such signal sequence is SEQ ID NO: 90.
[0052] In certain embodiments, vaccines or pharmaceutical compositions described herein include a fusion protein comprising a polypeptide encoded by a polynucleotide from Table 1, 2, or 3, a polypeptide from Table 1, 2 or 3, or an immunogenic fragment or variant thereof, and a tag. A tag may be N-terminal or C-terminal. For instance, tags may be added to the nucleic acid or polypeptide to facilitate purification, detection, solubility, or confer other desirable characteristics on the protein or nucleic acid. For instance, a purification tag may be a peptide, oligopeptide, or polypeptide that may be used in affinity purification. Examples include His, GST, TAP, FLAG, myc, HA, MBP, VSV-G, thioredoxin, V5, avidin, streptavidin, BCCP, Calmodulin, Nus, S tags, lipoprotein D, and β galactosidase. Particular exemplary His tags include HHHHHH (SEQ ID NO: 87) and MSYYHHHHHH (SEQ ID NO: 88). In other embodiments, the polypeptide is free of tags such as protein purification tags, and is purified by a method not relying on affinity for a purification tag. In some embodiments, the fused portion is short. This, in some instances, the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids on one or both termini of the polypeptide of Table 1, 2 or 3.
[0053] In certain embodiments, this application provides nucleic acids encoding one or more of the fusion proteins described herein, such as DNA (e.g., cDNA), RNA, or an analog thereof. The underlying DNA sequences for the polypeptides described above may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention. For instance, the DNA sequence may be codon-optimized to improve expression in a host such as E. coli, an insect cell line (e.g., using the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
[0054] In certain embodiments, this application provides polynucleotides (such as
DNA, cDNA, RNA, or an analog thereof) that are at least 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to in the polynucleotide sequences of Table 3, or a variant or portion of said sequence. In certain embodiments, the polynucleotide is 600-2000, 800-1800, 1000- 1600, 1200-1400 nucleotides in length. In some embodiments, the polynucleotide is 600- 1600, 800-1800, 1000-2000, 2000-3000, or 3000-4000 nucleotides in length. The nucleic acids may be used, for example, for recombinant production of the polypeptides of Table 1 or 2, or the fusion proteins of Table 3, or immunogenic fragments thereof. [0055] Polynucleotides encoding peptides of Tables 1, 2 or 3 or fragments thereof can be cloned into any of a variety of expression vectors, under the control of a variety of regulatory elements, and fusions can be created with other peptides of Tables 1, 2, or 3 or with other sequences of interest. Methods of cloning nucleic acids are routine and conventional in the art. For general references describing methods of molecular biology which are mentioned in this application, e.g., isolating, cloning, modifying, labeling, manipulating, sequencing and otherwise treating or analyzing nucleic acids and/or proteins, see, e.g., Sambrook, J. et al. (1989). Molecular Cloning, a Laboratory Manual. Cold Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (1995). Current Protocols in Molecular Biology, N.Y., John Wiley & Sons; Davis et al. (1986), Basic Methods in Molecular Biology, Elsevir Sciences Publishing,, Inc., New York; Hames et al. (1985), Nucleic Acid Hybridization, IL Press; Dracopoli, N.C. et al. Current Protocols in Human Genetics, John Wiley & Sons, Inc.; and Coligan, J. E., et al. Current Protocols in Protein Science, John Wiley & Sons, Inc.
B. Specific polypeptides and nucleic acids for use in S. pneumoniae vaccines and immunogenic compositions
[0056] This application describes S. pneumoniae fusion proteins. The fusion proteins include one, two or more of the polypeptides (or polypeptides encoded by the genes) listed in Table 1 and/or 2, or variants or fragments thereof as described below. In some embodiments, the fusion proteins contain one or more polypeptides from Table 1 and one or more polypeptides from Table 2. The DNA and protein sequence of each gene and polypeptide may be found by searching for the Locus Tag in the publicly available database, Entrez Gene (on the NCBI NIH web site on the World Wide Web, at
www.ncbi.nlm.nih.gov/sites/entrez?db=gene), in the Streptococcus pneumoniae TIGR4 genome, and the indicated sequences are also included in this application.
[0057] Certain polypeptides of Table 1 and 2, and variants thereof, are described in greater detail below.
1. SP0148 (SEQ ID NO: 2) and variants thereof
[0058] The protein SP0148 is named "ABC transporter, substrate-binding protein".
Proteins of this class are typically extracellular proteins that interact transiently with a transmembrane protein complex. Such complexes use energy generated by ATP hydrolysis to translocate specific substrates across a cell membrane. SP0148 is a 276 or 277 (depending on the isolate) amino acid protein that contains a conserved PBPb (periplasmic binding protein) domain, spanning amino acids 40-246, which is typical of membrane-bound transport complexes. In addition, SP0148 has a bacterial extracellular solute-binding proteins family 3 domain which is largely co-extensive with the PBPb domain and extends from amino acid 40 to 244. In some embodiments, a vaccine or other composition comprises a truncation mutant of SP0148 comprising or lacking one or more of said domains and motifs.
[0059] In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a fusion protein containing a polypeptide containing at least 20 consecutive amino acid residues selected from SP0148. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 275, 250, 225, 200, 175, 150, 125, or 100 consecutive amino acids from SP0148.
[0060] Endogenous SP0148 comprises a signal sequence that directs its secretion and potential lipidation. In some embodiments, the signal sequence of the polypeptide of SEQ ID NO: 2 is partially or fully processed by an expression host, e.g. E. coli. In some
embodiments, a variant of SP0148 that lacks the signal sequence (SEQ ID NO: 1) is used. The polypeptide of SEQ ID NO: 1 is encoded by the nucleic acid of SEQ ID NO: 37, although other nucleic acid sequences (including codon-optimized sequences) may be used. SEQ ID NO: 38 encodes the full length sequence of SP0148 used in the screens herein.
[0061] Variants of the amino acid sequence and nucleotide sequence of SP0148 may be found in U.S. Patent Application Publication No. 2005/0020813, U.S. Patent Nos.
7,378,514 and 7,504, 110, and European Patent Application No. EP1572868 and EP 1855717.
[0062] Consensus sequences illustrating combinations of SP0148 sequences from different S. pneumoniae serotypes are provided as SEQ ID NOS: 3 and 4. Accordingly, in certain embodiments, the vaccine formulation comprises a fusion protein that includes a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 3-4, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 1 or 2).
[0063] In some embodiments a fusion protein comprises or consists of an
immunogenic fragment of SP0148. Exemplary immunogenic fragments of SP0148 include ALGLVAAGV (SEQ ID NO: 97), ELTGYEIEV (SEQ ID NO: 98), AVNNLSYTK (SEQ ID NO: 99), TYLPAEADI (SEQ ID NO: 100), RYNMAVNNL (SEQ ID NO: 101),
DFQQIMVRL (SEQ ID NO: 102), EHTDNPTIL (SEQ ID NO: 103), APIAQNPNV (SEQ ID NO: 104), LPSDQQPYV (SEQ ID NO: 105), YVYPLLAQG (SEQ ID NO: 106), QGLDNLKVI (SEQ ID NO: 107), KYLYAAPI (SEQ ID NO: 108), GELTGYEI (SEQ ID NO: 109), NPNVLVVKK (SEQ ID NO: 1 10), KLSKQFFGD (SEQ ID NO: 1 11),
GSPRPFIYE (SEQ ID NO: 1 12), AVNNLSYTK (SEQ ID NO: 1 13), KIFDKIGVE (SEQ ID NO: 1 14), MVRLSDGQF (SEQ ID NO: 1 15), YVYPLLAQG (SEQ ID NO: 116),
WQATTSAK (SEQ ID NO: 117), TLEKLSKQF (SEQ ID NO: 1 18), VAAGVLAAC (SEQ ID NO: 119), LDNLKVIEL (SEQ ID NO: 120), and NMAVNNLSY (SEQ ID NO: 121). 2. SP2108 (SEQ ID NO: 6) and variants thereof
[0064] The polypeptide SP2108 is 423 amino acids in length and is alternatively known as MalX, maltose/maltodextrin ABC transporter, or maltose/maltodextrin-binding protein. Much of the protein (amino acids 3-423) is classified as a MalE (Maltose-binding periplasmic) domain. In addition, SP2108 contains a signal sequence that directs its secretion and potential lipidation. In some embodiments, the signal sequence of the polypeptide of SEQ ID NO: 6 is partially or fully processed by an expression host, e.g. E. coli. In some embodiments, a vaccine or other composition comprises a truncation mutant of SP2108 comprising one or more of said domains and motifs.
[0065] In some embodiments, the compositions and methods herein call for the use of an SP2108 variant that lacks the signal sequence. This variant is represented by polypeptide sequence SEQ ID NO: 5 and may be encoded by, for example, a nucleic acid according to SEQ ID NO: 39, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) may be used.
[0066] In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a fusion protein containing a polypeptide containing at least 20 consecutive amino acid residues selected from SP2108. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids from SP2108.
[0067] Consensus sequences illustrating combinations of SP2108 sequences from different serotypes are provided as SEQ ID NOS: 7 and 8. Thus, in certain embodiments, the vaccine formulation comprises a fusion protein containing a polypeptide having an amino acid sequence comprising, or consisting of, either of SEQ ID NOS: 7-8, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 5 or 6).
[0068] In some embodiments a fusion protein comprises or consists of an
immunogenic fragment of SP2108. Exemplary immunogenic fragments of SP2108 include AIIDGPWKA (SEQ ID NO: 122), VMMAPYDRV (SEQ ID NO: 123), SIAGINYAK (SEQ ID NO: 124), VWDPAKNML (SEQ ID NO: 125), QPLPNISQM (SEQ ID NO: 126), APYDRVGSL (SEQ ID NO: 127), APAVIESLV (SEQ ID NO: 128), FYYTYGLLA (SEQ ID NO: 129), SKYAFAGE (SEQ ID NO: 130), TEGAGNLI (SEQ ID NO: 131),
LADWTNFYY (SEQ ID NO: 132), SLVMYYNKD (SEQ ID NO: 133), KEAGVKVTL (SEQ ID NO: 134), KSTAVLGTV (SEQ ID NO: 135), GAKTDDTTK (SEQ ID NO: 136), SQKFVDFLV (SEQ ID NO: 137), QAFKDAKVN (SEQ ID NO: 138), AVIESLVMY (SEQ ID NO: 139), DAKTAANDA (SEQ ID NO: 140), YGVATIPTL (SEQ ID NO: 141), KTAAIIDGP (SEQ ID NO: 142), KAYEKEAGV (SEQ ID NO: 143), AGNGAYVFG (SEQ ID NO: 144), and AWVIPQAVK (SEQ ID NO: 145).
3. SP1912 (SEQ ID NO: 9) and variants thereof
[0069] SP 1912 is a hypothetical protein of 99 amino acids. While the protein function is not definitively known, sequence analysis suggests it is a putative thioredoxin.
[0070] In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP1912. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 90, 75, 60, 45 or 30 consecutive amino acids from SP1912.
[0071] In some embodiments, the compositions and methods herein call for the use of an SP 1912 variant that comprises an exogenous lipidation sequence. In some embodiments, a signal sequence directs lipidation. Thus, the lipidation signal may be, e.g., the signal sequence of SP2108 (SEQ ID NO: 90) or SP0148, or an E. coli signal sequence. The exemplary variant SP1912L, comprising the signal sequence of the E. coli gene RlpB (SEQ ID NO: 91) is represented by polypeptide sequence SEQ ID NO: 10. SP 1912 (SEQ ID NO: 9) and SP1912L (SEQ ID NO: 10) may be encoded, respectively, by nucleic acids according to SEQ ID NO: 40 and 41, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) may be used. In some embodiments, the lipidation sequence is provided by fusing a full-length polypeptide such as SP2108 (SEQ ID NO: 6) or SP0148 (SEQ ID NO: 2) to SP1912. Exemplary fusions are SEQ ID NOS: 49-54.
[0072] Consensus sequences illustrating combinations of SP 1912 sequences from different serotypes are provided as SEQ ID NO: 11. Thus, in certain embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, SEQ ID NO: 11, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising SEQ ID NO: 9). [0073] In some embodiments a fusion protein comprises or consists of an
immunogenic fragment of SP1912. Exemplary immunogenic fragments of SP1912 include KMWMAGLALLGIGSL (SEQ ID NO: 146), LLGIGSLALATKKVA (SEQ ID NO: 147), MAGLALLGIGSLALA (SEQ ID NO: 148), WMAGLALLGIGSLAL (SEQ ID NO: 149), GLALLGIG SL ALATK (SEQ ID NO: 150), LALLGIGSLALATKK (SEQ ID NO: 151), FSDMGEIATLYVQVY (SEQ ID NO: 152), KAKKMWMAGLALLGI (SEQ ID NO: 153), ALLGIGSLALATKKVAK (SEQ ID NO: 154), SDMGEIATLYVQVYE (SEQ ID NO: 155), DMGEIATLYVQVYES (SEQ ID NO: 156), AGLALLGIGSLALAT (SEQ ID NO: 157), MGEIATLYVQVYESS (SEQ ID NO: 158), KKMWMAGLALLGIGS (SEQ ID NO: 159), GMKAKKMWMAGLALL (SEQ ID NO: 160), MKAKKMWMAGLALLG (SEQ ID NO: 161), HF SDMGEI ATL YVQV (SEQ ID NO: 162), MNGMKAKKMWMAGLA (SEQ ID NO: 163), MWMAGLALLGIGSLA (SEQ ID NO: 164), DHF SDMGEI ATL YVQ (SEQ ID NO: 165), RDHFSDMGEIATLYV (SEQ ID NO: 166), NGMKAKKMWMAGLAL (SEQ ID NO: 167), and KMWMAGLALLGIG (SEQ ID NO: 168).
4. Additional Antigens
[0074] In certain embodiments, the vaccine formulations provided herein comprise or further comprise one or more, or two or more, known S. pneumoniae antigens. In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a fusion protein containing a known S. pneumoniae antigen. In some instances, the known 5*. pneumoniae antigens are predominantly antibody targets. In some instances, the known S. pneumoniae antigens protect from S pneumoniae colonization, or from S pneumoniae-m' duced sepsis. One appropriate art-recognized class of S. pneumoniae antigen is Pneumococcal surface protein A (PspA) (SEQ ID NO: 27) and derivatives of PspA. Derivatives of PspA include proline-rich segments with the non-proline block (PR+NPB, also referred to as PRN and further described below as well as in Daniels, C.C. et al. (2010) Infection and Immunity 78:2163-72) and related constructs comprising all or a fragment of the proline-rich region of PspA (e.g., regions containing one or more of the sequences PAPAP, PKP, PKEPEQ and PEKP and optionally including a non-proline block). In some embodiments, fragments or variants of PspA comprise proline-rich segments with the non-proline block and 10, 20 30, 40 or more additional amino acids of PspA sequence. H70 (SEQ ID NO: 33) is one exemplary sequence which includes the proline-rich region and non-proline-block encompassing amino acids 290-410 of PspA. An example of the non- proline-block has the exemplary sequence EKSADQQAEEDYARRSEEEYNRLTQQQ (SEQ ID NO: 34), which generally has no proline residues in an otherwise proline-rich area of the non-coiled region of PspA. Other embodiments of non-proline block (NPB) sequences include SEQ ID NOS: 35 and 36 and PspA and its derivatives can include genes expressing similar proline-rich structures (i.e. PKP, PKEPEQ and PEKP), with or without the NPB. The amino acids at either end of the NPB mark the boundaries of the proline-rich region. In one example, the amino-terminal boundary to the PR-region is DLKKAVNE (SEQ ID NO: 95), and the carboxy-terminal boundary is (K/G)TGW(K/G)QENGMW (SEQ ID NO: 96).
Peptides containing the NPB are particularly immunogenic, suggesting that the NPB may be an important epitope. Exemplary immunogenic PspA polypeptide derivatives containing the coiled-coil structure include SEQ ID NOS: 28 and 31. Particular embodiments of the immunogenic PspA polypeptide derivatives lacking the coiled-coil structure have the amino acid sequences shown as SEQ ID NOS: 29, 30 and 32. Immunogenic PspA polypeptides SEQ ID NO: 28-30 include both PR and NPB sequences (PR+NPB). Immunogenic PspA polypeptides of SEQ ID NOS: 31 and 32 include only a PR sequence (PR only) and lack the NPB.
[0075] Another appropriate art-recognized class of S. pneumoniae antigen is the pneumolysoids. Pneumolysoids have homology to the S. pneumoniae protein pneumolysin (PLY), but have reduced toxicity compared to pneumolysin. Pneumolysoids can be naturally occurring or engineered derivatives of pneumolysin. In some embodiments, a pneumolysoid has at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to pneumolysin. In some embodiments, the pneumolysoid demonstrates less than 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500, or 1/1000 the toxicity of pneumolysin in an assay for one or both of hemolytic activity towards erythrocytes and inhibition of polymorphonuclear leukocytes. Both assays are described in Saunders F.K. et al. ("Pneumolysin, the thiol-activated toxin of
Streptococcus pneumoniae, does not require a thiol group for in vitro activity" Infect Immun. 1989 Aug;57(8):2547-52). Exemplary pneumolysoids include PdT (a triple mutant further described in Berry, A.M. et al. (1995) Infection and Immunity 63: 1969-74); Pd-A and Pd-B (Paton J.C. et al. "Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide" Infect Immun. 1991 Jul;59(7):2297-304); rPd2 and rPd3 (Ferreira et al. "DNA vaccines based on genetically detoxified derivatives of pneumolysin fail to protect mice against challenge with Streptococcus pneumoniae" FEMS Immunol Med Microbiol (2006) 46: 291-297); Ply8, A6PLY, L460D (see, e.g., US 2009/0285846 and L. Mitchell, Protective Immune Responses to Streptococcus pneumoniae Pneumolysoids, ASM2011 conference abstract, 201 1), or a variant thereof. In some embodiments, the pneumolysin has a mutation in the catalytic center, such as at amino acid 428 or 433 or the vicinity.
[0076] Other appropriate S. pneumoniae antigens include Choline-binding protein A
(CbpA) and derivatives thereof (AD Ogunniyi et ah, "Protection against Streptococcus pneumoniae elicited by immunization with pneumolysin and CbpA," Infect Immun. 2001 Oct;69(10):5997-6003); Pneumococcal surface adhesin A (PsaA); caseinolytic protease; sortase A (SrtA); pilus 1 RrgA adhesin; PpmA; PrtA; PavA; LytA; Stk-PR; PcsB; RrgB and derivatives thereof. CpbA derivatives include fusion proteins described in WO 2012/134975. Such fusion proteins may comprise one or more copies of the R2 domain, R2i and/or R¾ subdomains of CpbA, or active variants and fragments thereof, or any combination thereof. Such fusion proteins may further comprise a pneumolysoid. The construct YLN, for example, comprises CpbA polypeptides YPT and NEEK, and pneumolysoid L460D.
[0077] In some cases, the antigen is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the corresponding wild-type S. pneumoniae protein. Sequences of the above-mentioned polypeptides, and nucleic acids that encode them, are known; see, for example, the S. pneumoniae ATCC 700669 complete genome sequence under GenBank accession number FM21 1187.1 and linked polypeptide sequences therein.
[0078] Typically, fusion proteins described herein include polypeptides, and fragments and variants thereof, that are immunogenic. These polypeptides may be immunogenic in mammals, for example mice, guinea pigs, or humans. An immunogenic polypeptide is typically one capable of raising a significant immune response in an assay or in a subject. The immune response may be innate, adaptive, humoral, cell-mediated, or mucosal (combining elements of innate, adaptive, humoral and cell-mediated immunity). For instance, an immunogenic polypeptide may increase the amount of IL-17 produced by T cells. Alternatively or additionally, an immunogenic polypeptide may (i) induce production of antibodies, e.g., neutralizing antibodies, that bind to the polypeptide and/or the whole bacteria, (ii) induce TH17 immunity, (iii) activate the CD4+ T cell response, for example by increasing CD4+ T cells and/or increasing localization of CD4+ T cells to the site of infection or reinfection, (iv) activate the CD8+ CTL response, for example by increasing CD8+ T cells and/or increasing localization of CD8+ T cells to the site of infection or reinfection, (v) induce THI immunity, and/or (vi) activate innate immunity. In some embodiments, an immunogenic polypeptide causes the production of a detectable amount of antibody specific to that antigen. [0079] In certain embodiments, polypeptides have less than 20%, 30%, 40%, 50%,
60% or 70% identity to human autoantigens and/or gut commensal bacteria (e.g., certain Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus , Bifidobacterium, Escherichia and Lactobacillus species). Examples of human autoantigens include insulin, proliferating cell nuclear antigen, cytochrome P450, and myelin basic protein.
[0080] A polypeptide may comprise one or more immunogenic portions and one or more non- immunogenic portions. The immunogenic portions may be identified by various methods, including protein microarrays, ELISPOT/ELISA techniques, and/or specific assays on different deletion mutants (e.g., fragments) of the polypeptide in question. Immunogenic portions may also be identified by computer algorithms. Some such algorithms, like
EpiMatrix (produced by EpiVax), use a computational matrix approach. Other computational tools for identifying antigenic epitopes include PEPVAC (Promiscuous EPitope-based VACcine, hosted by Dana Farber Cancer Institute on the world wide web at
immunax.dfci.harvard.edu/PEPVAC), MHCPred (which uses a partial least squares approach and is hosted by The Jenner Institute on the world wide web at www.jenner.ac.uk/MHCPred), and Immune Epitope Database algorithms on the world wide web at
tools.immuneepitope.org. An immunogenic fragment of a polypeptide described herein comprises at least one immunogenic portion, as measured experimentally or identified by algorithm.
[0081] Thus, in some aspects, this application provides a fusion protein that includes one or more immunogenic fragments of an antigen described herein. The fragments, in some instances, are close in size to the full-length polypeptide or the polypeptide of Table 1, 2 or 3. For example, they may lack at most one, two, three, four, five, ten, twenty, or thirty amino acids from one or both termini. In certain embodiments, the polypeptide is 100-500 amino acids in length, or 150-450, or 200-400, or 250-250 amino acids in length. In some embodiments, the polypeptide is 100-200, 150-250, 200-300, 250-350, 300-400, 350-450, or 400-500 amino acids in length. In certain embodiments, the fragments result from
processing, or partial processing, of signal sequences by an expression host, e.g. E. coli, an insect cell line (e.g., the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line. The fragments described above or sub-fragments thereof (e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues) preferably have one of the biological activities described below, such as increasing the amount of IL-17 released by at least 1.5 fold or 2 fold or more (e.g., either as an absolute measure or relative to an immunologically inactive protein). A fragment may be used as the polypeptide in the vaccines described herein or may be fused to another protein, protein fragment or a polypeptide.
[0082] In some embodiments, the fragment is a truncated fragment of any of SEQ ID
NOS: 1-36 or 47-66, having from 1-5, 1-10, or 1-20 amino acid residues removed from the N-terminus, C-terminus, or both. In some such embodiments, the same number of residues is removed from the N-terminus and the C-terminus, while in other embodiments, a different number of residues is removed from the N-terminus compared to the C-terminus.
[0083] In certain aspects, this application provides fusion proteins that include one or more immunogenic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to a polypeptide of Table 1, 2 or 3. In certain embodiments, the fusion protein comprises at least two different polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 1-36 or 47-66, or an immunogenic fragment thereof.
[0084] In certain embodiments, this application provides nucleic acids encoding one or more of the polypeptides described above, such as cDNA, DNA, RNA, or an analog thereof. The underlying DNA sequences for the polypeptides described above may be modified in ways that do not affect the sequence of the protein product, and such sequences are included in the invention. For instance, the DNA sequence may be codon-optimized to improve expression in a host such as E. coli, an insect cell line (e.g., using the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line.
[0085] In certain embodiments, this application provides nucleic acids (such as cDNA, DNA, RNA, or an analog thereof) that are at least 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a gene in Table 1, 2 or 3, or a variant or portion of said gene. In certain embodiments, the nucleic acid is 600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In some embodiments, the nucleic acid is 600-1600, 800-1800, or 1000-2000 nucleotides in length. The nucleic acids may be used, for example, for recombinant production of the polypeptides of Tables 1, 2 and 3, or immunogenic fragments or fusion proteins thereof.
C. Immunogenic compositions
[0086] The present disclosure also provides pharmaceutical compositions containing immunogenic polypeptides or polynucleotides encoding these immunogenic polypeptides, including fusion proteins, together with a pharmaceutical carrier. The S. pneumoniae antigens used were identified by screening immune cells from mice either immunized with unencapsulated killed whole S. pneumoniae or infected with S. pneumoniae, or from healthy human donors, that elicit a T cell specific immune response. The human donors had almost certainly been exposed to S. pneumoniae at some point during their lifetimes, because S. pneumoniae is a very common disease and colonizing pathogen. In some embodiments, these antigens are fused to known S. pneumoniae capsular antigens, known to stimulate a B- cell specific immunity. Thus, the present disclosure contemplates compositions of (e.g., fusion proteins including) 5*. pneumoniae antigens that elicit an immune response in all age groups that is strong and long-lived in immunized or infected mice or humans, for counteracting infection by S. pneumoniae.
[0087] In certain embodiments, a vaccine composition described herein induces a
TH17 cell response at least 1.5-fold greater than that induced by a control unrelated antigen (such as the HSV-2 protein ICP47 with the gene name US 12) after contacting TH17 cells. In some embodiments, a vaccine formulation described herein inhibits infection by S.
pneumoniae in an uninfected subject. In certain embodiments, a vaccine formulation reduces occurrence or duration of S. pneumoniae nasopharyngeal colonization in an individual infected by S. pneumoniae. In some embodiments, a vaccine formulation inhibits development of sepsis in an individual infected by S. pneumoniae. In some embodiments, a vaccine formulation inhibits development of invasive diseases such as pneumonia, meningitis, otitis media, sinusitis or infection of other sites or organs with S. pneumoniae.
[0088] An immunogenic composition may also comprise portions of said
Streptococcus polypeptides, including fusion proteins, for example deletion mutants, truncation mutants, oligonucleotides, and peptide fragments. In some embodiments, the portions of said polypeptides are immunogenic. The immunogenicity of a portion of a protein is readily determined using the same assays that are used to determine the immunogenicity of the full-length protein. In some embodiments, the portion of the polypeptide has substantially the same immunogenicity as the full-length proteins. In some embodiments, the immunogenicity is no more than 10%, 20%, 30%, 40%, or 50% less than that of the full-length protein (e.g., polypeptides of Tables 1, 2 and 3). The peptide fragments may be, for example, linear, circular, or branched.
[0089] In some embodiments, a vaccine formulation contains one or more fusion proteins described herein in combination with one or more, e.g., two, three, four, or more polypeptides from Table 1 or 2 or immunogenic fragments or variants thereof in a mixture. In some embodiments, the mixture contains both full-length polypeptides and fragments resulting from processing, or partial processing, of signal sequences by an expression host, e.g. E. coli, an insect cell line (e.g., the baculovirus expression system), or a mammalian (e.g., human or Chinese Hamster Ovary) cell line. In some embodiments, a vaccine formulation contains one or more fusion proteins of any of SEQ ID NOS: 55-66 in the absence of any other antigens. In some embodiments, a vaccine formulation contains one or more fusion proteins of any of SEQ ID NOS: 55-66 in combination with one or more additional proteins of any of SEQ ID NOS: 1-26 and 47-54 for eliciting a T cell response, in the absence of other antigens.
[0090] In some embodiments, polypeptides described in Tables 1 and 2 may be used without modification in conjunction with the fusion proteins described herein. In certain embodiments, when smaller related polypeptides are used, such as fragments or the like, and their molecular weight is less than about 5000 daltons, e.g., 1500 to 5000 daltons, modification may be useful in eliciting the desired immune response. For example, the smaller polypeptides can be conjugated to an appropriate immunogenic carrier such as tetanus toxoid, keyhole limpet hemocyanin or the like. In some embodiments, polypeptides described herein may be conjugated to S. pneumoniae polysaccharides. The conjugated polysaccharides may be, for example, as described in US Patent 5,623,057, US Patent 5,371, 197, or PCT/US2011/023526.
[0091] Some embodiments of the vaccine formulations and immunogenic
compositions described herein include an immunogenic polypeptide, including a fusion protein (e.g., a polypeptide of Table 1, 2 or 3) that contains a membrane translocating sequence (MTS), to facilitate introduction of the polypeptide into the mammalian cell and subsequent stimulation of the cell-mediated immune response. Exemplary membrane translocating sequences include hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of a-synuclein, β-synuclein, or γ-synuclein, the third helix of the Antennapedia homeodomain, SN50, integrin β3 h-region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and those MTSs described in US Patents 6,248,558, 6,432,680 and 6,248,558.
[0092] In certain embodiments, an antigen (e.g., a polypeptide of Table 1, 2 or 3) is covalently bound to another molecule. This may, for example, increase the half-life, solubility, bioabailability, affinity, or immunogenicity of the antigen, or raise an immune reaction against more than one pathogen. Molecules that may be covalently bound to the antigen include a carbohydrate, biotin, avidin or avidin derivatives, poly(ethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA. There are many different types of PEG, ranging from molecular weights of below 300 g/mol to over 10,000,000 g/mol. PEG chains can be linear, branched, or with comb or star geometries. In some embodiments, a polypeptide described herein is covalently bound to a moeity that stimulates the immune system. An example of such a moeity is a lipid moeity. In some instances, lipid moieties are recognized by a Toll-like receptor (TLR) such as TLR-2 or TLR-4, and activate the innate immune system.
D. Components of a vaccine or immunogenic composition comprising S.
pneumoniae antigens or antibodies recognizing the same
[0093] In certain embodiments, a vaccine or immunogenic composition described herein comprises one or more fusion proteins described herein and one or more of the following: an adjuvant, vaccine scaffold, stabilizer, buffer, surfactant, controlled release component, salt, preservative, and/or an antibody specific to a polypeptide included in such composition.
1. Adjuvants
[0094] The vaccine formulations and immunogenic compositions described herein may include an adjuvant. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action: vaccine delivery systems and immunostimulatory adjuvants (see, e.g., Singh et ah, Curr. HIV Res. 1 :309-20, 2003). In most vaccine formulations, the adjuvant provides a signal to the immune system so that it generates a response to the antigen, and the antigen is required for driving the specificity of the response to the pathogen. Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune-stimulating complexes (ISCOMs), nanoparticles, which may be, for example, particles and/or matrices, and liposomes. In contrast, immunostimulatory adjuvants are sometimes derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system.
[0095] Alternatively, adjuvants may be classified as organic and inorganic. Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous aluminum
hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules including macromolecules. An example of an organic adjuvant is cholera toxin. [0096] Adjuvants may also be classified by the response they induce. In some embodiments, the adjuvant induces the activation of ¾1 cells or TH2 cells. In other embodiments, the adjuvant induces the activation of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.
[0097] In certain embodiments, the adjuvant induces the activation of TH17 cells. It may promote the CD4+ or CD8+ T cells to secrete IL-17. In some embodiments, an adjuvant that induces the activation of TH17 cells is one that produces at least a 2-fold, and in some cases a 10-fold, experimental sample to control ratio in the following assay. In the assay, an experimenter compares the IL-17 levels secreted by two populations of cells: (1) cells from animals immunized with the adjuvant and a polypeptide known to induce TH17 activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide. An adjuvant that induces the activation of TH17 cells may cause the cells of population (1) to produce more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2). IL-17 may be measured, for example, by ELISA or ELISPOT. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E. coli, or ETEC), activate a TH17 response. Thus, in some embodiments, the adjuvant is a toxin. Cholera toxin was successfully used in the mouse model to induce protective immunity in conjunction with certain polypeptides from Table 1 (see Examples 1-2). One form of labile toxin is produced by Intercell. Mutant derivates of labile toxin that are active as adjuvants but significantly less toxic can be used as well. Exemplary detoxified mutant derivatives of labile toxin include mutants lacking ADP-ribosyltransferase activity. Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et ah, "Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants" PNAS Vol. 92, pp. 1644-1648, February 1995) and LTK63 (Williams et ah, "Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease" The Journal of Immunology, 2004, 173: 7435-7443), LT- G192 (Douce et al. "Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants" Infect Immun. 1999 Sep;67(9):4400-6), and LTR72 ("Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity." J Exp Med. 1998 Apr 6; 187(7): 1123-32).
[0098] In some embodiments, the adjuvant comprises a VLP (virus-like particle).
One such adjuvant platform, Alphavirus replicons, induces the activation of TH17 cells using alphavirus and is produced by Alphavax. In certain embodiments of the Alphavirus replicon system, alphavirus may be engineered to express an antigen of interest, a cytokine of interest (for example, IL-17 or a cytokine that stimulates IL-17 production), or both, and may be produced in a helper cell line. More detailed information may be found in U.S. Patent Nos. 5,643,576 and 6,783,939. In some embodiments, a vaccine formulation is administered to a patient in combination with a nucleic acid encoding a cytokine.
[0099] Certain classes of adjuvants activate toll-like receptors (TLRs) in order to activate a TH17 response. TLRs are well known proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). Administering a known TLR ligand together with an antigen of interest (for instance, as a fusion protein) can promote the development of an immune response specific to the antigen of interest. One exemplary adjuvant that activates TLRs comprises Monophosphoryl Lipid A (MPL).
Traditionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin obtained from Gram negative bacteria, such as S. Minnesota. In particular, sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction (which is MPL), and lacks the saccharide groups and all but one of the phosphates present in LPS. A number of synthetic TLR agonists (in particular, TLR-4 agonists) are disclosed in Evans JT et al.
"Enhancement of antigen-specific immunity via the TLR-4 ligands MPL adjuvant and Ribi.529." Expert Rev Vaccines 2003 Apr;2(2):219-29. Like MPL adjuvants, these synthetic compounds activate the innate immune system via TLR. Another type of TLR agonist is a synthetic phospholipid dimer, for example E6020 (Ishizaka ST et al. "E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant" Expert Rev. Vaccines 2007 Oct;
6(5):773-84). Various TLR agonists (including TLR-4 agonists) have been produced and/or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate (TDM), and
dioctadecyldimethylammonium bromide (DDA). Another TLR-activating adjuvant is R848 (resiquimod).
[0100] In some embodiments, the adjuvant is or comprises a saponin. Typically, the saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree. A saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C. [0101] A particular form of saponins that may be used in vaccine formulations described herein is immunostimulating complexes (ISCOMs). ISCOMs are an art-recognized class of adjuvants, that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline). In certain embodiments, an ISCOM is assembled together with a polypeptide or nucleic acid of interest. However, different saponin fractions may be used in different ratios. In addition, the different saponin fractions may either exist together in the same particles or have substantially only one fraction per particle (such that the indicated ratio of fractions A and C are generated by mixing together particles with the different fractions). In this context, "substantially" refers to less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or even 1%. Such adjuvants may comprise fraction A and fraction C mixed into a ratio of 70-95 A: 30-5 C, such as 70 A : 30 C to 75 A : 5 C, 75 A : 5 C to 80 A : 20 C, 80 A : 20 C to 85 A : 15 C, 85 A : 15 C to 90 A : 10 C, 90 A : 10 C to 95 A : 5 C, or 95 A : 5 C to 99 A : 1 C.
[0102] In certain embodiments, combinations of adjuvants are used. Three exemplary combinations of adjuvants are MPL and alum, E6020 and alum, and MPL and an ISCOM.
[0103] Adjuvants may be covalently bound to antigens (e.g., to one or more polypeptides included in a fusion protein described herein). In some embodiments, the adjuvant may comprise a protein which induces inflammatory responses through activation of antigen-presenting cells (APCs). In some embodiments, one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (see Wu et al, Cancer Res 2005; 65(1 1), pp 4947-4954). In certain embodiments, a polypeptide, including a fusion protein, described herein is presented in the context of the trivalent conjugate system, comprising a fusion protein of S. pneumoniae Pneumococcal surface adhesin A (PsaA) with the pneumolysoid PdT and a cell wall polysaccharide (PsaA:PdT-CPs), described in Lu et al. ("Protection against Pneumococcal colonization and fatal pneumonia by a trivalent conjugate of a fusion protein with the cell wall polysaccharide." Infect Immun. 2009 May;77(5):2076-83). PdT carries three amino acid substitutions (W433F, D385N, and C428G) which render the molecule nontoxic but do not interfere with its TLR-4-mediated inflammatory properties. Conjugation of a
polysaccharide to the fusion of a polypeptide to the TLR-4-agonist PdT enhances
immunological response to the polypeptide. In some embodiments, one or more polypeptides described herein are used in place of PsaA in the trivalent conjugate. The trivalent conjugate system typically includes alum and is usually administered parenterally. Other exemplary adjuvants that may be covalently bound to antigens comprise polysaccharides, pneumolysin, synthetic peptides, lipopeptides, and nucleic acids.
[0104] Typically, the same adjuvant or mixture of adjuvants is present in each dose of a vaccine. Optionally, however, an adjuvant may be administered with the first dose of vaccine and not with subsequent doses (i.e., booster shots). Alternatively, a strong adjuvant may be administered with the first dose of vaccine and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses. The adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human patients, non-human animals, or both.
2. Additional components of a vaccine or immunogenic composition
[0105] In addition to one or more fusion proteins and adjuvants described above, a vaccine formulation or immunogenic composition may include one or more additional components.
[0106] In certain embodiments, the vaccine formulation or immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L- glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
[0107] In certain embodiments, the vaccine formulation or immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.
[0108] In certain embodiments, the vaccine formulation or immunogenic composition includes one or more surfactants such as polysorbate 80 (Tween 80), Triton X-100,
Polyethylene glycol tert-octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-( 1, 1,3,3- Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100); Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN 20); and 4-(l, 1,3,3- Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic or nonionic.
[0109] In certain embodiments, the vaccine formulation or immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.
[0110] In certain embodiments, a preservative is included in the vaccine or immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.
[0111] In certain embodiments, the vaccine formulation or immunogenic composition is a controlled release formulation.
E. Use of Vaccines
[0112] The S. pneumoniae vaccines described herein may be used for prophylactic and/or therapeutic treatment of S. pneumoniae. In some embodiments, vaccine compositions may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce risk of infection by, and reduce severity of, and/or reduce incidence of one or more symptoms or features of a pneumococcal disease, disorder, and/or condition. In some embodiments, vaccine compositions may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of pneumococcal infection (e.g., but not limited to sepsis, pneumonia, acute otitis media, bacteremia, and bacterial meningitis). In some
embodiments, vaccine compositions may be used to reduce the incidence of pneumococcal infection in subjects administered the composition. In some embodiments, vaccine compositions may be used to reduce the incidence of nasopharyngeal colonization by a pneumococcal organism. In some embodiments, vaccine compositions may be used to reduce the likelihood of chronic infection by a pneumococcal organism. In some embodiments, vaccine compositions may be used to reduce the likelihood of suffering from pelvic inflammatory disease or infertility subsequent to a pneumococcal infection.
[0113] In some embodiments, prophylactic and/or therapeutic protocols involve administering a therapeutically effective amount of one or more vacc ine compositions de s c rib e d h e re in to a healthy subject (i.e., a subject who does not display any symptoms of, and/or who has not been diagnosed with, pneumococcal infection). For example, healthy individuals may be vaccinated using inventive vaccine compositions prior to pneumococcal colonization, development of pneumococcal infection and/or onset of symptoms of pneumococcal infection; at risk individuals (e.g., patients exposed to individuals suffering from pneumococcal infection, patients with HIV or a compromised immune system, or a subject who is susceptible is to pneumococcal infection such as a subject with Job's syndrome (subject lacking TH17 cell-mediated response) or a subject with aggamaglobunemic (a subject lacking antibody-mediated response) can be treated substantially contemporaneously with (e.g., within 48 hours, within 24 hours, or within 12 hours of) the onset of symptoms of and/or exposure to pneumococcal infection. Of course individuals known to have pneumococcus infection or sepsis may receive treatment at any time.
[0114] In some embodiments, prophylactic and/or therapeutic protocols involve administering a therapeutically effective amount of one or more inventive vaccine compositions to a subject such that an immune response is stimulated in both T cells and B cells.
[0115] In some embodiments, by combining one or more antigens and adjuvants, immune responses (e.g. T cell and/or B cell responses) can be tailored to preferentially elicit the most desirable type of immune response for a given indication, e.g., humoral response, mucosal response, THl-biased T cell response, TH17-biased T cell response, IFN-γ secretion by antigen-specific T cells, IL- 17A secretion by antigen-specific T cells, cytotoxic T cell response, antibody response, B cell response, ad aptiv e immune re sp o n s e , innate immune response, or a combination of these responses .
[0116] Accordingly, this application provides a method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering an effective amount of any of the vaccine formulations described herein. In some aspects, the method inhibits S. pneumoniae colonization in an individual. In some aspects, the method inhibits S. pneumoniae symptoms, invasive disease or sequelae, such as sepsis, pneumonia, meningitis, otitis media, sinusitis or infection of other sites or organs with S. pneumoniae. The subject receiving the vaccination may be a male or a female, and may be a child or adult. In some embodiments, the subject being treated is a human. In other embodiments, the subject is a non-human animal. 1. Prophylactic use
[0117] In prophylactic embodiments, the vaccine is administered to a subject to induce an immune response that can help protect against the establishment of S. pneumoniae, for example by protecting against colonization, the first and necessary step in disease. Thus, in some aspects, the method inhibits infection by S. pneumoniae in a non-colonized or uninfected subject. In another aspect, the method may reduce the duration of colonization in an individual who is already colonized.
[0118] In some embodiments, the vaccine compositions described herein confer protective immunity, allowing a vaccinated individual to exhibit delayed onset of symptoms or sequelae, or reduced severity of symptoms or sequelae, as the result of his or her exposure to the vaccine. In certain embodiments, the reduction in severity of symptoms or sequelae is at least 25%, 40%, 50%, 60%, 70%, 80% or even 90%. In particular embodiments, vaccinated individuals may display no symptoms or sequelae upon contact with S.
pneumoniae, do not become colonized by S. pneumoniae, or both. Protective immunity is typically achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. The sIGA antibodies are generated after a series of events, mediated by antigen-processing cells, B and T lymphocytes, that results in sIGA production by B lymphocytes on mucosa- lined tissues of the body. Humoral immunity is typically the result of IgG antibodies and IgM antibodies in serum. Cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies. In particular, cellular immunity may be mediated by THI or TH17 cells.
[0119] Essentially any individual has a certain risk of becoming infected with 5*. pneumoniae. However, certain sub-populations have an increased risk of infection. In some embodiments, a vaccine formulation as described herein is administered to patients that are immunocompromised.
[0120] An immunocompromising condition, e.g., arising from a medical treatment, can expose the individual in question to a higher risk of infection with 5*. pneumoniae. It is possible to treat an infection prophylactically in an individual having an
immunocompromised condition before or during treatments known to compromise immune function. By prophylactically treating with an antigenic composition (e.g., a fusion protein described herein), or with antibodies reactive to two or more antigens from Table 1 or 2, or to an antigen from Table 3, before or during a treatment known to compromise immune function, it is possible to prevent a subsequent S. pneumoniae infection or to reduce the risk of the individual contracting an infection due to the immunocompromised condition. Should the individual contract an 5*. pneumoniae infection e.g., following a treatment leading to an immunocompromised condition it is also possible to treat the infection by administering to the individual an antigen composition.
[0121] The following groups are at increased risk of pneumococcal disease or its complications, and therefore it is advantageous for subjects falling into one or more of these groups to receive a vaccine formulation described herein: children, especially those from 1 month to 5 years old or 2 months to 2 years old; children who are at least 2 years of age with asplenia, splenic dysfunction or sickle-cell disease; children who are at least 2 years of age with nephrotic syndrome, chronic cerebrospinal fluid leak, HIV infection or other conditions associated with immunosuppression.
[0122] In another embodiment, at least one dose of a vaccine composition described helrein is given to adults in the following groups at increased risk of pneumococcal disease or its complications: all persons 65 years of age; adults with asplenia, splenic dysfunction or sickle-cell disease; adults with the following conditions: chronic cardiorespiratory disease, cirrhosis, alcoholism, chronic renal disease, nephrotic syndrome, diabetes mellitus, chronic cerebrospinal fluid leak, HIV infection, AIDS and other conditions associated with immunosuppression (Hodgkin's disease, lymphoma, multiple myeloma, immunosuppression for organ transplantation), individuals with cochlear implants; individuals with long-term health problems such as heart disease and lung disease, as well as individuals who are taking any drug or treatment that lowers the body's resistance to infection, such as long-term steroids, certain cancer drugs, radiation therapy; Alaskan natives and certain Native
American populations.
2. Therapeutic use
[0123] In therapeutic applications, a vaccine formulation described herein may be administered to a patient suffering from 5*. pneumoniae infection, in an amount sufficient to treat the patient. Treating the patient, in this case, refers to reducing S. pneumoniae symptoms and/or bacterial load and/or sequelae in an infected individual. In some embodiments, treating the patient refers to reducing the duration of symptoms or sequelae, or reducing the intensity of symptoms or sequelae. In some embodiments, treating the patient refers to eliminating pneumococcal organisms in the nasopharynx or reducing the duration of nasopharyngeal colonization. In some embodiments, the vaccine reduces transmissibility of S. pneumoniae from the vaccinated patient. In certain embodiments, the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or even 90%.
[0124] In therapeutic embodiments, a vaccine is administered to an individual postinfection. The vaccine may be administered shortly after infection, e.g. before symptoms or sequelae manifest, or may be administered during or after manifestation of symptoms or sequelae.
[0125] A vaccine formulation described herein can reduce the intensity and/or duration of the various symptoms or sequelae of S. pneumoniae infection. Symptoms or sequelae of S. pneumoniae infection can take many forms. In some cases, an infected patient develops pneumonia, acute sinusitis, otitis media (ear infection), meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brain abscess.
[0126] Sepsis is a rare but life-threatening complication of S. pneumoniae infection, where the bacterium invades the bloodstream and systemic inflammation results. Typically, fever is observed and white blood cell count increases. A further description of sepsis is found in Goldstein, B. et al. "International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics." Pediatr Crit Care Med. Jan 2005;6 (l):2-8. 3. Assaying vaccination efficacy
[0127] The efficacy of vaccination with vaccines disclosed herein may be determined in a number of ways, in addition to the clinical outcomes described above. First, one may assay IL-17 levels (particularly IL-17A) by stimulating T cells derived from the subject after vaccination with a vaccine composition described herein. The IL-17 levels may be compared to IL-17 levels in the same subject before vaccination. Increased IL-17 (e.g., IL-17 A) levels, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine. Alternatively (or in combination), one may assay neutrophils in the presence of T cells or antibodies from the patient for pneumococcal killing. Increased pneumococcal killing, such as a 1.5 fold, 2-fold, 5-fold, 10- fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine. In addition, one may measure TH17 cell activation, where increased ¾17 cell activation, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates with an increased response to the vaccine. One may also measure levels of an antibody specific to the vaccine, where increased levels of the specific antibody, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, are correlated with increased vaccine efficacy. In certain embodiments, two or more of these assays are used. For example, one may measure IL-17 levels and the levels of vaccine- specific antibody. In certain embodiments, one may assay for additional cytokines and transcription factors in the IL-17 lineage, e.g., cytokines IL-22, IL-23, 11-6 and TGF-B, and transcription factors STAT3, RORyt and RORa. Alternatively, one may follow
epidemiological markers such as incidence of, severity of, or duration of pneumococcal infection in vaccinated individuals compared to unvaccinated individuals.
[0128] Vaccine efficacy may also be assayed in various model systems such as the mouse model. For instance, BALB/c or C57BL/6 strains of mice may be used. After administering a vaccine formulation described herein to a subject (as a single dose or multiple doses), the experimenter administers a challenge dose of S. pneumoniae. In some cases, a challenge dose administered intranasally is sufficient to cause S. pneumoniae colonization (especially nasal colonization) in an unvaccinated animal, and in some cases a challenge dose administered via aspiration is sufficient to cause sepsis and a high rate of lethality in unvaccinated animals. One can then measure the reduction in colonization or the reduction in lethality in vaccinated animals. Examples 1-2 and 4 show the efficacy of polypeptides of Table 1 in inhibiting 5*. pneumoniae nasal colonization following intranasal challenge in the mouse model. Examples 5 and 6 show the efficacy of polypeptides of Table 1 in protecting against sepsis and death following infection with 5*. pneumoniae via aspiration in the mouse model.
F. Use of Immunogenic Compositions
1. Defense against S. pneumoniae infection
[0129] The immunogenic compositions (e.g., vaccine formulations) of the present disclosure are designed to elicit an immune response against S. pneumoniae. Compositions described herein (e.g., ones comprising one or more fusion proteins described herein) may stimulate an antibody response or a cell-mediated immune response, or both, in the mammal to which it is administered. In some embodiments, the composition stimulates a ¾1 -biased CD4+ T cell response, a TH17-biased CD4+ T cell response and/or a CD8+ T cell response. In some embodiments, the composition elicits IFN-γ secretion by antigen-specific T cells. In some embodiments, the composition elicits IL-17A secretion by antigen-specific T cells. In some embodiments, the composition stimulates an antibody response (e.g., an IgG, IgM and/or IgA response). In some embodiments, the composition stimulates a B cell response. In some embodiments, the composition stimulates a cytotoxic T cell (CTL) response. In some embodiments, the composition stimulates an adaptive immune response. In some embodiments, the composition stimulates an innate immune response. In some embodiments, the composition stimulates both a T cell response and a B cell response. In some embodiments, the composition stimulates a THl-biased CD4+ T cell response, TH17-biased CD4+ T cell response and/or a CD8+ T cell response, and an antibody and/or B cell response.
[0130] In certain embodiments, the composition (e.g., one comprising one or more fusion proteins described herein) includes a cytokine or nucleotide coding region encoding a cytokine such as IL- 17, to provide additional stimulation to the immune system of the mammal. In certain embodiments, the composition comprises a cytokine such as IL-17.
[0131] While not wishing to be bound by theory, in some embodiments a TH17 cell response is desirable in mounting an immune response to the compositions disclosed herein. In certain embodiments, an active TH17 response is beneficial in clearing a pneumococcal infection. For instance, mice lacking the IL-17A receptor show decreased whole cell vaccine-based protection from a pneumococcal challenge (Lu et ah, "Interleukin-17A mediates acquired immunity to pneumococcal colonization." PLoS Pathog. 2008 Sep 19;4(9)).
[0132] Thus, in some embodiments, the present disclosure includes methods of increasing IL-17 production by administering one or more compositions described herein (e.g., ones comprising one or more fusion proteins described herein) to a subject.
Furthermore, the present disclosure includes methods of activating TH17 cells by
administering said compositions to a subject. In certain embodiments, increased IL-17A levels result in increased pneumococcal killing by neutrophils or neutrophil-like cells, for instance by inducing recruitment and activation of neutrophils of neutrophil-like cells. In certain embodiments, this pneumococcal killing is independent of antibodies and
complement. However, specific antibody production and complement activation may be useful additional mechanisms that contribute to clearing of a pneumococcal infection.
[0133] Immunogenic compositions containing one or more fusion proteins described herein (or nucleic acids encoding a fusion protein described herein) together with a pharmaceutical carrier are also provided.
[0134] In some instances, the immunogenic composition comprises one or more nucleic acids (e.g., cDNA molecules) encoding one or more polypeptides of SEQ ID NOS: 1- 66, such as one or more polypeptides selected from SEQ ID NOS 55-66. In some embodiments these nucleic acids are expressed in the immunized individual, producing the encoded S. pneumoniae antigens, and the S. pneumoniae antigens so produced can produce an immunostimulatory effect in the immunized individual.
[0135] Such a nucleic acid-containing immunostimulatory composition may comprise, for example, an origin of replication, and a promoter that drives expression of one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 55-66. Such a composition may also comprise a bacterial plasmid vector into which is inserted a promoter (sometimes a strong viral promoter), one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 55-66, and a polyadenylation/transcriptional termination sequence. In some instances, the nucleic acid is DNA.
G. Doses and Routes of Administration
1. Dosage forms, amounts, and timing
[0136] The amount of a fusion protein described herein in each vaccine or immunogenic composition dose is selected as an effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses. Preferably, the dose is without significant adverse side effects in typical vaccinees. Such amount will vary depending upon which specific antigen is employed. Generally, it is expected that a dose will comprise 1-1000 μg of each protein, in some instances 2-100 μg, for instance 4-40 μg. In some aspects, the vaccine formulation comprises 1-1000 μg of the polypeptide and 1-350 μg of the adjuvant. In some embodiments, the appropriate amount of antigen to be delivered will depend on the age, weight, and health (e.g. immune-compromised status) of a subject. When present, typically an adjuvant will be present in amounts from 1 μg - 350 μg per dose, for example 50-150 μg, 75-125 μg or 100 μ&
[0137] In some embodiments, only one dose of the vaccine is administered to achieve the results described above. In other embodiments, following an initial vaccination, subjects receive one or more boost vaccinations, for a total of two, three, four or five vaccinations. Advantageously, the number is three or fewer. A boost vaccination may be administered, for example, about 1 month, 2 months, 4 months, 6 months, or 12 months after the initial vaccination, such that one vaccination regimen involves administration at 0, 0.5-2 and 4-8 months. It may be advantageous to administer split doses of vaccines which may be administered by the same or different routes. [0138] The vaccines and immunogenic compositions described herein may take on a variety of dosage forms. In certain embodiments, the composition is provided in solid or powdered (e.g., lyophilized) form; it also may be provided in solution form. In certain embodiments, a dosage form is provided as a dose of lyophilized composition and at least one separate sterile container of diluent.
[0139] In some embodiments, the composition will be administered in a dose escalation manner, such that successive administrations of the composition contain a higher concentration of composition than previous administrations. In some embodiments, the composition will be administered in a manner such that successive administrations of the composition contain a lower concentration of composition than previous administrations.
[0140] In therapeutic applications, compositions are administered to a patient suffering from a disease in an amount sufficient to treat the patient. Therapeutic applications of a composition described herein include reducing transmissibility, slowing disease progression, reducing bacterial viability or replication, or inhibiting the expression of proteins required for toxicity, such as by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the levels at which they would occur in individuals who are not treated with the composition.
[0141] In prophylactic embodiments, compositions are administered to a human or other mammal to induce an immune response that can inhibit the establishment of an infectious disease or other condition. In some embodiments, a composition may partially block the bacterium from establishing an infection.
[0142] A therapeutically effective amount may be determined on a population basis, and is not required to be an amount that naturally induces a protective response in a particular subject.
[0143] In some embodiments, the compositions are administered in combination with antibiotics. This co-administration is particularly appropriate for a patient who has recently been exposed (or is suspected of having been recently exposed) to, colonized or infected by S. pneumoniae. Many antibiotics are used to treat pneumococcal infections, including penicillin, amoxicillin, amoxicillin/clavulanate, cefuroxime, cefotaxime, ceftriaxone, and vancomycin. The appropriate antibiotic may be selected based on the type and severity of the infection, as well as any known antibiotic resistance of the infection (Jacobs MR "Drug- resistant Streptococcus pneumoniae: rational antibiotic choices" Am J Med. 1999 May 3; 106(5A): 19S-25S).
[0144] In some embodiments, the compositions are administered in combination with one or more additional vaccines, such as Prevnar-13 (Wyeth Pharmaceuticals, Inc., Philadelphia, PA) or Pneumovax (Merck & Co., Inc., Whitehouse Station, NJ ) . This coadministration can be used, for example, to treat a patient who has been exposed to, colonized or infected by S. pneumoniae serotypes that are not represented in said existing vaccine.
2. Routes of administration
[0145] The vaccine formulations and pharmaceutical compositions herein can be delivered by administration to an individual, typically by systemic administration (e.g., by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, or buccal routes; by inhalation; or intratracheal or bronchial instillation) or they can be administered by topical application. In some embodiments, the route of administration is intramuscular. In other embodiments, the route of administration is subcutaneous. In yet other embodiments, the route of administration is mucosal. In certain embodiments, the route of administration is transdermal or intradermal.
[0146] Certain routes of administration are particularly appropriate for vaccine formulations and immunogenic compositions comprising specified adjuvants. In particular, transdermal administration is one suitable route of administration for 5*. pneumoniae vaccines comprising toxins (e.g. cholera toxin or labile toxin); in other embodiments, the
administration is intranasal. Vaccines formulated with Alphavirus replicons may be administered, for example, by the intramuscular or the subcutaneous route. Vaccines comprising aluminum hydroxide or aluminum phosphate (alum), Monophosphory Lipid A (MPL), Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide (DDA) are suitable (inter alia) for intramuscular and subcutaneous administration. A vaccine comprising resiquimod may be administered topically or subcutaneously, for example.
3. Formulations
[0147] The vaccine formulation or immunogenic composition may be suitable for administration to a human patient, and vaccine or immunogenic composition preparation may conform to USFDA guidelines. In some embodiments, the vaccine formulation or immunogenic composition is suitable for administration to a non-human animal. In some embodiments, the vaccine or immunogenic composition is substantially free of either endotoxins or exotoxins. Endotoxins may include pyrogens, such as lipopolysaccharide (LPS) molecules. The vaccine or immunogenic composition may also be substantially free of inactive protein fragments which may cause a fever or other side effects. In some embodiments, the composition contains less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/or inactive protein fragments. In some embodiments, the vaccine or immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water. Other vaccine or
immunogenic composition components may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation. In other embodiments, the pyrogens may be inactivated or destroyed prior to administration to a patient. Raw materials for vaccines, such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in the vaccine may be sterile, and each lot of the vaccine may be tested for sterility. Thus, in certain embodiments the endotoxin levels in the vaccine fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU kg of product for a non- intrathecal injectable composition, and 0.25-0.5 EU/mL for sterile water.
[0148] In certain embodiments, the preparation comprises less than 50%, 20%, 10%, or 5% (by dry weight) contaminating protein. In certain embodiments, a fusion protein described herein is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture). In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). In some embodiments, the vaccine or immunogenic composition comprising purified subunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of protein from host cells in which the subunit proteins were expressed, relative to the amount of purified subunit. In some embodiments, the desired polypeptides are substantially free of nucleic acids and/or carbohydrates. For instance, in some embodiments, the vaccine or immunogenic composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNA and/or RNA. In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of biological macromolecules of the same type are present in the preparation (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
[0149] It is preferred that the vaccine or immunogenic composition has low or no toxicity, within a reasonable risk-benefit ratio. In certain embodiments, the vaccine or immunogenic composition comprises ingredients at concentrations that are less than LD50 measurements for the animal being vaccinated. LD50 measurements may be obtained in mice or other experimental model systems, and extrapolated to humans and other animals.
Methods for estimating the LD50 of compounds in humans and other animals are well-known in the art. A vaccine formulation or immunogenic composition, and any component within it, might have an LD50 value in rats of greater than 100 g/kg, greater than 50g/kg, greater than 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2 g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200 mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20 mg/kg, or greater than 10 mg/kg. A vaccine formulation or immunogenic composition that comprises a toxin such as botulinum toxin (which can be used as an adjuvant) should contain significantly less than the LD50 of botulinum toxin.
[0150] The formulations suitable for introduction of the vaccine formulations or pharmaceutical composition vary according to route of administration. Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
[0151] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. In the case of adoptive transfer of therapeutic T cells, the cells can be administered intravenously or parenterally.
[0152] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of a fusion protein described herein or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. The pharmaceutical compositions can be encapsulated, e.g., in liposomes, or in a formulation that provides for slow release of the active ingredient.
[0153] A fusion protein described herein, alone or in combination with other suitable components, can be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
[0154] Suitable formulations for vaginal or rectal administration include, for example, suppositories, which consist of the polypeptides or packaged nucleic acids with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the polypeptides or packaged nucleic acids with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
H. Preparation and Storage of Vaccine Formulations and Immunogenic
Compositions
[0155] The S. pneumoniae vaccines and immunogenic compositions described herein may be produced using a variety of techniques. For example, a polypeptide (e.g., a fusion protein) may be produced using recombinant DNA technology in a suitable host cell. A suitable host cell may be bacterial, yeast, mammalian, or other type of cell. The host cell may be modified to express an exogenous copy of one of the relevant polypeptide genes. Typically, the gene is operably linked to appropriate regulatory sequences such as a strong promoter and a polyadenylation sequence. In some embodiments, the promoter is inducible or repressible. Other regulatory sequences may provide for secretion or excretion of the polypeptide of interest or retention of the polypeptide of interest in the cytoplasm or in the membrane, depending on how one wishes to purify the polypeptide. The gene may be present on an extrachromosomal plasmid, or may be integrated into the host genome. One of skill in the art will recognize that it is not necessary to use a nucleic acid 100% identical to the naturally-occurring sequence. Rather, some alterations to these sequences are tolerated and may be desirable. For instance, the nucleic acid may be altered to take advantage of the degeneracy of the genetic code such that the encoded polypeptide remains the same. In some embodiments, the gene is codon-optimized to improve expression in a particular host. The nucleic acid may be produced, for example, by PCR or by chemical synthesis.
[0156] Once a recombinant cell line has been produced, a polypeptide (e.g., fusion protein) may be isolated from it. The isolation may be accomplished, for example, by affinity purification techniques or by physical separation techniques (e.g., a size column).
[0157] In a further aspect of the present disclosure, there is provided a method of manufacture comprising mixing one or more fusion proteins described herein with a carrier and/or an adjuvant.
[0158] In some embodiments, fusion proteins for inclusion the vaccine formulations and immunogenic compositions may be produced in cell culture. One method comprises providing one or more expression vectors and cloning nucleotides encoding one or more fusion proteins described herein, then expressing and isolating the fusion proteins.
[0159] The immunogenic fusion proteins described herein, and nucleic acid compositions that express the fusion proteins, can be packaged in packs, dispenser devices, and kits for administering nucleic acid compositions to a mammal. For example, packs or dispenser devices that contain one or more unit dosage forms are provided. Typically, instructions for administration of the composition will be provided with the packaging, along with a suitable indication on the label that the composition is suitable for treatment of an indicated condition, such as those disclosed herein.
[0160] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
[0161] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way. EXAMPLES
Example 1
SP2108, SP0148 and SP1634 polypeptides
[0162] The SP2108 polypeptide (SEQ ID NO: 6), SP0148 polypeptide (SEQ ID NO:
2) and SP1634 polypeptide (SEQ ID NO: 25) were formulated as vaccine compositions using 4 μg of the polypeptide in combination with 1 μg cholera toxin adjuvant (CT). For combinations, 4 μg of each polypeptide was used. The compositions were administered intranasally to C57BL/6 mice three times, one week apart. The mice were then allowed to rest for 3 weeks, and bled at that time for immunogenicity. For this assay, heparinized whole blood was collected from the retrograde orbital sinus. The total PBMC were stimulated with either killed, unencapsulated whole cell S. pneumoniae (WCC) or a combination of the three polypeptides in round bottomed tubes for three days. The supernatants were then harvested and evaluated by ELISA for IL-17 levels. Cholera toxin alone (CT) or an unrelated antigen from HSV (003) were used as negative controls. Results of the IL-17 immunogenicity assay are shown in FIGS. 1 and 2, where the left panels show data in scatter format, and the right panels show data as averages with standard deviations. The subjects were allowed to rest an additional 2 weeks, at which time they were challenged with intranasal administration of live, encapsulated S. pneumoniae. The subjects were sacrificed a week later, and the number of colony-forming units (CFU) was counted from nasal washes. Results of the colonization assay are shown in FIG. 3.
Example 2
SP0148, SP0314, SP0882, and SP2108 polypeptides tested in the BALB/c mouse
[0163] To determine whether similar immune responses were seen across different mouse genotypes, vaccine compositions were administered to BALB/c mice. Vaccine compositions comprised the polypeptides SP0148 (SEQ ID NO: 2), SP0314 (SEQ ID NO: 26), SP0882 (SEQ ID NO: 17) or SP2108 (SEQ ID NO: 6), and cholera toxin adjuvant (CT). Using a protocol similar to that of Example 1, the mice were immunized, challenged intranasally with S. pneumoniae, and the number of CFU was recorded. The results of this colonization experiment are shown in FIG. 4.
Example 3
SP1912, SP2108 and SP0148 polypeptides: IL-17A immunogenicity assay
[0164] The polypeptides SP1912 (SEQ ID NO: 9), SP2108 (SEQ ID NO: 6) or
SP0148 (SEQ ID NO: 2) were formulated as vaccine compositions with cholera toxin adjuvant (CT). The vaccine compositions were administered to mice two times, one week apart. The positive control was killed, unencapsulated whole cell S. pneumoniae + CT (WCB), and the negative controls were CT alone or recombinant proteins without CT (with the exception of SP1912). Three weeks after the last immunization, peripheral blood was collected from the retroorbital sinus and evaluated in a whole blood assay. Briefly, the heparizined whole blood was diluted in media and then cultured in duplicate with A) the protein of immunization, or B) the whole cell vaccine for six days. The supernatants were harvested and IL-17A levels measured by ELISA. Results of the IL-17A immunogenicity assay are shown in FIG. 5. Each symbol in the graph represents responses from individual mice, and the line indicates the median response of the group.
Example 4
SP1912, SP2108 and SP0148 polypeptides: colonization assay
[0165] Animals were immunized with vaccine formulations comprising the polypeptides SP1912 (SEQ ID NO:9), SP2108 (SEQ ID NO: 6) or SP0148 (SEQ ID NO: 2) and cholera toxin adjuvant (CT) as described in Example 3, and then challenged intranasally with 107 live type 6B 5*. pneumoniae four weeks after the last immunization (and one week after retroorbital blood collection). Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Results are shown in FIG. 6 as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. (*** = p-value <0.05).
Example 5
SP1912 polypeptide: aspiration challenge (sepsis assay)
[0166] Polypeptide SP1912 was evaluated for its ability to protect mice from sepsis.
Groups of ten mice were subcutaneously immunized three times, two weeks apart with vaccine compositions comprising either the SP1912 polypeptide (SEQ ID NO: 9) or pneumolysoid (PdT) adsorbed to alum. The positive control was killed, unencapsulated whole cell S. pneumoniae + alum (WCB), and the negative control was alum alone. Three weeks after the final immunization, blood was collected for evaluation of IL-17A response and antibody levels, and then one week later, the mice underwent aspiration challenge with 107 live strain 0603 (type 6B) 5*. pneumoniae. Animals were monitored for survival for eight days. Results of the aspiration challenge are shown in FIG. 7 as survival curves for each immunized group. Example 6
Pneumolysoid PdT, SP0148 and SP0641N polypeptides: aspiration challenge (sepsis assay)
[0167] Polypeptide SP0148 was evaluated for its ability to protect mice from sepsis when immunized singly or in combination with SP0641N and/or pneumolysoid (PdT).
Groups of ten mice were subcutaneously immunized three times, two weeks apart with vaccine compositions comprising polypeptide SP0148 (SEQ ID NO: 2), singly or in combination with polypeptide SP0641N (SEQ ID NO: 13) and/or PdT, adsorbed to alum. The positive control was killed, unencapsulated whole cell S. pneumoniae + alum (WCB), and the negative control was alum alone. Three weeks after the final immunization, blood was collected for evaluation of IL- 17 and antibody, and then one week later, the mice underwent aspiration challenge with 107 live strain 0603 (type 6B) S. pneumoniae. Animals were monitored for survival for eight days. The data are shown in FIG. 8 as survival curves for each immunized group.
Example 7
SP1912, SP2108 and SP0148 polypeptides: colonization assay
[0168] Additional studies were performed essentially as described in Example 4, for a total of four separate studies. Briefly, animals were immunized with vaccine formulations comprising the polypeptides SP 1912 (SEQ ID NO: 9), SP2108 (SEQ ID NO: 6), SP0148 (SEQ ID NO: 2), or additionally SP2108 plus SP0148, and cholera toxin adjuvant (CT) as described in Example 3. Control animals were immunized with killed, unencapsulated whole cell S. pneumoniae plus CT (WCV), or CT alone. Immunized animals were challenged intranasally with 107 live type 6B 5*. pneumoniae four weeks after the last immunization. Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Pooled results of four studies are shown in FIG. 9 as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. (*** = p-value <0.05). N indicates the total number of animals evaluated. Percentages refer to the number of animals protected from colonization. Example 8
SP1912 and SP0148 polypeptides: IL-17A immunogenicity assay
[0169] Groups of ten mice were subcutaneously immunized twice, two weeks apart with vaccine compositions comprising either SP 1912 polypeptide (SEQ ID NO: 9), SP0148 polypeptide (SEQ ID NO: 2), or both adsorbed to alum. Control animals were immunized with alum alone. Three weeks after the last immunization, heparinized blood was collected by cardiac puncture and evaluated for IL-17A levels in a whole blood assay. Briefly, the heparizined whole blood was diluted in media and then cultured for six days with the protein(s) of immunization. The supernatants were harvested and IL-17A levels measured by ELISA. Results of the IL-17A immunogenicity assay are shown in FIG. 10. Each symbol in the graph represents responses from individual mice, and the line indicates the median response of the group.
Example 9
SP1912 and SP0148 polypeptides: colonization assay
[0170] Animals were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 2) at different doses plus and minus SP 1912 (SEQ ID NO: 9), adsorbed to alum. Control animals were immunized with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), or alum alone. Immunized animals were challenged intranasally with 107 live type 6B 5*. pneumoniae four weeks after the last immunization. Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Results are shown in FIG. 1 1 as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. The number of animals protected from colonization out of the number of animals in the group is indicated at the top of the figure. Example 10
SP1912, SP0148, and SP2108 polypeptides: colonization assay
[0171] In two separate studies, animals were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 2) and SP0148 plus SP 1912 (SEQ ID NO: 9), or additionally with SP2108 (SEQ ID NO: 6), SP2108 plus SP0148, and SP2108 plus SP1912, adsorbed to alum. Control animals were immunized with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), or alum alone. Immunized animals were challenged intranasally with 107 live type 6B 5*. pneumoniae four weeks after the last immunization. Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Pooled results of the two studies are shown in FIG. 12 as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. The number of animals protected from colonization out of the number of animals in the group and corresponding percentage of animals protected from colonization are indicated at the top of the figure. (*p<0.05, **p<0.01, ***p<0.001 Dunn's Multiple Comparison Test compared to Alum control)
Example 11
Pneumolysoid L460D, PspA derivative CD2, SP1912, SP0148, and SP2108 polypeptides: colonization assay
[0172] Animals were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 2), SP2108 (SEQ ID NO: 6), SP0148 plus SP2108, and SP0148 plus SP2108 in combination with SP 1912 (SEQ ID NO: 9) or known S. pneumoniae antigens L460D plus CD2, adsorbed to alum. Two separate studies were conducted. Control animals were immunized with alum alone.
Immunized animals were challenged intranasally with 107 live type 6B S. pneumoniae four weeks after the last immunization. Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Results of the second study are shown in FIG. 13 as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. The number of animals protected from colonization out of the number of animals in the group is indicated at the top of the figure.
The chart below shows the absolute number and corresponding percentage of animals protected from colonization in the four studies described in Examples 10 and 1 1.
Figure imgf000050_0001
0148 + 2108 + L460D +
2/10 6/10 8/20 40% CD2
Example 12
PspA, SP0148 and SP2108 passive antibody transfer and aspiration challenge (sepsis assay)
[0173] Groups of ten mice were injected with monoclonal antibodies specific for
PspA, heat- inactivated rabbit sera specific for SP0148, SP2108, or combinations of these. Antibody and antisera concentrations and total injection volumes were adjusted with normal rabbit serum (NRS) and PBS. Control animals were injected with NRS, or serum against killed, unencapsulated whole cell S. pneumoniae (WCV). One day after injection, the mice underwent aspiration challenge with 106 live S. pneumoniae type WU-2 (ST-3). Animals were monitored for survival for eight days. The data are shown in FIG. 14 as survival curves for each immunized group.
[0174] FIG. 15 shows the percent of animals protected from sepsis in six separate aspiration challenge studies, two of which aredescribed in more detail in Examples 6 and 12. Example 13
SP0148, SP2108, and SP1912 fusion proteins: IL-17A immunogenicity assay
[0175] Groups of four mice were subcutaneously immunized three times, two weeks apart with vaccine compositions comprising the fusion proteins SP2108/0148/1912 (SEQ ID NO: 53), SP0148/2108 (SEQ ID NO: 48), SP0148/1912 (SEQ ID NO: 50) or the comparable combinations of the single antigens SP2108 (SED ID NO: 6), SP0148 (SEQ ID NO: 2) and SP1912 (SEQ ID NO: 9), adsorbed to alum. Control animals were immunized with alum alone. Two weeks after the last immunization, peripheral blood was collected from the retroorbital sinus and evaluated for IL-17A levels in a whole blood assay. Briefly, the heparizined whole blood was diluted in media and then cultured for six days with single (non-fusion) proteins of immunization. The supernatants were harvested and IL- 17A levels measured by ELISA. Results of the IL-17A immunogenicity assay are shown in FIG. 16, Panels A-C. Each bar represents the mean ± SD IL-17A response from each group of four mice in response to the indicated antigen.
Example 14
SP0148, SP2108, and SP1912 fusion proteins: colonization assay
[0176] Animals are subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the fusion proteins SP2108/0148 (SEQ ID NO: 47), SP0148/2108 (SEQ ID NO: 48), SP2108/1912 (SEQ ID NO: 49), SP0148/1912 (SEQ ID NO: 50), SP2108/1912/0148 (SEQ ID NO: 51), SP0148/1912/2108 (SEQ ID NO: 52),
SP2108/0148/1912 (SEQ ID NO: 53), or SP0148/2108/1912 (SEQ ID NO: 54), adsorbed to alum. Control animals are immunized with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), or alum alone. Immunized animals are challenged intranasally with 107 live type 6B 5*. pneumoniae four weeks after the last immunization. Seven days after challenge, animals are euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Results are shown as the colony forming units of bacteria (CFU) per lavage. Each symbol represents a titer from an individual mouse response, and the horizontal line represents the median of the group. The absolute number and corresponding percentage of animals protected from colonization are also indicated.
Example 15
SP0148, SP2108, and SP1912 fusion proteins: aspiration challenge (sepsis assay)
[0177] Groups of ten mice are subcutaneously immunized three times, two weeks apart with vaccine compositions comprising the fusion proteins SP2108/0148 (SEQ ID NO: 47), SP0148/2108 (SEQ ID NO: 48), SP2108/1912 (SEQ ID NO: 49), SP0148/1912 (SEQ ID NO: 50), SP2108/1912/0148 (SEQ ID NO: 51), SP0148/1912/2108 (SEQ ID NO: 52), SP2108/0148/1912 (SEQ ID NO: 53), or SP0148/2108/1912 (SEQ ID NO: 54), adsorbed to alum. Control animals are immunized with killed, unencapsulated whole cell S. pneumoniae plus alum (WCV), or alum alone. Three weeks after the final immunization, blood is collected for evaluation of IL-17A and antibody, and then one week later, the mice undergo aspiration challenge with 107 live strain 0603 (type 6B) 5*. pneumoniae. Animals are monitored for survival for eight days. Results are reported as survival curves for each immunized group.
Example 16
SP0148, SP2108, SP1912, CD2 and H70 fusion proteins: colonization assay
[0178] In two separate studies, mice were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the fusion proteins SP0148/1912 (SEQ ID NO: 50), SP0148/1912 (SEQ ID NO: 50) plus SP2108 (SEQ ID NO: 6), SP2108/1912 (SEQ ID NO: 49), SP2108/1912 (SEQ ID NO: 49) plus SP0148 (SEQ ID NO: 2), SP2108/CD2 (SEQ ID NO: 57) plus SP0148/1912 (SEQ ID NO: 50), SP0148/CD2 (SEQ ID NO: 57) plus SP2108/1912 (SEQ ID NO: 49), SP2108/H70 (SEQ ID NO: 58) plus SP0148/1912 (SEQ ID NO: 50), and SP0148/H70 (SEQ ID NO: 56) plus SP2108/1912 (SEQ ID NO: 49), adsorbed to alum. Control animals were immunized with SP2108 (SEQ ID NO: 6) plus SP0148 (SEQ ID NO: 2) plus SP 1912 (SEQ ID NO: 9) adsorbed to alum, or alum alone. Immunized animals were challenged intranasally with 107 live type 6B 5*. pneumoniae four weeks after the last immunization. Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S.
pneumoniae titers. Pooled results of the two studies are shown in FIG. 17 as the colony forming units of bacteria (CFU) per nasal wash. Each symbol represents the number of colonies cultured from an individual mouse, and the horizontal line represents the median of the group. The difference in mean log number ofCFUs recovered from immunized mice relative to alum-only control mice is indicated at the top of the figure.
Example 17
SP0148, SP2108, SP1912, CD2 and H70 fusion proteins: intravenous challenge (sepsis assay)
[0179] Mice were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the fusion proteins SP2108/H70 (SEQ ID NO: 58), SP2108/CD2 (SEQ ID NO: 57), SP0148/H70 (SEQ ID NO: 56), SP0148/CD2 (SEQ ID NO: 55), SP2108/H70 (SEQ ID NO: 58) plus SP0148/1912 (SEQ ID NO: 50), and SP0148/H70 (SEQ ID NO: 56) plus SP2108/1912 (SEQ ID NO: 49), adsorbed to alum. Control animals were immunized with H70 (SEQ ID NO: 33) or CD2 (SEQ ID NO: 29) adsorbed to alum, or alum alone. Immunized animals were challenged intravenously with 368 CFUs of strain A66.1 S. pneumoniae three weeks after the last immunization. Mice were observed daily for fourteen days. Results are shown in FIG. 18 as hours to moribund. [NB: In this figure, CD2 is labeled PRN.] Each symbol represents the survival hours of an individual mouse, and the horizontal line represents the median hours to moribund of the group. The table below the figure indicates the mean and median hours to moribund and the standard error.
Example 18
SP0148, SP2108, SP1912, CD2 and H70 fusion proteins: IL-17A immunogenicity assay
[0180] Mice were immunized in two separate studies as described in Example 16.
Two weeks after the last immunization, peripheral blood was collected from the retroorbital sinus and evaluated for IL-17A levels in a whole blood assay. Briefly, the heparizined whole blood was diluted in media and then cultured for six days with killed, unencapsulated whole cell S. pneumoniae (WCA). The supernatants were harvested and IL-17A levels measured by ELISA. Results of the IL-17A immunogenicity assay for each study are shown in FIG. 19, Panels A and B. Individual symbols represent the cytokine secretion in whole blood from each mouse. Each horizontal line represents the mean IL-17A response from each group of animals in response to the indicated antigen.
Example 19
SP0148, SP2108, SP1912, CD2 and H70 fusion proteins: antibody response
[0181] Mice were immunized as described in Example 18 and bled 16-20 days after the last immunization. Total IgG antibody titers against CD2 or H70 were measured by standard ELISA. FIG. 20 shows the mean (± SD) IgG titer for the group expressed as the log 10 μ§ πύ. [NB: In this figure, CD2 is labeled PRN.]
EQUIVALENTS
[0182] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
SEQUENCES
SEQ ID NO: 1
SP0148 lacking signal sequence
MCSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEWRAIFKDSDKYDVKFEKTEWSGVFAGLDADR YNMAVNNLSYTKERAEKYLYAAPIAQNPNVLWKKDDSS IKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPT ILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRI KELYKDGTLEKLSKQFFGDTYLPAEADIKE
SEQ ID NO: 2
SP0148 including signal sequence (277 amino acids with N-terminal E) MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKE
SEQ ID NO: 3
SP0148 consensus lacking signal sequence
MCSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEWRAIFKDSDKYDVKFE Q S R N N X
KTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSS IKSLDD
I E
IGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADLQQIMVRLSDGQFDYKIFDKIG
F
VETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQ Y S
FFGDTYLPAEADIK (E)
SEQ ID NO: 4
SP0148 consensus including signal sequence
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELT G L Q S R N GYEIEVVRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAP
N X I
IAQNPNVLWKKDDSS IKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKAD
E
LQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELK
F Y S
SFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIK (E)
SEQ ID NO: 5
SP2108 lacking signal sequence
MCGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVM MAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLA KDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGS IVGINYAKSWYEKWPKGMQ DTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEYAAFGGGKAWVI PQAVKNLEASQK FVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSG QKDAKTAANDAVTLIKETIKQKFGE
SEQ ID NO: 6
SP2108 including signal sequence
>gi I 14973620 |gb|AAK76167.1 I maltose/maltodextrin ABC transporter, maltose/maltodextrin-binding protein (Streptococcus pneumoniae TIGR4) MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE
SEQ ID NO: 7
SP2108 consensus lacking signal sequence
MCGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLD
A I
KLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGA
I X T
PAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGL
A LAGNGAYVFGQNGKDAKDIGLANDGS IVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQE G P A X H
GKTAA11DGPWKAQAFKDAKVNYGVATI PTLPNGKEYAAFGGGKAWVIPQAVKNLEASQK
A
FVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSA S A S
VWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE
SEQ ID NO: 8
SP2108 consensus including signal sequence
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVA T T V A
KAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLS I I X
DGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAF T
AGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGS IVGINYAKSWY A G P A X
EKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY H
AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDE
A S A
LTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQK S
FGE
SEQ ID NO: 9
SP1912 MNGMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGV IFEDGRHYTFVYENEDLVYEEEVL
SEQ ID NO: 10
SP1912L
MRYLATLLLSLAVLITAGCKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRH YTFVYENEDLVYEEEVL
SEQ ID NO: 11
SP1912 consensus
MNGMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLY
H A L
VQVYESSLESLVGGVIFEDGRHYTFVYENEDLVYEEEVL I L S I
SEQ ID NO: 12
SP0641
>gi 114972117 |gb|AAK74791.1 I serine protease, subtilase family
[Streptococcus pneumoniae TIGR4]
MKKSTVLSLTTAAVILAAYAPNEVVLADTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEK EWSKNPVIDNNTSNEEAKIKEENSNKSQGDYTDSFVNKNTENPKKEDKWYIAEFKDKESGEKAIKELSSLKNT KVLYTYDRIFNGSAIETTPDNLDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRG MVISNIDTGTDYRHKAMRIDDDAKASMRFKKEDLKGTDKNYWLSDKI PHAFNYYNGGKITVEKYDDGRDYFDPHG MHIAGILAGNDTEQDIKNFNGIDGIAPNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDWSVSSGFTGTG LVGEKYWQAIRALRKAGI PMWATGNYATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFD KVNIGGESFKYRNIGAFFDKSKITTNEDGTKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKK AMDKGARAIMWNTVNYYNRDNWTELPAMGYEADEGTKSQVFS ISGDDGVKLWNMINPDKKTEVKRNNKEDFKDK LEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDTDKELYKEDI IVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLN VINGKSTYGYMSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKE KSQYFASPRQQGAGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSA SAITTDSLTDRLKLDETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKF VESFIHFESVEEMEALNSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTL NKGIGGEHGIDKFNPAGVIQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGL TPSPLVLRSAEEGLIS IVNTNKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRG REENAPESKDNQDPATKIRGQFEPIAEGQYFYKFKYRLTKDYPWQVSYI PVKIDNTAPKIVSVDFSNPEKIKLIT KDTYHKVKDQYKNETLFARDQKEHPEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTI YEIKGAGDLRGKI IEVIALDGSSNFTKIHRIKFANQADEKGMISYYLVDPDQDSSKYQKLGEIAESKFKNLGNGK EGSLKKDTTGVEHHHQENEESIKEKSSFTIDRNISTIRDFENKDLKKLIKKKFREVDDFTSETGKRMEEYDYKYD DKGNI IAYDDGTDLEYETEKLDEIKSKIYGVLSPSKDGHFEILGKISNVSKNAKVYYGNNYKS IEIKATKYDFHS KTMTFDLYANINDIVDGLAFAGDMRLFVKDNDQKKAEIKIRMPEKIKETKSEYPYVSSYGNVIELGEGDLSKNKP DNLTKMESGKIYSDSEKQQYLLKDNI ILRKGYALKVTTYNPGKTDMLEGNGVYSKEDIAKIQKANPNLRALSETT IYADSRNVEDGRSTQSVLMSALDGFNI IRYQVFTFKMNDKGEAIDKDGNLVTDSSKLVLFGKDDKEYTGEDKFNV EAIKEDGSMLFIDTKPVNLSMDKNYFNPSKSNKIYVRNPEFYLRGKISDKGGFNWELRVNESVVDNYLIYGDLHI DNTRDFNIKLNVKDGDIMDWGMKDYKANGFPDKVTDMDGNVYLQTGYSDLNAKAVGVHYQFLYDNVKPEVNIDPK GNTSIEYADGKSWFNINDKRNNGFDGEIQEQHIYINGKEYTSFNDIKQI IDKTLNIKIVVKDFARNTTVKEFIL NKDTGEVSELKPHRVTVTIQNGKEMSSTIVSEEDFILPVYKGELEKGYQFDGWEISGFEGKKDAGYVINLSKDTF IKPVFKKIEEKKEEENKPTFDVSKKKDNPQVNHSQLNESHRKEDLQREEHSQKSDSTKDVTATVLDKNNISSKST TNNPNKLPKTGTASGAQTLLAAGIMFIVGIFLGLKKKNQD
SEQ ID NO: 13
SP0641N
MWLADTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEKEWSKNPVIDNNTSNEEAKIKE ENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDNL DKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDD AKASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGI DGIAPNAQIFSYKMYSDAGSGFAGDETMFHAIEDS IKHNVDWSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVV ATGNYATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSK ITTNEDGTKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMWNTVNYYNRDN WTELPAMGYEADEGTKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDE KEIDFKFAPDTDKELYKEDI IVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYG
SEQ ID NO: 14
SP0641M
MSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQ QGAGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTD RLKLDETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESV EEMEALNSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGI DKFNPAGVIQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGLTPSPLVLRSA EEGLIS IVNTNKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGREENAPESKD NQDPATKIRGQFEPIAEGQYFYKFKYRLTKDYPWQVSYI PVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQ YKNETLFARDQKEHPEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTIYEIKGAGDLR GKI IEVIALDGSSNFTKIHRIKFANQADEKGMISYYLVDPDQDSSKYQ
SEQ ID NO: 15
SP641N consensus
MWLADTSSSEDALNISDKEKVA ENKEKHENIHSAMETS
N S WDKET KD N I L
QDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAK K TE TI EG A T TK R
IKEENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESG
D- Q H Q
NAH SA G RL
EKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDN S Q N G Q
G
LDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYL T I
KSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDA
KASMRFKKEDLKGTDKNYWLSDKI PHAFNYYNGGKITV
EKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGI
APNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVV
SVSSGFTGTGLVGEKYWQAIRALRKAGI PMVVATGNY
ATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVA
SAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDG Q N
TKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDL KNAFKKAMDKGARAIMWNTVNYYNRDNWTELPAMGY
EADEGTKSQVFS ISGDDGVKLWNMINPDKKTEVKRNNKE
N
DFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDT
DKELYKEDI IVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYG
SEQ ID NO: 16
SP641M consensus
MSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKI
K T
DLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQG
AGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDK
KYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKL
V
DETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSS Y
FDLNAVINVGEAKNKNKFVESFIHFESVEEMEAL R A
NSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLG S TD K ME
GYDDDGKPKI PGTLNKGIGGEHGIDKFNPAGV
IQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYP R D D Q VH E T LDSNGNPQDAQLERGLTPSPLVLRSAEEGLISIVNT
NKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSK K V G
LKVWGDLKWDGLIYNPRGREENAPESKDNQDPATKIRGQF
EPIAEGQYFYKFKYRLTKDYPWQVSYIPVKIDNTAPKIVS
VDFSNPEKIKLITKDTYHKVKDQYKNETLFARDQKEH
PEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQ
GRNRKLDKDGNTIYEIKGAGDLRGKI IEVIALDGSSNFT S A
KIHRIKFANQADEKGMISYYLVDPDQDSSKYQ DH K A E
SEQ ID NO: 17
SP0882
>gi 114972356 I gb I AAK75009.1 I conserved hypothetical protein (Streptococcus pneumoniae TIGR4)
MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPWYFHDGQNVFNSKESFIGHSWKI I PAIKRNP DISRMIWAIDNDGMGRMNEYAAWKFQESPI PGQQFGGKGVEYAEFVMEWKPFIDETYRTKADCQHTAMIGSSL GGNITQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYID SSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW
SEQ ID NO: 18
SP0882N
MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPWYFHDGQNVFNSKESFIGHSWKI I PAIKRNP DISRMIWAIDNDGMGRMNEYAAWKFQESPI PGQQFGGKGVEYAEFVMEWKPFI SEQ ID NO: 19
SP0882 with exogenous signal sequence
MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPWYFHDG QNVFNSKESFIGHSWKI I PAIKRNPDISRMIWAIDNDGMGRMNEYAAWKFQESPI PGQQFGGKGVEYAEFVMEV VKPFIDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFI YVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW
SEQ ID NO: 20
SP0882N with exogenous signal sequence
MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPWYFHDG QNVFNSKESFIGHSWKI I PAIKRNPDISRMIWAIDNDGMGRMNEYAAWKFQESPI PGQQFGGKGVEYAEFVMEV VKPFI
SEQ ID NO: 21
SP0882 consensus
MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPWYFHDGQNVFNSKESF I Y
IGHSWKI I PAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAE
Y H E E
FVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQ
EK
EAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVH I H R
LDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW
SEQ ID NO: 22
SP0882N consensus
MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPWYFHDGQNVFNSKESF I Y
IGHSWKI I PAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAE
Y H E E
FVMEVVKPFI SEQ ID NO: 23
SP0882 consensus with exogenous signal sequence
MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYE T T V I
KDTDRSYPWYFHDGQNVFNSKESFIGHSWKI I PAIKRNPDISRMIVVAIDNDGMGRMNE
Y Y H
YAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNIT E E
QFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLM EK I H
DGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW
R
SEQ ID NO: 24
SP0882N consensus with exogenous signal sequence
MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYE T T V I
KDTDRSYPWYFHDGQNVFNSKESFIGHSWKI I PAIKRNPDISRMIVVAIDNDGMGRMNE
Y Y H
YAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFI E E
SEQ ID NO: 25
SP1634
>gi 114973124 |gb|AAK75714.1 I hypothetical protein SP_1634 Streptococcus pneumoniae TIGR4
MANIFDYLKDVAYDSYYDLPLNELDILTLIEITYLSFDNLVSTLPQRLLDLAPQVPRDPTMLTSKNRLQLLDELA QHKRFKNCKLSHFINDIDPELQKQFAAMTYRVSLDTYLIVFRGTDDS I IGWKEDFHLTYMKEI PAQKHALRYLKN FFAHHPKQKVILAGHSKGGNLAIYAASQIEQSLQNQITAVYTFDAPGLHQELTQTAGYQRIMDRSKIFIPQGSI I GMMLEI PAHQI IVQSTALGGIAQHDTFSWQIEDKHFVQLDKTNSDSQQVDTTFKEWVATVPDEELQLYFDLFFGT ILDAGISS INDLASLKALEYIHHLFVQAQSLTPEERETLGRLTQLLIDTRYQAWKNR SEQ ID NO: 26
SP0314
>gi 114971788 I gb I AAK74491.1 I hyaluronidase Streptococcus pneumoniae
TIGR4MQTKTKKLIVSLSSLVLSGFLLNHYMTIGAEETTTNTIQQSQKEVQYQQRDTKNLVENGDFGQTEDGSSP
WTGSKAQGWSAWVDQKNSADASTRVIEAKDGAITISSHEKLRAALHRMVPIEAKKKYKLRFKIKTDNKIGIAKVR
IIEESGKDKRLWNSATTSGTKDWQTIEADYSPTLDVDKIKLELFYETGTGTVSFKDIELVEVADQLSEDSQTDKQ
LEEKIDLPIGKKHVFSLADYTYKVENPDVASVKNGILEPLKEGTTNVIVSKDGKEVKKIPLKILASVKDAYTDRL
DDWNGI IAGNQYYDSKNEQMAKLNQELEGKVADSLSS ISSQADRTYLWEKFSNYKTSANLTATYRKLEEMAKQVT
NPSSRYYQDETVVRTVRDSMEWMHKHVYNSEKS IVGNWWDYEIGTPRAINNTLSLMKEYFSDEEIKKYTDVIEKF
VPDPEHFRKTTDNPFKALGGNLVDMGRVKVIAGLLRKDDQEISSTIRSIEQVFKLVDQGEGFYQDGSYIDHTNVA
YTGAYGNVLIDGLSQLLPVIQKTKNPIDKDKMQTMYHWIDKSFAPLLVNGELMDMSRGRS ISRANSEGHVAAVEV
LRGIHRIADMSEGETKQCLQSLVKTIVQSDSYYDVFKNLKTYKDISLMQSLLSDAGVASVPRPSYLSAFNKMDKT
AMYNAEKGFGFGLSLFSSRTLNYEHMNKENKRGWYTSDGMFYLYNGDLSHYSDGYWPTVNPYKMPGTTETDAKRA
DSDTGKVLPSAFVGTSKLDDANATATMDFTNWNQTLTAHKSWFMLKDKIAFLGSNIQNTSTDTAATTIDQRKLES
GNPYKVYVNDKEASLTEQEKDYPETQSVFLESFDSKKNIGYFFFKKSSISMSKALQKGAWKDINEGQSDKEVENE
FLTISQAHKQNRDSYGYMLI PNVDRATFNQMIKELESSLIENNETLQSVYDAKQGVWGIVKYDDSVSTISNQFQV
LKRGVYTIRKEGDEYKIAYYNPETQESAPDQEVFKKLEQAAQPQVQNSKEKEKSEEEKNHSDQKNLPQTGEGQS I
LASLGFLLLGAFYLFRRGKNN
SEQ ID NO: 27
PspA
MNKKKMILTSLASVAILGAGFVASSPTFVRAEEAPVANQSKAEKDYDAAVKKSEAAKKDYETAKKKAEDA QKKYDEDQKKTEAKAEKERKASEKIAEATKEVQQAYLAYLQASNESQRKEADKKIKEATQRKDEAEAAFA TIRTTIWPEPSELAETKKKAEEATKEAEVAKKKSEEAAKEVEVEKNKILEQDAENEKKIDVLQNKVADL EKGIAPYQNEVAELNKEIARLQSDLKDAEENNVEDYIKEGLEQAITNKKAELATTQQNIDKTQKDLEDAE LELEKVLATLDPEGKTQDELDKEAAEAELNEKVEALQNQVAELEEELSKLEDNLKDAETNNVEDYIKEGL EEAIATKKAELEKTQKELDAALNELGPDGDEEETPAPAPQPEKPAEEPENPAPAPKPEKSADQQAEEDYA RRSEEEYNRLTQQQPPKAEKPAPAPQPEQPAPAPKIGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNS NGAMATGWLQYNGSWYYLNANGAMATGWLQYNGSWYYLNANGAMATGWLQYNGSWYYLNANGDMATGWLQ YNGSWYYLNANGDMATGWAKVHGSWYYLNANGSMATGWVKDGETWYYLEASGSMKANQWFQVSDKWYYVN GLGSLSVNTTVDGYKVNANGEWV
SEQ ID NO: 28
Immunogenic PspA/PspC polypeptides including the coiled-coil structure (PR + NPB)
MSDKI IHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVA
KLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSG
HMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNE
PEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQ
QPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMWCRQACGRTRAPPPPPLRSGC SEQ ID NO: 29
Immunogenic PspA/PspC polypeptide CD2
ADLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARR SEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMW
SEQ ID NO: 30
Immunogenic PspA/PspC polypeptides lacking the coiled-coil structure (PR NPB)
MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRLTQQQPPKA SEQ ID NO: 31
Immunogenic PspA/PspC polypeptides including the coiled-coil structure ( only)
MSDKI IHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVA
KLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSG
HMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNE
PETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKP
APAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMWCRQACGRTRAPPPPPLRSG
SEQ ID NO: 32
Immunogenic PspA/PspC polypeptides lacking the coiled-coil structure (PR only)
DLKKAVNEPETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPA PAPKPAPAPKPAPAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMW
SEQ ID NO: 33
Immunogenic PspA/PspC polypeptide H70 (aa 290-410 of PspA)
YFKEGLEKTIAAKKAELEKTEADLKKAVNEPEKPAPAPETPAPEAPAEQPKPAPAPQPAPAPK
PEKPAEQPKPEKTDDQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPKTGWKQENGM
SEQ ID NO: 34
Non-proline Block (NPB)
EKSADQQAEEDYARRSEEEYNRLTQQQ
SEQ ID NO: 35
Non-proline Block (NPB)
DQQAEEDYARRSEEEYNRLTQQQ
SEQ ID NO: 36
Non-proline Block (NPB) MEKSADQQAEEDYARRSEEEYNRLTQQQ
SEQ ID NO: 37
SP0148 lacking signal sequence (nucleotides)
ATGTGCTCAGGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCA CCAAAGCCATTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAA GATTCTGACAAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGT TACAATATGGCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCC CAAAATCCTAATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCG ACGGAAGTCGTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACT ATCCTTAACTATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATT TTTGATAAAATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGC GACCAACAACCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATC AAAGAACTTTATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAA GCTGATATTAAAGAGTAA
SEQ ID NO: 38
SP0148 including signal sequence (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAGAGTAA
SEQ ID NO: 39
SP2108 lacking signal sequence (nucleotides)
ATGTGCGGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTA GACGAGGGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTT AAAACTGGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATG ATGGCTCCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCT AAAACAGACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAG TCACTTGTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCT AAAGATAGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTAC TATACATATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGT CTTGCAAACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAA GATACAGAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCT TGGAAAGCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAA GAATATGCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAA TTTGTAGACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACT GAGGCTCGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAA CCACTGCCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGT CAAAAAGATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAA TAA
SEQ ID NO: 40
SP1912 (nucleotides)
ATGAATGGTATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCT ACGAAAAAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTT TCCGACATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTC ATTTTTGAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTATGA
SEQ ID NO: 41
SP1912L (nucleotides)
ATGAGATACCTGGCAACATTGTTGTTATCTCTGGCGGTGTTAATCACCGCCGGGTGCAAAAAAGTTGCAGATGAC CGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCGACATGGGGGAAATTGCG ACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTTGAGGATGGCCGTCAT TATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTATGA
SEQ ID NO: 42
SP0641N (nucleotides)
ATGGTAGTCTTAGCAGACACATCTAGCTCTGAAGATGCTTTAAACATCTCTGATAAAGAAAAAGTAGCAGAAAAT AAAGAGAAACATGAAAATATCCATAGTGCTATGGAAACTTCACAGGATTTTAAAGAGAAGAAAACAGCAGTCATT AAGGAAAAAGAAGTTGTTAGTAAAAATCCTGTGATAGACAATAACACTAGCAATGAAGAAGCAAAAATCAAAGAA GAAAATTCCAATAAATCCCAAGGAGATTATACGGACTCATTTGTGAATAAAAACACAGAAAATCCCAAAAAAGAA GATAAAGTTGTCTATATTGCTGAATTTAAAGATAAAGAATCTGGAGAAAAAGCAATCAAGGAACTATCCAGTCTT AAGAATACAAAAGTTTTATATACTTATGATAGAATTTTTAACGGTAGTGCCATAGAAACAACTCCAGATAACTTG GACAAAATTAAACAAATAGAAGGTATTTCATCGGTTGAAAGGGCACAAAAAGTCCAACCCATGATGAATCATGCC AGAAAGGAAATTGGAGTTGAGGAAGCTATTGATTACCTAAAGTCTATCAATGCTCCGTTTGGGAAAAATTTTGAT GGTAGAGGTATGGTCATTTCAAATATCGATACTGGAACAGATTATAGACATAAGGCTATGAGAATCGATGATGAT GCCAAAGCCTCAATGAGATTTAAAAAAGAAGACTTAAAAGGCACTGATAAAAATTATTGGTTGAGTGATAAAATC CCTCATGCGTTCAATTATTATAATGGTGGCAAAATCACTGTAGAAAAATATGATGATGGAAGGGATTATTTTGAC CCACATGGGATGCATATTGCAGGGATTCTTGCTGGAAATGATACTGAACAAGACATCAAAAACTTTAACGGCATA GATGGAATTGCACCTAATGCACAAATTTTCTCTTACAAAATGTATTCTGACGCAGGATCTGGGTTTGCGGGTGAT GAAACAATGTTTCATGCTATTGAAGATTCTATCAAACACAACGTTGATGTTGTTTCGGTATCATCTGGTTTTACA GGAACAGGTCTTGTAGGTGAGAAATATTGGCAAGCTATTCGGGCATTAAGAAAAGCAGGCATTCCAATGGTTGTC GCTACGGGTAACTATGCGACTTCTGCTTCAAGTTCTTCATGGGATTTAGTAGCAAATAATCATCTGAAAATGACC GACACTGGAAATGTAACACGAACTGCAGCACATGAAGATGCGATAGCGGTCGCTTCTGCTAAAAATCAAACAGTT GAGTTTGATAAAGTTAACATAGGTGGAGAAAGTTTTAAATACAGAAATATAGGGGCCTTTTTCGATAAGAGTAAA ATCACAACAAATGAAGATGGAACAAAAGCTCCTAGTAAATTAAAATTTGTATATATAGGCAAGGGGCAAGACCAA GATTTGATAGGTTTGGATCTTAGGGGCAAAATTGCAGTAATGGATAGAATTTATACAAAGGATTTAAAAAATGCT TTTAAAAAAGCTATGGATAAGGGTGCACGCGCCATTATGGTTGTAAATACTGTAAATTACTACAATAGAGATAAT TGGACAGAGCTTCCAGCTATGGGATATGAAGCGGATGAAGGTACTAAAAGTCAAGTGTTTTCAATTTCAGGAGAT GATGGTGTAAAGCTATGGAACATGATTAATCCTGATAAAAAAACTGAAGTCAAAAGAAATAATAAAGAAGATTTT AAAGATAAATTGGAGCAATACTATCCAATTGATATGGAAAGTTTTAATTCCAACAAACCGAATGTAGGTGACGAA AAAGAGATTGACTTTAAGTTTGCACCTGACACAGACAAAGAACTCTATAAAGAAGATATCATCGTTCCAGCAGGA TCTACATCTTGGGGGCCAAGAATAGATTTACTTTTAAAACCCGATGTTTCAGCACCTGGTAAAAATATTAAATCC ACGCTTAATGTTATTAATGGCAAATCAACTTATGGC
SEQ ID NO: 43
SP0882N DNA
ATGAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGT GTACGTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGG CAAAATGTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCG GATATCAGTCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAG TTCCAAGAATCTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTG GTCAAGCCTTTTATC
SEQ ID NO: 44
SP0882 with exogenous signal sequence (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC ATGAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGT GTACGTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGG CAAAATGTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCG GATATCAGTCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAG TTCCAAGAATCTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTG GTCAAGCCTTTTATCGATGAGACCTATCGTACAAAAGCAGACTGCCAGCATACGGCTATGATTGGTTCCTCACTA GGAGGCAATATTACCCAGTTTATCGGTTTGGAATACCAAGACCAAATTGGTTGCTTGGGCGTTTTTTCATCTGCA AACTGGCTCCACCAAGAAGCCTTTAACCGCTATTTCGAGTGCCAGAAACTATCGCCTGACCAGCGCATCTTCATC TATGTAGGAACAGAAGAAGCAGATGATACAGACAAGACCTTGATGGATGGCAATATCAAACAAGCCTATATCGAC TCGTCGCTTTGCTATTACCATGATTTGATAGCAGGGGGAGTACATCTGGATAATCTTGTGCTAAAAGTTCAGTCT GGTGCCATCCATAGTGAAATCCCTTGGTCAGAAAATCTACCAGATTGTCTGAGATTTTTTGCAGAAAAATGGTAA
SEQ ID NO: 45
SP0882N with exogenous signal sequence (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC ATGAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGT GTACGTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGG CAAAATGTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCG GATATCAGTCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAG TTCCAAGAATCTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTG GTCAAGCCTTTTATC
SEQ ID NO: 46
PSPA (nucleotides)
TTGACAAATATTTACGGAGGAGGCTTATGCTTAATATAAGTATAGGCTAAAAATGATTATCAGAAAAGAG GTAAATTTAGATGAATAAGAAAAAAATGATTTTAACAAGCCTAGCCAGCGTCGCTATCTTAGGGGCTGGT TTTGTTGCGTCTTCGCCTACTTTTGTAAGAGCAGAAGAAGCTCCTGTAGCTAACCAGTCTAAAGCTGAGA AAGACTATGATGCAGCAGTGAAAAAATCTGAAGCTGCTAAGAAAGATTACGAAACGGCTAAAAAGAAAGC AGAAGACGCTCAGAAGAAATATGATGAGGATCAGAAGAAAACTGAGGCAAAAGCGGAAAAAGAAAGAAAA GCTTCTGAAAAGATAGCTGAGGCAACAAAAGAAGTTCAACAAGCGTACCTAGCTTATCTACAAGCTAGCA ACGAAAGTCAGAGAAAAGAGGCAGATAAGAAGATAAAAGAAGCTACGCAACGCAAAGATGAGGCGGAAGC TGCATTTGCTACTATTCGAACAACAATTGTAGTTCCTGAACCAAGTGAGTTAGCTGAGACTAAGAAAAAA GCAGAAGAGGCAACAAAAGAAGCAGAAGTAGCTAAGAAAAAATCTGAAGAGGCAGCTAAAGAGGTAGAAG TAGAGAAAAATAAAATACTTGAACAAGATGCTGAAAACGAAAAGAAAATTGACGTACTTCAAAACAAAGT CGCTGATTTAGAAAAAGGAATTGCTCCTTATCAAAACGAAGTCGCTGAATTAAATAAAGAAATTGCTAGA CTTCAAAGCGATTTAAAAGATGCTGAAGAAAATAATGTAGAAGACTACATTAAAGAAGGTTTAGAGCAAG CTATCACTAATAAAAAAGCTGAATTAGCTACAACTCAACAAAACATAGATAAAACTCAAAAAGATTTAGA GGATGCTGAATTAGAACTTGAAAAAGTATTAGCTACATTAGACCCTGAAGGTAAAACTCAAGATGAATTA GATAAAGAAGCTGCTGAAGCTGAGTTGAATGAAAAAGTTGAAGCTCTTCAAAACCAAGTTGCTGAATTAG AAGAAGAACTTTCAAAACTTGAAGATAATCTTAAAGATGCTGAAACAAACAACGTTGAAGACTACATTAA AGAAGGTTTAGAAGAAGCTATCGCGACTAAAAAAGCTGAATTGGAAAAAACTCAAAAAGAATTAGATGCA GCTCTTAATGAGTTAGGCCCTGATGGAGATGAAGAAGAGACTCCAGCGCCGGCTCCTCAACCAGAAAAAC CAGCTGAAGAGCCTGAGAATCCAGCTCCAGCACCAAAACCAGAGAAGTCAGCAGATCAACAAGCTGAAGA AGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACCCAACAGCAACCGCCAAAAGCAGAAAAA CCAGCTCCTGCACCACAACCAGAGCAACCAGCTCCTGCACCAAAAATAGGTTGGAAACAAGAAAACGGTA TGTGGTACTTCTACAATACTGATGGTTCAATGGCGACAGGTTGGCTACAAAACAACGGTTCATGGTACTA CCTCAACAGCAATGGCGCTATGGCTACAGGTTGGCTCCAATACAATGGTTCATGGTATTACCTAAACGCT AACGGCGCTATGGCGACAGGCTGGCTCCAATACAATGGCTCATGGTACTACCTCAACGCTAACGGCGCTA TGGCGACAGGCTGGCTCCAATACAATGGCTCATGGTACTACCTCAACGCTAATGGTGATATGGCGACAGG ATGGCTCCAATACAACGGTTCATGGTATTACCTCAACGCTAATGGTGATATGGCTACAGGTTGGGCTAAA GTCCACGGTTCATGGTACTACCTCAACGCTAACGGTTCAATGGCAACAGGTTGGGTGAAAGATGGAGAAA CCTGGTACTATCTTGAAGCATCAGGTTCTATGAAAGCAAACCAATGGTTCCAAGTATCAGATAAATGGTA CTATGTCAATGGTTTAGGTTCCCTTTCAGTCAACACAACTGTAGATGGCTATAAAGTCAATGCCAATGGT GAATGGGTTTAAGCCG
SEQ ID NO: 47
SP2108/SP0148 fusion protein (675 amino acids) MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGESGGAKKEGEAASKKEI IVATNGSPKPF IYEENGELTGYEIEWRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPN VLVVKKDDSS IKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKI GVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIK
SEQ ID NO: 48
SP0148/SP2108 fusion protein (674 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKGSKTADKPADSGSSEVKELTVYVD EGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAK TDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYY TYGLLAGNGAYVFGQNGKDAKDIGLANDGS IVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPW KAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVI PQAVKNLEASQKFVDFLVATEQQKVLYDKTNEI PANTE ARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE
SEQ ID NO: 49
SP2108/SP1912 fusion protein (521 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGENGMKAKKMWMAGLALLGIGSLALATKK VADDRKLMKTQE ELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRHYTFVYENEDLVYEEEVL
SEQ ID NO: 50
SP0148/SP1912 fusion protein (374 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKNGMKAKKMWMAGLALLGIGSLALA TKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRHYTFVYENEDLVYEEEVL
SEQ ID NO: 51 SP2108/SP1912/SP0148 fusion protein (773 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGENGMKAKKMWMAGLALLGIGSLALATKK VADDRKLMKTQE
ELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRHYTFVYENEDLVYEEEVLSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKY LYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSD GQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFG DTYLPAEADIK
SEQ ID NO: 52
SP0148/SP1912/SP2108 fusion protein (772 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKNGMKAKKMWMAGLALLGIGSLALA TKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRHYTFVYENEDLVYEEEVLG SKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAP YDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDS KYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGS IVGINYAKSWYEKWPKGMQDTE GAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVI PQAVKNLEASQKFVD FLVATEQQKVLYDKTNEI PANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKD AKTAANDAVTLIKETIKQKFGE
SEQ ID NO: 53
SP2108/SP0148/SP1912 fusion protein (773 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGESGGAKKEGEAASKKEI IVATNGSPKPF IYEENGELTGYEIEWRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPN VLVVKKDDSS IKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKI GVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIK NGMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVI FEDGRHYTFVYENEDLVYEEEVL SEQ ID NO: 54
SP0148/SP2108/SP1912 fusion protein (772 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKGSKTADKPADSGSSEVKELTVYVD EGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAK TDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYY TYGLLAGNGAYVFGQNGKDAKDIGLANDGS IVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPW KAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVI PQAVKNLEASQKFVDFLVATEQQKVLYDKTNEI PANTE ARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGEN GMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIF EDGRHYTFVYENEDLVYEEEVL
SEQ ID NO: 55
SP0148/CD2 (389 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKMAMADLKKAVNEPEKPAEEEPENP APAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTG WKQENGMWHHHHHH
SEQ ID NO: 56
SP0148/H70 (404 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKYFKEGLEKTIAAKKAELEKTEADL KKAVNEPEKPAPAPETPAPEAPAEQPKPAPAPQPAPAPKPEKPAEQPKPEKTDDQQAEEDYARRSEEEYNRLTQQ QPPKAEKPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 57
SP2108/CD2 (536 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGEMAMADLKKAVNEPEKPAEEEPENPAPA PKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWKQ ENGMWHHHHHH
SEQ ID NO: 58
SP2108/H70 (551 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGEYFKEGLEKTIAAKKAELEKTEADLKKA VNEPEKPAPAPETPAPEAPAEQPKPAPAPQPAPAPKPEKPAEQPKPEKTDDQQAEEDYARRSEEEYNRLTQQQPP KAEKPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 59
SP0148/LC/CD2 (403 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKGPKPHRIQSTPKGSMAMADLKKAV NEPEKPAEEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAP VPKPEQPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 60
SP0148/LC/H70 (418 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKGPKPHRIQSTPKGSYFKEGLEKTI AAKKAELEKTEADLKKAVNEPEKPAPAPETPAPEAPAEQPKPAPAPQPAPAPKPEKPAEQPKPEKTDDQQAEEDY ARRSEEEYNRLTQQQPPKAEKPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 61
SP2108/LC/CD2 (550 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGEGPKPHRIQSTPKGSMAMADLKKAVNEP EKPAEEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPVPK PEQPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 62
SP2108/LC/H70 (565 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGEGPKPHRIQSTPKGSYFKEGLEKTIAAK KAELEKTEADLKKAVNEPEKPAPAPETPAPEAPAEQPKPAPAPQPAPAPKPEKPAEQPKPEKTDDQQAEEDYARR SEEEYNRLTQQQPPKAEKPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 63
SP0148/LR/CD2 (409 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKLAEATAKEATAKEATAKATAMAMA DLKKAVNEPEKPAEEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAE KPAPAPVPKPEQPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 64
SP0148/LR/H70 (424 amino acids)
MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEI IVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSD KYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEV VQATTSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQ PYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKLAEATAKEATAKEATAKATAYFKE GLEKTIAAKKAELEKTEADLKKAVNEPEKPAPAPETPAPEAPAEQPKPAPAPQPAPAPKPEKPAEQPKPEKTDDQ QAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 65
SP2108/LR/CD2 (565 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGELAEATAKEATAKEATAKATAMAMADLK KAVNEPEKPAEEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPA PAPVPKPEQPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 66
SP2108/LR/H70 (571 amino acids)
MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKT GDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESL VMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLA NDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAI IDGPWKAQAFKDAKVNYGVATI PTLPNGKEY AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPL PNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGELAEATAKEATAKEATAKATAYFKEGLE KTIAAKKAELEKTEADLKKAVNEPEKPAPAPETPAPEAPAEQPKPAPAPQPAPAPKPEKPAEQPKPEKTDDQQAE EDYARRSEEEYNRLTQQQPPKAEKPAPAPKTGWKQENGMWHHHHHH
SEQ ID NO: 67
SP2108/SP0148 fusion (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAATCAGGG GGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCATTT ATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGACAAA TATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATGGCT GTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCTAAT GTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCGTT CAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTAT ACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATC GGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAACCG TACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTAT AAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAA
SEQ ID NO: 68
SP0148/SP2108 fusion (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTSCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAGGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGAC GAGGGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAA ACTGGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATG GCTCCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAA ACAGACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCA CTTGTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAA GATAGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTAT ACATATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTT GCAAACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGAT ACAGAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGG AAAGCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAA TATGCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTT GTAGACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAG GCTCGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCA CTGCCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAA AAAGATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAA
SEQ ID NO: 69
SP2108/SP1912 fusion (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAAAATGGT ATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCTACGAAAAAA GTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCGACATG GGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTTGAG GATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTA
SEQ ID NO: 70
SP0148/SP1912 fusion (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAAATGGTATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCT ACGAAAAAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTT TCCGACATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTC ATTTTTGAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTA
SEQ ID NO: 71
SP2108/SP1912/SP0148 fusion (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAAAATGGT ATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCTACGAAAAAA GTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCGACATG GGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTTGAG GATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTATGCTCAGGGGGT GCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCATTTATC TATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGACAAATAT GATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATGGCTGTC AACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCTAATGTC CTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCGTTCAA GCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTATACT AAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATCGGT GTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAACCGTAC GTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTATAAA GATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAA
SEQ ID NO: 72
SP0148/SP1912/SP2108 fusion (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAAATGGTATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCT ACGAAAAAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTT TCCGACATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTC ATTTTTGAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTAGGA AGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAGGGA TATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACTGGT GATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCTCCA TACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACAGAC GACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTTGTT ATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGATAGC AAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACATAT GGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCAAAC GACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACAGAA GGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAAGCT CAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATATGCT GCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTAGAC TTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCTCGT TCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTGCCA AACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAAGAT GCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAACACCACCAC CACCACCACTGA
SEQ ID NO: 73
SP2108/SP0148/SP1912 fusion (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAATCAGGG GGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCATTT ATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGACAAA TATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATGGCT GTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCTAAT GTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCGTT CAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTAT ACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATC GGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAACCG TACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTAT AAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAA AATGGTATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCTACG AAAAAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCC GACATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATT TTTGAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTA
SEQ ID NO: 74
SP0148/SP2108/SP1912 fusion (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAGGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGAC GAGGGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAA ACTGGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATG GCTCCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAA ACAGACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCA CTTGTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAA GATAGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTAT ACATATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTT GCAAACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGAT ACAGAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGG AAAGCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAA TATGCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTT GTAGACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAG GCTCGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCA CTGCCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAA AAAGATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAAAAT GGTATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCTACGAAA AAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCGAC ATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTT GAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTA
SEQ ID NO: 75
SP0148/CD2 (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAATGGCCATGGCCGACCTGAAGAAGGCCGTGAACGAGCCGGAGAAACCGGCCGAAGAGGAGCCGGAGAACCCG GCACCGGCACCTAAACCTGCACCTGCACCGCAGCCTGAGAAGCCTGCCCCTGCACCGGCACCGAAACCGGAGAAG AGCGCCGACCAGCAGGCAGAGGAAGACTACGCCCGCCGCAGCGAAGAGGAGTACAATCGCCTGACCCAGCAGCAA CCGCCGAAGGCAGAAAAGCCGGCCCCGGCCCCGGTTCCGAAACCTGAACAGCCGGCACCTGCACCTAAAACAGGC TGGAAGCAGGAAAACGGCATGTGGCACCACCACCACCACCACTGA
SEQ ID NO: 76
SP0148/H70 (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAATACTTCAAAGAGGGCTTAGAGAAGACCATCGCCGCCAAGAAGGCCGAGTTAGAGAAAACCGAGGCCGACCTG AAGAAAGCCGTGAACGAGCCGGAGAAGCCTGCACCTGCACCTGAAACACCTGCCCCTGAAGCACCGGCCGAACAG CCTAAGCCGGCACCGGCACCTCAACCTGCCCCGGCACCGAAGCCGGAAAAACCGGCCGAGCAGCCGAAACCGGAG AAAACCGACGACCAGCAGGCAGAAGAGGACTATGCCCGCCGCAGCGAGGAAGAGTACAACCGCCTGACCCAGCAG CAGCCTCCGAAAGCCGAGAAACCGGCCCCGGCCCCGAAAACCGGCTGGAAGCAGGAGAACGGCATGTGGCACCAC CACCACCACCACTGA
SEQ ID NO: 77
SP2108/CD2 (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAAATGGCC ATGGCCGACCTGAAGAAGGCCGTGAACGAGCCGGAGAAACCGGCCGAAGAGGAGCCGGAGAACCCGGCACCGGCA CCTAAACCTGCACCTGCACCGCAGCCTGAGAAGCCTGCCCCTGCACCGGCACCGAAACCGGAGAAGAGCGCCGAC CAGCAGGCAGAGGAAGACTACGCCCGCCGCAGCGAAGAGGAGTACAATCGCCTGACCCAGCAGCAACCGCCGAAG GCAGAAAAGCCGGCCCCGGCCCCGGTTCCGAAACCTGAACAGCCGGCACCTGCACCTAAAACAGGCTGGAAGCAG GAAAACGGCATGTGGCACCACCACCACCACCACTGA
SEQ ID NO: 78
SP2108/H70 (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAATACTTC AAAGAGGGCTTAGAGAAGACCATCGCCGCCAAGAAGGCCGAGTTAGAGAAAACCGAGGCCGACCTGAAGAAAGCC GTGAACGAGCCGGAGAAGCCTGCACCTGCACCTGAAACACCTGCCCCTGAAGCACCGGCCGAACAGCCTAAGCCG GCACCGGCACCTCAACCTGCCCCGGCACCGAAGCCGGAAAAACCGGCCGAGCAGCCGAAACCGGAGAAAACCGAC GACCAGCAGGCAGAAGAGGACTATGCCCGCCGCAGCGAGGAAGAGTACAACCGCCTGACCCAGCAGCAGCCTCCG AAAGCCGAGAAACCGGCCCCGGCCCCGAAAACCGGCTGGAAGCAGGAGAACGGCATGTGGCACCACCACCACCAC CACTGA
SEQ ID NO: 79
SP0148/LC/CD2 (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAGGCCCGAAGCCGCACCGTATCCAGAGCACCCCGAAGGGCAGCATGGCCATGGCAGACCTGAAGAAGGCCGTG AACGAGCCGGAAAAACCTGCCGAAGAGGAGCCGGAAAATCCTGCCCCTGCACCTAAACCTGCACCTGCCCCTCAG CCGGAGAAACCGGCACCTGCACCGGCACCTAAGCCGGAGAAGAGCGCCGATCAGCAGGCCGAGGAGGACTATGCC CGTCGCAGCGAAGAGGAGTACAACCGCCTGACCCAGCAACAGCCTCCGAAGGCCGAAAAACCGGCCCCTGCACCG GTGCCTAAGCCTGAGCAACCTGCCCCGGCCCCGAAAACCGGCTGGAAGCAGGAAAACGGCATGTGGCACCACCAC CACCACCACTGA
SEQ ID NO: 80
SP0148/LC/H70 (nucleotides) ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAAGGCCCGAAGCCGCACCGYATCCAGAGCACACCGAAAGGCAGCTACTTCAAAGAGGGCTTAGAGAAGACCATC GCCGCCAAGAAGGCCGAGTTAGAGAAAACCGAGGCCGACCTGAAGAAGGCCGTGAACGAGCCGGAGAAACCTGCA CCTGCACCGGAGACACCGGCACCTGAAGCCCCTGCAGAGCAGCCTAAACCTGCCCCTGCACCTCAACCGGCACCT GCACCTAAGCCGGAAAAACCTGCCGAGCAGCCGAAGCCGGAGAAAACCGACGACCAGCAGGCCGAAGAGGACTAT GCACGCCGCAGCGAAGAGGAGTACAACCGCTTAACCCAACAGCAGCCGCCGAAGGCAGAAAAACCGGCCCCTGCC CCTAAGACCGGTTGGAAGCAGGAGAACGGCATGTGGCACCACCACCACCACCACTGA
SEQ ID NO: 81
SP2108/LC/CD2 (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAAGGCCCG AAGCCGCACCGTATCCAGAGCACCCCGAAGGGCAGCATGGCCATGGCAGACCTGAAGAAGGCCGTGAACGAGCCG GAAAAACCTGCCGAAGAGGAGCCGGAAAATCCTGCCCCTGCACCTAAACCTGCACCTGCCCCTCAGCCGGAGAAA CCGGCACCTGCACCGGCACCTAAGCCGGAGAAGAGCGCCGATCAGCAGGCCGAGGAGGACTATGCCCGTCGCAGC GAAGAGGAGTACAACCGCCTGACCCAGCAACAGCCTCCGAAGGCCGAAAAACCGGCCCCTGCACCGGTGCCTAAG CCTGAGCAACCTGCCCCGGCCCCGAAAACCGGCTGGAAGCAGGAGAATGGCATGTGGCACCACCACCACCACCAC TGA
SEQ ID NO: 82
SP2108/LC/H70 (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAAGGCCCG AAGCCGCACCGCATCCAGAGCACACCGAAAGGCAGCTACTTCAAAGAGGGCTTAGAGAAGACCATCGCCGCCAAG AAGGCCGAGTTAGAGAAAACCGAGGCCGACCTGAAGAAGGCCGTGAACGAGCCGGAGAAACCTGCACCTGCACCG GAGACACCGGCACCTGAAGCCCCTGCAGAGCAGCCTAAACCTGCCCCTGCACCTCAACCGGCACCTGCACCTAAG CCGGAAAAACCTGCCGAGCAGCCGAAGCCGGAGAAAACCGACGACCAGCAGGCCGAAGAGGACTATGCACGCCGC AGCGAAGAGGAGTACAACCGCTTAACCCAACAGCAGCCGCCGAAGGCAGAAAAACCGGCCCCTGCCCCTAAGACC GGTTGGAAGCAGGAGAACGGCATGTGGCACCACCACCACCACCACTGA
SEQ ID NO: 83
SP0148/LR/CD2 (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAACTGGCCGAAGCAACCGCCAAAGAAGCCACCGCCAAGGAAGCCACAGCCAAGGCCACCGCAATGGCCATGGCC GACCTGAAGAAGGCCGTGAATGAGCCGGAGAAGCCGGCCGAAGAAGAGCCGGAGAATCCTGCACCGGCCCCTAAG CCTGCACCGGCACCTCAGCCTGAAAAACCGGCACCTGCACCTGCCCCGAAGCCTGAAAAGAGCGCCGACCAGCAG GCCGAAGAGGACTATGCCCGCCGCAGCGAAGAGGAGTACAACCGTCTGACCCAGCAGCAGCCGCCGAAAGCCGAG AAACCTGCCCCGGCACCGGTGCCTAAGCCGGAACAGCCTGCCCCTGCACCGAAGACCGGTTGGAAGCAGGAAAAC GGCATGTGGCACCACCACCACCACCACTGA
SEQ ID NO: 84
SP0148/LR/H70 (nucleotides)
ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCA GGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCA TTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGAC AAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATG GCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCT AATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTC GTTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAAC TATACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAA ATCGGTGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAA CCGTACGTTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTT TATAAAGATGGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATT AAACTGGCCGAAGCAACCGCCAAAGAGGCCACCGCCAAGGAAGCCACCGCCAAAGCCACCGCCTACTTCAAAGAG GGCTTAGAGAAGACCATCGCCGCCAAGAAGGCCGAGTTAGAGAAAACCGAGGCCGACCTGAAGAAGGCCGTGAAC GAGCCGGAGAAGCCTGCACCTGCCCCTGAAACCCCTGCACCGGAAGCACCTGCAGAGCAGCCTAAACCTGCACCT GCACCGCAACCTGCCCCTGCACCTAAACCGGAGAAACCGGCAGAGCAGCCGAAGCCGGAGAAAACCGACGACCAG CAGGCCGAGGAAGACTACGCCCGTCGTAGCGAGGAAGAGTACAACCGCCTGACCCAGCAGCAACCGCCGAAAGCC GAAAAGCCGGCCCCTGCACCGAAAACCGGCTGGAAGCAGGAGAACGGCATGTGGCACCACCACCACCACCACTGA
SEQ ID NO: 85
SP2108/LR/CD2 (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAACTGGCC GAGGCAACCGCCAAAGAAGCCACCGCCAAGGAAGCCACAGCCAAGGCCACCGCAATGGCCATGGCCGACCTGAAG AAGGCCGTGAATGAGCCGGAGAAGCCGGCCGAAGAAGAGCCGGAGAATCCTGCACCGGCCCCTAAGCCTGCACCG GCACCTCAGCCTGAAAAACCGGCACCTGCACCTGCCCCGAAGCCTGAAAAGAGCGCCGACCAGCAGGCCGAAGAG GACTATGCCCGCCGCAGCGAAGAGGAGTACAACCGTCTGACCCAGCAGCAGCCGCCGAAAGCCGAGAAACCTGCC CCGGCACCGGTGCCTAAGCCGGAACAGCCTGCCCCTGCACCGAAGACCGGTTGGAAGCAGGARAACGGCATGTGG CACCACCACCACCACCACTGA
SEQ ID NO: 86
SP2108/LR/H70 (nucleotides)
ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGC GGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGACGAG GGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAACT GGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACA GACGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTT GTTATGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGAT AGCAAATACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACA TATGGACTTCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCA AACGACGGTTCTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACA GAAGGTGCTGGAAACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAA GCTCAAGCCTTTAAAGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATAT GCTGCATTCGGTGGTGGTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTA GACTTCCTTGTTGCAACTGAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCT CGTTCATACGCTGAAGGTAAAAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTG CCAAACATCTCTCAAATGTCTGCAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAA GATGCTAAAACAGCTGCTAACGATGCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAACTGGCC GAAGCAACCGCCAAAGAGGCCACCGCCAAGGAAGCCACCGCCAAAGCCACCGCCTACTTCAAAGAGGGCTTAGAG AAGACCATCGCCGCCAAGAAGGCCGAGTTAGAGAAAACCGAGGCCGACCTGAAGAAGGCCGTGAACGAGCCGGAG AAGCCTGCACCTGCCCCTGAAACCCCTGCACCGGAAGCACCTGCAGAGCAGCCTAAACCTGCACCTGCACCGCAA CCTGCCCCTGCACCTAAACCGGAGAAACCGGCAGAGCAGCCGAAGCCGGAGAAAACCGACGACCAGCAGGCCGAG GAAGACTACGCCCGTCGTAGCGAGGAAGAGTACAACCGCCTGACCCAGCAGCAACCGCCGAAAGCCGAAAAGCCG GCCCCTGCACCGAAAACCGGCTGGAAGCAGGAGAACGGCATGTGGCACCACCACCACCACCACTGA
SEQ ID NO: 87
HHHHHH SEQ ID NO: 88
MSYYHHHHHH
SEQ ID NO: 89
Canonical lipobox motif
[LIVMFESTAGPC] - [LVIAMFTG] - [IVMSTAGCP] - [AGS] -C
SEQ ID NO: 90
SP2108 signal sequence
MSSKFMKSAAVLGTATLASLLLVAC
SEQ ID NO: 91
E. coli RlpB signal sequence
MRYLATLLLSLAVLITAG [C]
SEQ ID NO: 92
Fusion protein linker (19-mer)
LGGGGSGGGGSGGGGSAAA
SEQ ID NO: 93
Fusion protein LR linker (20-mer)
LAEATAKEATAKEATAKATA
SEQ ID NO: 94
Fusion protein LC linker (14-mer)
GPKPHRIQSTPKGS
SEQ ID NO: 95
Amino-terminal boundary to the PR-region DLKKAVNE
SEQ ID NO: 96
Carboxy-terminal boundary to the PR-region (K/G) TGW (K/G) QENGMW

Claims

We claim:
1. A fusion protein comprising:
(a) a first polypeptide comprising an immunogenic polypeptide having the amino acid sequence of one of SEQ ID NOS: 1-8, or an immunogenic fragment thereof; and
(b) a second polypeptide comprising a Streptococcus pneumoniae antigen that is predominantly an antibody target.
2. The fusion protein of claim 1, wherein the first polypeptide is N-terminal to the
second polypeptide.
3. The fusion protein of claim 2, wherein the first polypeptide is lipidated.
4. The fusion protein of any of the preceding claims, wherein the first polypeptide
comprises the amino acid sequence of SEQ ID NO: 2.
5. The fusion protein of any of the preceding claims, wherein the first polypeptide
comprises the amino acid sequence of SEQ ID NO: 6.
6. The fusion protein of any of the preceding claims, wherein the first polypeptide is directly bonded to the second polypeptide.
7. The fusion protein of any of the preceding claims, further comprising a linker
polypeptide.
8. The fusion protein of claim 7, wherein the linker polypeptide is positioned between the first polypeptide and the second polypeptide.
9. The fusion protein of claim 8, wherein the linker polypeptide is rigid.
10. The fusion protein of claim 9, wherein the linker polypeptide comprises SEQ ID NO:
93.
11. The fusion protein of claim 8, wherein the linker polypeptide is semi-rigid.
12. The fusion protein of claim 1 1, wherein the linker polypeptide comprises SEQ ID NO: 94.
13. The fusion protein of any of claims 1-12, wherein the second polypeptide is
Pneumococcal surface protein A (PspA) or a derivative thereof.
14. The fusion protein of claim 13, wherein the PspA derivative comprises all or a
fragment of the proline-rich region of PspA.
15. The fusion protein of claim 14, wherein the PspA derivative includes a non-proline block.
16. The fusion protein of claim 15, wherein the PspA derivative comprises SEQ ID NO:
29.
17. The fusion protein of claim 15, wherein the PspA derivative comprises SEQ ID NO:
33.
18. The fusion protein of any of claims 1-12, wherein the second polypeptide is a
pneumolysoid or a derivative thereof.
19. A vaccine formulation comprising:
the fusion protein of any of claims 1-18; and
a pharmaceutically acceptable carrier.
20. The vaccine formulation of claim 19, further comprising an immunogenic polypeptide comprising the amino acid sequence of one of SEQ ID NOS: 1-1 1, or an
immunogenic fragment thereof.
21. The vaccine formulation of claim 19, further comprising a second fusion protein comprising two immunogenic polypeptides, each immunogenic polypeptide of the second fusion protein comprising the amino acid sequence of one of SEQ ID NOS: 1- 11 , or an immunogenic fragment thereof.
22. The vaccine formulation of any of claims 19-21, further comprising an adjuvant.
23. The vaccine formulation of claim 22, wherein the adjuvant is an agonist of toll-like receptors (TLRs).
24. The vaccine formulation of claim 22, wherein the adjuvant is alum.
25. The vaccine formulation of claim 22, wherein the vaccine formulation comprises 1- 1000 μg of each fusion protein and 25-500 μg of the adjuvant.
26. The vaccine formulation of any of claims 19-25, characterized in that, upon
contacting TH17 cells, the vaccine formulation induces a TH17 cell response at least 1.5 -fold greater than that induced by a control.
27. The vaccine formulation of any of claims 19-26, characterized in that, upon
administration to an uninfected subject, the vaccine formulation inhibits infection by S. pneumoniae in the uninfected subject.
28. The vaccine formulation of any of claims 19-27, characterized in that, upon
administration to a subject, the vaccine formulation inhibits S. pneumoniae colonization in the subject.
29. The vaccine formulation of any of claims 19-28, characterized in that, upon
administration to a subject, the vaccine formulation inhibits S. pneumoniae symptoms in the subject.
30. The vaccine formulation of any of claims 19-29, characterized in that, upon
administration to a subject, the vaccine formulation inhibits S. pneumoniae-' dxxcQd invasive disease in the subject.
31. The vaccine formulation of any of claims 19-29, characterized in that, upon
administration to a subject, the vaccine formulation inhibits S. pneumoniae-m' dxxcQd sepsis in the subject.
32. A method for treating a subject suffering from or susceptible to S. pneumoniae infection, comprising administering to the subject an effective amount of a vaccine formulation according to any of claims 19-31, thereby treating the subject.
33. The method of claim 32, wherein the subject is an uninfected subject, and the vaccine formulation inhibits infection by S. pneumoniae in the uninfected subject.
34. The method of claim 32, wherein the vaccine formulation inhibits S. pneumoniae colonization in the subject.
35. The method of claim 32, wherein the vaccine formulation inhibits one or more S. pneumoniae symptoms in the subject.
36. The method of claim 32, wherein the vaccine formlulation inhibits S. pneumonia- induced invasive disease in the subject.
37. The method of claim 32, wherein the vaccine formulation inhibits S. pneumoniae- induced sepsis in the subject.
38. The method of claim 32, wherein one dose of the vaccine formulation is adminstered.
39. The method of claim 32, wherein three doses of the vaccine formulation are
administered.
40. The method of any one of claims 32-39, wherein the subject is a human.
PCT/US2013/052352 2012-07-26 2013-07-26 Fused antigen vaccines and compositions against streptococcus pneumoniae WO2014018904A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261676276P 2012-07-26 2012-07-26
US61/676,276 2012-07-26

Publications (1)

Publication Number Publication Date
WO2014018904A1 true WO2014018904A1 (en) 2014-01-30

Family

ID=49997872

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/052352 WO2014018904A1 (en) 2012-07-26 2013-07-26 Fused antigen vaccines and compositions against streptococcus pneumoniae

Country Status (1)

Country Link
WO (1) WO2014018904A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017060660A (en) * 2015-09-25 2017-03-30 株式会社日立ハイテクノロジーズ Biological optical measurement apparatus, information processing program, and information processing method
US10406221B2 (en) 2015-12-10 2019-09-10 National Research Council Of Canada Lipidated Streptococcus pneumoniae antigen compositions, methods of preparation and use
WO2020056127A1 (en) * 2018-09-12 2020-03-19 The Children's Medical Center Corporation Pneumococcal fusion protein vaccines
US11013793B2 (en) 2018-09-12 2021-05-25 Affinivax, Inc. Multivalent pneumococcal vaccines
US11305001B2 (en) 2017-03-28 2022-04-19 The Children's Medical Center Corporation Multiple antigen presenting system (MAPS)-based Staphylococcus aureus vaccine, immunogenic composition, and uses thereof
US11560410B2 (en) 2011-05-11 2023-01-24 Children's Medical Center Corporation Modified biotin-binding protein, fusion proteins thereof and applications
US11576958B2 (en) 2013-02-07 2023-02-14 Children's Medical Center Corporation Protein antigens that provide protection against pneumococcal colonization and/or disease
US11612647B2 (en) 2017-06-23 2023-03-28 University Of Maryland, Baltimore Immunogenic compositions

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004092209A2 (en) * 2003-04-15 2004-10-28 Intercell Ag S. pneumoniae antigens
WO2007089866A2 (en) * 2006-02-02 2007-08-09 University Of Alabama Research Foundation Non-coiled protective regions of pneumococcal surface proteins pspa and pspc
WO2008127179A1 (en) * 2007-04-16 2008-10-23 Lunds Universitets Utvecklingsaktiebolag Fusion protein vaccine
WO2010080424A1 (en) * 2008-12-18 2010-07-15 E. I. Du Pont De Nemours And Company Peptide linkers for effective multivalent peptide binding
WO2011008548A1 (en) * 2009-06-29 2011-01-20 Genocea Biosciences, Inc. Vaccines and compositions against streptococcus pneumoniae
EP2278013A1 (en) * 2001-04-16 2011-01-26 Wyeth Holdings Corporation Streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2278013A1 (en) * 2001-04-16 2011-01-26 Wyeth Holdings Corporation Streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof
WO2004092209A2 (en) * 2003-04-15 2004-10-28 Intercell Ag S. pneumoniae antigens
WO2007089866A2 (en) * 2006-02-02 2007-08-09 University Of Alabama Research Foundation Non-coiled protective regions of pneumococcal surface proteins pspa and pspc
WO2008127179A1 (en) * 2007-04-16 2008-10-23 Lunds Universitets Utvecklingsaktiebolag Fusion protein vaccine
WO2010080424A1 (en) * 2008-12-18 2010-07-15 E. I. Du Pont De Nemours And Company Peptide linkers for effective multivalent peptide binding
WO2011008548A1 (en) * 2009-06-29 2011-01-20 Genocea Biosciences, Inc. Vaccines and compositions against streptococcus pneumoniae

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATION JAMES C.: "Purification and Immunogenicity of Genetically Obtained Pneumolysin Toxoids and Their Conjugation to Streptococcus pneumoniae Type 19F Polysaccharide.", INFECTION AND IMMUNITY, vol. 59, no. 7, 1991, pages 2297 - 2304 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11560410B2 (en) 2011-05-11 2023-01-24 Children's Medical Center Corporation Modified biotin-binding protein, fusion proteins thereof and applications
US11576958B2 (en) 2013-02-07 2023-02-14 Children's Medical Center Corporation Protein antigens that provide protection against pneumococcal colonization and/or disease
JP2017060660A (en) * 2015-09-25 2017-03-30 株式会社日立ハイテクノロジーズ Biological optical measurement apparatus, information processing program, and information processing method
US10406221B2 (en) 2015-12-10 2019-09-10 National Research Council Of Canada Lipidated Streptococcus pneumoniae antigen compositions, methods of preparation and use
US11305001B2 (en) 2017-03-28 2022-04-19 The Children's Medical Center Corporation Multiple antigen presenting system (MAPS)-based Staphylococcus aureus vaccine, immunogenic composition, and uses thereof
US11612647B2 (en) 2017-06-23 2023-03-28 University Of Maryland, Baltimore Immunogenic compositions
WO2020056127A1 (en) * 2018-09-12 2020-03-19 The Children's Medical Center Corporation Pneumococcal fusion protein vaccines
US11013793B2 (en) 2018-09-12 2021-05-25 Affinivax, Inc. Multivalent pneumococcal vaccines
US11701416B2 (en) 2018-09-12 2023-07-18 Affinivax, Inc. Multivalent pneumococcal vaccines

Similar Documents

Publication Publication Date Title
US11207375B2 (en) Vaccines and compositions against Streptococcus pneumoniae
US10188717B2 (en) Vaccines and compositions against Streptococcus pneumoniae
WO2014018904A1 (en) Fused antigen vaccines and compositions against streptococcus pneumoniae
AU2013209513B2 (en) Fused antigen vaccines and compositions against Streptococcus pneumoniae
US20150079132A1 (en) Evoking protection against streptotoccus pneumoniae incorporating b-cell and t-cell pneumococcal protein antigens and pneumococcal polysaccharides delivered concomitantly
NZ614460B2 (en) Vaccines and compositions against streptococcus pneumoniae

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13822111

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13822111

Country of ref document: EP

Kind code of ref document: A1