NZ614460B2 - Vaccines and compositions against streptococcus pneumoniae - Google Patents

Vaccines and compositions against streptococcus pneumoniae Download PDF

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Publication number
NZ614460B2
NZ614460B2 NZ614460A NZ61446012A NZ614460B2 NZ 614460 B2 NZ614460 B2 NZ 614460B2 NZ 614460 A NZ614460 A NZ 614460A NZ 61446012 A NZ61446012 A NZ 61446012A NZ 614460 B2 NZ614460 B2 NZ 614460B2
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seq
vaccine formulation
polypeptide
pneumoniae
vaccine
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NZ614460A
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NZ614460A (en
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Todd Gierahn
Richard Malley
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Children's Medical Center Corporation
Genocea Biosciences Inc
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Priority claimed from PCT/US2012/022128 external-priority patent/WO2012100234A1/en
Publication of NZ614460A publication Critical patent/NZ614460A/en
Publication of NZ614460B2 publication Critical patent/NZ614460B2/en

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    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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

Abstract

Disclosed is a vaccine formulation comprising a pharmaceutically acceptable carrier and a first polypeptide comprising an SP1912 amino acid sequence in combination with an amino acid sequence of either SP2108 or SP0148, of S. pneumoniae. Also disclosed is the use of the vaccine for treating a subject suffering from or susceptible to S. pneumoniae infection. bject suffering from or susceptible to S. pneumoniae infection.

Description

Vaccines and Compositions Against ococcus Pneumoniae Related Application This ation claims the benefit of the filing date of U.S. ional Application No. 61/434,818, filed January 20, 2011. The entire teachings of the referenced application are expressly incorporated herein by reference. ment Support This work was made with Government support under Grant AI066013 awarded by the National Institutes of Health. Therefore, the U.S. Government has certain rights in this invention.
I. Background 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, itis, bacteremia, and otitis media are attributed to infection with the pathogen ococcus 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 infectious when S. pneumoniae is carried into the Eustachian tubes, nasal sinuses, lungs, bloodstream, meninges, joint spaces, bones and peritoneal cavity.
S. pneumoniae has several virulence factors that enable the organism to evade the immune . 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 dividing cocci into different serotypes. To date, 93 serotypes of S. pneumoniae have been identified.
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 ting pneumonia and other invasive diseases due to S. pneumoniae in the adult and aging populations. The vaccine contains ar polysaccharides (CPs) from 23 serotypes of S. niae. As T cell-independent antigens, these CPs induce only short-lived antibody responses, itating repeated doses, which increases the risk of immunological tolerance. The antibodies raised against S. pneumoniae, termed anticapsular antibodies, are ized 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 ndependent antigens and, therefore, are not afforded optimal protection by PPV-23.
Another S. pneumoniae e, Prevnar, includes bacterial polysaccharides from 7 S. pneumoniae strains conjugated to the eria 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. Serotype replacement has already been demonstrated in several clinical trials and iologic studies, itating development of different formulations of these vaccines. An example is the recently introduced Prevnar l3, directed to 13 pneumococcal pes. Furthermore, the two Prevnar formulations are expensive to cture, 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 t risk for pneumococcal disease.
Thus, there remains a need to design more effective pharmaceutical compositions than the current strategies offer. In particular, such compositions need to incorporate novel or ic antigens that elicit an immune response against S. pneumoniae. 11. Summary Streptococcus pneumoniae is a major health concern, especially in very young, elderly, or compromised patients. While DNA and protein sequence information for S. pneumoniae has been known for some time, and researchers have long attempted to produce es against S. pneumoniae, a major problem was how to fy protective ptides from among the approximately 2100 genes in the S. pneumoniae genome. The instant application presents the results of whole-genome screens designed to identify the most immunogenic proteins in the S. pneumoniae genome. Several of the hits from the screen have been shown to protect against S. pneumoniae colonization in a mouse model, and in some instances against both colonization and S. pneumoniae-induced sepsis. Accordingly, the present sure provides, inter alia, certain highly effective vaccines against Streptococcus pneumoniae. The vaccines may be used therapeutically or prophylactically. The present disclosure also provides specific antigens and methods for using the antigens to elicit an immune se against S. niae.
In certain aspects, the present disclosure provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a polypeptide having an amino acid sequence comprising (or consisting of) SEQ ID NO: 265 or 268 or an immunogenic nt thereof.
In some embodiments, the polypeptide comprises an exogenous signal sequence. For instance, the ptide may have an amino acid sequence comprising SEQ ID NO: 266 or an immunogenic fragment thereof. The polypeptide may have an amino acid sequence consisting of SEQ ID NO: 265, 266, or 268.
In some embodiments, the vaccine formulation further comprises a first polypeptide having an amino acid ce comprising (or consisting of) one of SEQ ID NOS: 1-23, 267, and 269-270 or an genic fragment thereof. In certain embodiments, the vaccine formulation further comprises a second polypeptide having an amino acid sequence sing any of SEQ ID NOS: 1-23, 267, and 0 or an immunogenic fragment thereof.
In certain embodiments, the first and the second ptides belong to a different group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenic nt thereof, (ii) one of SEQ ID NOS: 2-5 and 14-17 or an immunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 and 18-19 or an immunogenic fragment thereof, (iv) SEQ ID NO: 8 or an immunogenic fragment thereof, (v) one of SEQ ID NOS: 9-10 and 20-21 or an immunogenic fragment thereof, and (vi) one of SEQ ID NO: 11-13, 267, and 269-270 or an immunogenic fragment f.
In some such embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: . In some embodiments, the vaccine formulation comprises a polypeptide ting of SEQ ID NO: 6 or 7 and a polypeptide consisting of SEQ ID NO: 9 or 10.
In any of the aspects or embodiments herein, the vaccine formulation may comprise a polypeptide of SEQ ID NO: 265, 266, or 268 Which is a ted fragment having from 1-20 amino acid residues removed from the N-terminus, C—terminus, or both. In some embodiments, the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-21 and 265-270.
In certain embodiments, the vaccine formulation ses one or more polypeptides having an amino acid sequence comprising SEQ ID NOS: 22 or 23 or an immunogenic fragment thereof.
In another aspect, the invention provides vaccine formulations comprising a known S. niae antigen, such as a lysoid, Choline-binding protein A (Cpr), or Pneumococcal surface n A (PspA), or derivatives thereof, and one, two, or three polypeptides from Table 1 or Table 2. An exemplary vaccine formulation comprises: (i) a polypeptide having an amino acid sequence comprising (or consisting of) one or more of SEQ ID NOS: 1-23 and 265-270 or an immunogenic fragment thereof, (ii) a pneumolysoid, and (iii) a pharmaceutically acceptable carrier. A further exemplary vaccine ation ses: (i) a polypeptide having an amino acid sequence comprising (or consisting of) one or more of SEQ ID NOS: 1-23 and 265-270 or an immunogenic fragment thereof, (ii) Cpr or a derivative thereof, and (iii) a pharmaceutically acceptable carrier. A further exemplary vaccine formulation comprises: (i) a polypeptide having an amino acid sequence comprising (or ting of) one or more of SEQ ID NOS: 1-23 and 265-270 or an immunogenic fragment thereof, (ii) PspA or a derivative thereof, and (iii) a pharmaceutically acceptable carrier. In some such embodiments, the polypeptide of (i) comprises any one of SEQ ID NO: 2-5, 6, 7, 9-13, and 265-267. In some embodiments, the e formulation further comprises a second polypeptide having an amino acid sequence comprising one of SEQ ID NO: 1-23 and 265-270. In some embodiments, the pneumolysoid is PdT, Pd-A, Pd-B, rPd2, rPd3, Ply8, A6PLY, L460D (see, e.g., US 285846 and L. Mitchell, Protective Immune Responses to Streptococcus pneumoniae Pneumolysoids, ASM2011 conference abstract, 2011), or a variant thereof. In some embodiments, the derivative of PspA comprises all or a nt of the proline-rich region of PspA.
In certain embodiments, the polypeptide is conjugated to an genic carrier. In some embodiments, the vaccine formulation comprises at least one lipidated polypeptide.
In some embodiments, the vaccine ation further comprises conjugated S. pneumoniae polysaccharides. The conjugated ccharides may be, for example, as described in US Patent 5,623,057, US Patent 5,371,197, or 2011/023526.
In some embodiments, the vaccine formulation further comprises an nt. The adjuvant may be, for example, an agonist of toll-like receptors (TLRs). The adjuvant may be, for example, alum. In some ments, the vaccine formulation comprises 1-1000 ug of each ptide and 1-250 ug of the adjuvant.
In certain embodiments, the vaccine formulation induces a THl7 cell response at least l.5-fold greater than that d by a control ted antigen after contacting THl7 cells. In some ments, the vaccine formulation inhibits infection by S. pneumoniae in an cted subject. In certain embodiments, the vaccine formulation inhibits S. niae colonization in an individual. In some embodiments, the vaccine formulation ts S. pneumoniae symptoms or sequelae. For instance, the vaccine formulation inhibits S. pneumoniae-induced sepsis.
In certain aspects, the t disclosure provides a method for treating a subject suffering from or susceptible to S. niae infection, comprising administering an effective amount of any of the vaccine formulations bed herein.
In some embodiments, the method ts infection by S. pneumoniae in an uninfected subject.
In some embodiments, the method inhibits S. pneumoniae colonization in a subject. In some embodiments, the method inhibits S. pneumoniae symptoms or sequelae. An exemplary sequela is sepsis.
In certain embodiments, the method treats a subject With one dose. In other ments, the method treats a subject With two or three doses. In some embodiments, the method treats a subject Within three doses.
In certain embodiments, the subject is a human.
The present disclosure provides, for example, a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13, 265, 266 and 267, or an immunogenic fragment thereof.
The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and at least one polypeptide having an amino acid sequence comprising SEQ ID NO: 6, SEQ ID NO:10 or SEQ ID NO: 265, or an genic fragment thereof. The present disclosure further provides a vaccine formulation comprising a pharmaceutically acceptable carrier and at least one polypeptide having an amino acid sequence consisting of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 265, or an immunogenic fragment thereof. rmore, the instant application provides a vaccine ation comprising a pharmaceutically acceptable carrier and one or more polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-23, 268, 269 and 270, or an immunogenic fragment thereof.
The t disclosure r es an immunogenic composition comprising a pharmaceutically acceptable carrier and two or more polypeptides having amino acid sequences comprising any of SEQ ID NOS: 1-13, 265, 266 and 267, or an immunogenic fragment thereof.
The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and two or more polypeptides having amino acid ces comprising SEQ ID NO: 6, SEQ ID NO:10 or SEQ ID NO: 265, or an immunogenic fragment f. The present disclosure further es a vaccine formulation comprising a pharmaceutically acceptable carrier and two or more polypeptides having amino acid sequences consisting of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 265, or an immunogenic fragment thereof. This disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and two or more polypeptides having amino acid ces comprising SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 265, or an immunogenic fragment f. In addition, this disclosure provides a vaccine formulation comprising a pharmaceutically acceptable carrier and two or more polypeptides having amino acid sequences comprising SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 265, or an immunogenic fragment thereof. In some embodiments, the amino acid sequence comprising SEQ ID NO: 265 comprises an exogenous signal sequence.
The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and three or more polypeptides having amino acid sequences comprising SEQ ID NO: 6, SEQ ID NO:10 and SEQ ID NO: 265, respectively, or an genic fragment thereof. The present disclosure r provides a vaccine ation comprising a pharmaceutically acceptable carrier and three or more polypeptides having amino acid sequences consisting of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 265, respectively, or an immunogenic fragment thereof.
The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a first polypeptide comprising the amino acid ce of SEQ ID NO: 265, 266, or 268, or an immunogenic nt or homologue thereof, and a second polypeptide comprising the amino acid ce of SEQ ID NO: 9, 10, 20, or 21, or an immunogenic fragment or homologue thereof.
The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a first polypeptide comprising the amino acid sequence of SEQ ID NO: 265, 266, or 268, or an immunogenic fragment or homologue thereof, and a second polypeptide sing the amino acid sequence of SEQ ID NO: 6, 7, 18, or 19, or an immunogenic fragment or gue thereof.
The present disclosure also provides a e formulation according to the present disclosure, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6, or an immunogenic fragment or homologue thereof, and further comprising a third polypeptide comprising the amino acid sequence of SEQ ID NO: 7, or an immunogenic fragment or homologue f.
The present disclosure also provides a vaccine formulation according to the present disclosure, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 9, or an immunogenic fragment or homologue thereof, and further comprising a third polypeptide comprising the amino acid sequence of SEQ ID NO: 10, or an immunogenic nt or gue thereof.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general dge in the field nt to the t disclosure as it existed before the priority date of each claim of this application.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be tood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. - 6A - 111. Brief Description of the Drawings shows the tration of IL-17 generated by blood samples from mice that were zed with the indicated protein(s) and cholera toxin adjuvant, then stimulated with killed, unencapsulated whole cell S. pneumoniae, as described in Example 5. 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, , and SP1634. shows the concentration of IL-17 ted 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 ), as described in Example 5. shows the number of S. pneumoniae colonies ed from a nasal wash in mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then challenged with intranasal stration of S. niae, as described in Example 5. 003 represents a control unrelated antigen. shows the concentration of IL-17 generated by blood s 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 6. shows the concentration of IL-17 generated by blood samples from mice that were immunized with the indicated protein(s) and cholera toxin adjuvant, then stimulated by the indicated protein(s), as described in Example 6. 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 nt, then challenged with intranasal administration of S. pneumoniae, as described in Example 6. The HSV-2 protein ICP47 with the gene name USl2 4543.l, NC_001798.l; shown in the figure as 003) and ovalbumin (OVA) represent control antigens. 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 7. shows the number of S. pneumoniae colonies obtained from a nasal wash in BALB/c mice that were immunized with the indicated n(s) and cholera toxin adjuvant, then challenged with intranasal administration of S. pneumoniae, as described in Example 8. shows the tration of IL-17A generated by blood samples from mice that were immunized with the indicated proteins and a toxin adjuvant, then stimulated with the protein of immunization (left panel) or killed, unencapsulated whole cell S. pneumoniae (right , as described in e 9. shows the number of S. pneumoniae colonies ed from a nasal wash in mice that were immunized with the indicated proteins and cholera toxin adjuvant, then challenged with intranasal administration of S. pneumoniae, as described in Example 10. shows surVival of mice that were immunized with the indicated proteins and the adjuvant alum, then underwent aspiration challenge with S. pneumoniae as described in Example 1 1. shows al of mice that were immunized with the indicated proteins and the adjuvant alum, then underwent aspiration challenge with S. pneumoniae as described in Example 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 with intranasal administration of S. pneumoniae, as described in e 13. shows the concentration of IL-17A generated by blood samples form mice that were immunized with the indicated ns and alum, then ated with the proteins indicated at upper left, as bed in Example 14. shows the number of S. pneumoniae colonies obtained from a nasal wash in mice that were immunized with the ted proteins and alum or with killed, psulated whole cell S. pneumoniae plus alum (WCV), then challenged with intranasal administration of S. pneumoniae, as described in Example 15. 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 with intranasal administration of S. pneumoniae, in two pooled studies as described in Example 16. 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 with intranasal administration of S. pneumoniae, as described in Example 17. shows al of mice that were injected with antibodies or sera specific to the ted proteins, then underwent aspiration challenge with S. pneumoniae, as described in Example 18. shows the percent of animals ted from sepsis in six separate aspiration challenge studies, two of which are described in more detail in Examples 12 and 18.
IV. Detailed Description A. Specific polypeptides and nucleic acids for use in S. pneumoniae vaccines and genic compositions This application describes S. pneumoniae vaccines that include one or more of the polypeptides or genes listed in Table 1, or variants or fragments thereof as described below. The vaccine may include a polypeptide that comprises a sequence of Table 1 or a variant or immunogenic fragment thereof or a polypeptide that consists of a sequence of Table 1 or a t or immunogenic fragment thereof. 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 ed in this application.
Table 1. Immunogenic polypeptides for vaccine ations Locus tag name and description Protein DNA DNA GenBank SEQ ID SEQ ID Accession No.
. N0. (from March 30, 2010) SP0024 -1 — NC_003028.3|:27381— 27878 SP0882 -2 — NC_003028.3|:831804— 832628 N 24 _ SP0882 with exogenous signal sequence 25 - SP0882N with exogenous signal 26 - _ 9 _ sequence - SP0148 lacking signal ce _ 27 - SP0148 including signal sequence 7 28 NC_003028.3I:145,513— SP1072 NC_003028.3I:1008420— 1010180 SP2108 including signal sequence - - NC_003028.3I:2020750— 2022021 SP2108 lacking signal sequence 29 - SP0641M 30 — SP0641 -12 — NC_003028.3I:2020750— 2022021 SP0641N 31 — SP0882 sus 14 - - SP0882N consensus 15 - - SP0882 consensus With exogenous l—k Cfi leader SP0882N consensus With ous l—k \] leader SP0148 consensus lacking signal l—k OO sequence SP0148 consensus including signal 19 - — sequence SP2108 consensus lacking signal 20 - — sequence SP2108 sus including signal b) l—k sequence SP1634 b0b9 NC_003028.3I:1534348— 1535421 SP0314 b0 00 NC_003028.3I:287483— . l—k C) | - 290683 SP1912 265 271 NC_003028.3|:1824672— 1824971 SP1912L 266 272 — SP0641.1 267 273 — SP1912 consensus 268 — — N consensus 269 — — SP0641M consensus 270 - — >“NB: The database sequence incorrectly lists TTG (encoding Leu) at tide positions 541-543. The correct sequence, as shown in SEQ ID NO: 28, has TTC at that codon and encodes Phe. The database sequence further does not include a C—terminal Glu found in certain isolates.
Certain polypeptides of Table 1, and variants thereof, are described in r detail below. 1. SP1912 (SEQ ID NO: 265) and variants thereof SP1912 is a etical n of 99 amino acids. While the protein function is not definitively known, sequence analysis suggests it is a putative thioredoxin.
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 n embodiments, the polypeptide includes no more than 90, 75, 60, 45 or 30 consecutive amino acids from SP1912.
In some embodiments, the compositions and methods herein call for the use of an SP1912 variant that comprises an exogenous lipidation sequence. In some embodiments, a signal sequence s lipidation. Thus, the lipidation signal may be, e.g., the signal sequence of SP2108 (SEQ ID NO: 275) or SP0148, or an E. coli signal sequence. The exemplary variant SP1912L, sing the signal ce of the E. coli gene RlpB (SEQ ID NO: 276) is represented by polypeptide sequence SEQ ID NO: 266. SP1912 (SEQ ID NO: 265) and SP1912L (SEQ ID NO: 266) may be encoded, respectively, by nucleic acids according to SEQ -11_ ID NO: 271 and 272, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) may be used.
Consensus sequences illustrating combinations of SP1912 ces from different pes are ed as SEQ ID NO: 268. Thus, in certain embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence sing, or consisting of, SEQ ID NO: 268, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising SEQ ID NO: 265). 2. SP0024 (SEQ ID NO: 1) and variants thereof SP0024 represents a hypothetical protein of 165 amino acids, ning a conserved carbonic ase domain that extends from amino acid 27 to amino acid 163. Based on this consensus motif, SP0024 may be a zinc-binding protein.
In some ments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected from SP0024. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide es no more than 150, 125, or 100 consecutive amino acids from SP0024. 3. SP0882 (SEQ ID NO: 2) and variants thereof SP0882 is a conserved hypothetical protein of 274 amino acids. Much of the protein (amino acids 2-270) forms an esterase or lipase—like region.
In some ments, vaccines or pharmaceutical compositions sing an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid es selected from SP0882. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids from SP0882.
One particular truncation variant named SP0882N consists of the N—terminal 130 amino acids of SP0882, and is shown as SEQ ID NO: 3. SP0882N includes a region that is particularly well ved among different serotypes. In certain embodiments, a polypeptide comprising SP0882 or SP0882N, or an genic fragment of either, also comprises an exogenous signal sequence. In some embodiments, the signal sequence is an E. coli or S. pneumoniae signal sequence. The signal sequence may be, for example, the signal sequence of SP2108. Two exemplary such polypeptides are SEQ ID NOS: 4 and 5. -12_ Variants of DNA and protein sequences of SP0882 are described, inter alia, in US Patent Application Publication No. 2009/0215149 and International ations WO2002/07702l, WO98/18931, and WO2007/106407. A variant of SP0882N is disclosed in International ation WO2008/146164.
Sequence variation occurs at the protein level between different S. pneumoniae serotypes, and consensus sequences rating combinations of SP0882 sequences from different S. pneumoniae serotypes are provided as SEQ ID NOS: l4-l7. Accordingly, in certain ments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising, or consisting of, any of SEQ ID NOS: 14-17, or an genic fragment f (e. g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 2—5).
Nucleic acid sequences encoding different variants of SP0882 (SEQ ID NOS: 2-5) are provided as SEQ ID NOS: 24-26, although due to degeneracy in the genetic code, other DNA sequences (including codon-optimized sequences) could encode these polypeptides. 4. SP0148 (SEQ ID NO: 7) and variants f The protein SP0148 is named “ABC transporter, substrate-binding protein”. Proteins of this class are typically extracellular proteins that interact transiently With a embrane 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 xes. In on, SP0148 has a bacterial extracellular solute-binding proteins family 3 domain Which is y 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.
In some embodiments, es or pharmaceutical compositions comprising an S. pneumoniae polypeptide include 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 250, 275, 200, 175, 150, 125, or 100 utive amino acids from SP0148. -13_ Endogenous SP0148 comprises a signal sequence that directs its ion and ial lipidation. In some embodiments, the signal sequence of the polypeptide of SEQ ID NO: 7 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: 6) is used. The polypeptide of SEQ ID NO: 6 is encoded by the nucleic acid of SEQ ID NO: 27, although other nucleic acid sequences (including codon-optimized sequences) may be used. SEQ ID NO: 28 encodes the full length sequence of SP0148 used in the screens herein. ts of the amino acid sequence and nucleotide sequence of SP0148 may be found in U.S.Patent Application Publication No. 2005/0020813, US. Patent Nos. 7,378,514 and 7,504,110, and European Patent Application No. EP1572868 and EP1855717.
Consensus sequences illustrating combinations of SP0148 sequences from different S. pneumoniae serotypes are provided as SEQ ID NOS: 18 and 19. Accordingly, in certain embodiments, the vaccine formulation comprises a polypeptide having an amino acid ce comprising, or consisting of, either of SEQ ID NOS: 18-19, or an genic fragment thereof (e. g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 6 or 7).
. SP1072 (SEQ ID NO: 8) and variants thereof , also known as dnaG, is a DNA primase enzyme that catalyzes formation of an RNA primer Which allows DNA rase to initiate DNA replication. A protein of 586 amino acids, SP1072 contains several ved motifs. Beginning at the N-terminus, amino acids 2 — 96 form a zinc finger domain, the DNA primase catalytic core spans amino acids 122 — 250, and a highly conserved omerase-primase (TORPIM) nucleotidyl transferase/hydrolase domain region extends from amino acid 258 to 330. In some embodiments, a vaccine or other composition comprises a truncation mutant of SP1072 comprising or lacking one or more of said domains and motifs.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae polypeptide include a polypeptide containing at least 20 consecutive amino acid residues selected form SP1072. The polypeptide may also be a t of the at least 20 e fragment. In certain embodiments, the polypeptide includes no more than 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 utive amino acids from SP1072. -14_ 6. SP2108 (SEQ ID NO: 9) 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.
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: 9 is lly 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.
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 ce SEQ ID NO: 10 and may be encoded by, for example, a nucleic acid according to SEQ ID NO: 29, although due to degeneracy in the genetic code, other DNA ces (including codon-optimized sequences) may be used.
In some embodiments, vaccines or pharmaceutical compositions comprising an S. pneumoniae ptide include a polypeptide containing at least 20 consecutive amino acid residues ed from SP2108. The polypeptide may also be a variant of the at least 20 e fragment. In certain embodiments, the polypeptide includes no more than 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids from SP2108.
Consensus ces rating combinations of SP2108 sequences from different serotypes are provided as SEQ ID NOS: 20 and 21. Thus, in certain embodiments, the vaccine formulation ses a polypeptide having an amino acid ce comprising, or consisting of, either of SEQ ID NOS: 20-21, or an immunogenic fragment thereof (e. g., in place of a ptide having an amino acid sequence comprising one of SEQ ID NOS: 9 or 10). 7. SP0641 (SEQ ID NO: 12) and variants thereof At 2144 amino acids in length, SP0641 is also known as PrtA, a cell wall-associated serine protease. Full-length SP0641 contains a number of conserved motifs: the PA_2 motif, extending between amino acids 485 and 597, which may form a protein binding surface; the Fn3- like domain (amino acids 800 — 939); and two predicted catalytic domains of the S8 C5a type located at amino acids 226 — 449 and 639 — 777. In some embodiments, a vaccine or other -15_ ition comprises a truncation mutant of SP0641 comprising or lacking one or more of said domains and motifs.
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 SP0641. The polypeptide may also be a variant of the at least 20 residue fragment. In certain embodiments, the polypeptide includes no more than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 consecutive amino acids from SP0641.
Certain other truncation mutants of SP0641 may also be used. For ce, the polypeptide designated SP0641N (SEQ ID NO: 13) consists of 661 amino acids corresponding to amino acids 24-684 near the N-terminus of SP0641. Roughly adjacent to SP0641N (and corresponding to amino acids 686-1333 of SP0641) lies the 648 residue region captured by the truncation variant SP0641M (SEQ ID NO: 11). The ptide designated SP0641.1 (SEQ ID NO: 267) consists of 978 amino acids corresponding to amino acids 28-1006 of .
Variants of SP0641 are disclosed in, for example, US. Patents No. 7,338,786, 6,573,082, and 107, as well as International Application W000/0673 8.
SEQ ID NOS: 30, 31 and 273 display the DNA sequences of SP0641M (SEQ ID NO: 11), SP0641N (SEQ ID NO: 13) and SP641.1 (SEQ ID NO: 267), respectively, although due to degeneracy in the genetic code, other DNA sequences ding codon-optimized sequences) could encode these SP0641 variants.
Consensus ces illustrating combinations of SP0641N and SP0641M sequences from different S. pneumoniae pes are provided as SEQ ID NOS: 269 and 270.
Accordingly, in n embodiments, the vaccine formulation comprises a ptide having an amino acid ce comprising, or consisting of, either of SEQ ID NOS: 269 or 270, or an immunogenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NOS: 11 or 13).
Polypeptides homologous to the polypeptides of Tables 1 and 2 (for example, SP1912, SP1912L, SP0024, SP0882, SP0882N, SP0148 With or Without a signal sequence, SP1072, SP2108 With or Without a signal sequence, SP0641, SP0641M, SP0641N, or SP0641.1) may also be used in the compositions and methods disclosed herein. Individual s of S. pneumoniae contain numerous ons 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 -16— acid sequence, or a portion thereof, with the homologous amino acid sequence from a different S. pneumoniae strain. In certain aspects, this application provides immunogenic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% ty to the ptides of Tables 1 and 2 or an immunogenic fragment thereof. Serotypic variation may be used to design such variants of the ptides of Tables 1 and 2.
In some embodiments, the vaccine compositions herein comprise a fragment of a protein of Table l or 2 (for example, fragments of SPl9l2, SPl9l2L, SP0024, SP0882, SP0882N, 0SPl48 with or without a signal sequence, SP1072, SP2108 with or without a signal sequence, SP064l, SP0641M, SP0641N, or SP064l.l). In some embodiments, this ation es truncation mutants that are close in size to the polypeptide of Table l or 2 (for example, one of SEQ ID NOS: 1-13, 265, 266 or 267). For example, they may lack at most one, two three, four, five, ten, or twenty amino acids from one or both termini. Internal ons, e. g., of 1-10, 11-20, 21-30, or 31-40 amino acids, are also contemplated.
In certain ments the e formulation comprises one or more polypeptides having an amino acid sequence comprising, or consisting of, any of SEQ ID NOS: 14-21, 268, 269 and 270. In certain embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: 14-21, 268, 269 and 270, 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: 14-21, 268, 269 and 270, 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 inus and C—terminus resulting in a protein with amino acid es removed.
In certain embodiments, the vaccine formulations provided herein comprise or r comprise one or more, or two or more, known S. pneumoniae ns. In some instances, the known S. pneumoniae antigens are predominantly antibody targets. In some ces, the known S. pneumoniae antigens protect from S. pneumoniae colonization, or from S pneumoniae- induced sepsis. One 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 -17_ 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 niae, 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, AM. et al. (1995) Infection and Immunity 63: 1969-74); Pd-A and Pd-B (Paton J.C. et al. ication and genicity of cally obtained pneumolysin toxoids and their ation to Streptococcus pneumoniae type 19F polysaccharide” Infect Immun. 1991 Jul;59(7):2297-304); rPd2 and rPd3 ira 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, 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.
Other appropriate S. pneumoniae antigens for combination vaccines include Pneumococcal surface n A (PspA); derivatives of PspA, Choline-binding n A (Cpr) and derivatives thereof (AD Ogunniyi et al., “Protection against Streptococcus niae elicited by immunization with pneumolysin and Cpr,” Infect Immun. 2001 Oct;69(10):5997— 6003); Pneumococcal surface adhesin A ; caseinolytic protease; sortase A (SrtA); pilus 1 RrgA adhesin; PpmA; PrtA; PavA; LytA; Stk-PR; PcsB; RrgB and derivatives thereof.
Derivatives of PspA e proline-rich segments with the non-proline block (PR+NPB, 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 e-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 . An example of the non-proline-block has the exemplary sequence EKSADQQAEEDYARRSEEEYNRLTQQQ (SEQ ID NO: 306), 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: 307 and 308. PspA and its derivatives can include genes expressing similar e-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 .18_ the PR-region is DLKKAVNE (SEQ ID NO: 309), and the carboxy-terminal ry is (K/G)TGW(K/G)QENGMW (SEQ ID NO: 310). 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 e SEQ ID NOs: 301 and 302. Particular embodiments of the immunogenic PspA polypeptide derivatives lacking the coiled-coil structure have the amino acid sequences shown as SEQ ID NOS: 303—305.
Immunogenic PspA polypeptides SEQ ID NO: 301, 303 and 305 include both PR and NPB sequences (PR+NPB). Immunogenic PspA polypeptides of SEQ ID NOS: 302 and 304 e only a PR sequence (PR only) and lack the NPB.
In some cases, the other appropriate S. niae antigen is at least at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to the corresponding ype S. niae protein. Sequences of the above-mentioned polypeptides, and nucleic acids that encode them, are known; see, for example, the S. niae ATCC 700669 complete genome sequence under GenB anl< accession number FM211187.1 and linked polypeptide sequences therein.
Further S. pneumoniae antigens for combination vaccines include ated S. pneumoniae polysaccharides. The conjugated polysaccharides may be, for example, as described in US Patent 5,623,057, US Patent 5,371,197, or .
In addition to those nucleic acids and polypeptides described in Table 1 above, this application also es immunogenic compositions that include one or more of the polypeptides or genes listed in Table 2, or variants or fragments thereof as bed herein. The DNA and protein sequence of each gene and protein may be found by searching for the Locus Tag in the publicly available database, Entrez Gene, as described above.
Table 2. Immunogenic proteins identified in human and mouse screens Locus tag Protein accession DNA accession number (from name number March 30, 2010) SP1574 AAK75660.1 NC_003028.3|:c1481367-1480609 SP1655 AAK75734.1 NC_003028.3|:c1557922-1557230 SP2106 AAK76165.1 NC_003028.3I:c2018657-2016399 SP1473 67.1 NC_003028.3|:c1386534-1386277 -19_ SP0605 AAK74757.1 NC_003028.3|:571604—572485 SP1177 86.1 NC_003028.3|:01115580—1115317 SP0335 AAK74510.1 NC_003028.3|:306559—306876 SP0906 AAK75031.1 NC_003028.3|:0859160—859029 SP1828 AAK75901.1 NC_003028.3|:01740010—1739000 SP2157 11.1 NC_003028.3|:02072146—2070995 SP1229 AAK75335.1 NC_003028.3I:01163388—1161718 SP1128 AAK75238.1 NC_003028.3|:1061773—1063077 SP1836 AAK75909.1 NC_003028.3|:1746104—1746280 SP1865 AAK75937.1 028.3|:01772987—1771923 SP0904 AAK75029.1 NC_003028.3|:0858126—857311 SP0765 AAK74903.1 NC_003028.3|:724170—725207 SP1634 AAK75714.1 NC_003028.3|:1534348—1535421 SP0418 AAK74581.1 NC_003028.3|:396692—396916 SP1923 AAK75991.1 NC_003028.3|:01833311—1831896 SP1313 91.1 NC_003028.3|:01833311—1831896 SP0775 AAK74913.1 NC_003028.3|:731798—732070 SP0314 AAK74491.1 NC_003028.3|:287483—290683 SP0912 AAK75037.1 NC_003028.3|:864707—865465 SP0159 AAK74341.1 NC_003028.3|:0157554—156292 SP0910 AAK75035.1 NC_003028.3|:863462—863734 SP2148 AAK76205.1 NC_003028.3|:2062144—2063373 SP1412 AAK75510.1 028.3|:01332393—1331605 SP0372 AAK74539.1 NC_003028.3|:350268—350597 SP1304 AAK75407.1 NC_003028.3|:01232491—1232390 SP2002 AAK76069.1 NC_003028.3|:01906183—1905446 SP0612 64.1 NC_003028.3|:579708—579806 SP1988 AAK76055.1 NC_003028.3I:01892598—1890565 SP0484 AAK74643.1 NC_003028.3|:465572—466402 SP0847 AAK74978.1 NC_003028.3|:794144—795202 -20_ SP1527 AAK75616.1 NC_003028.3|:c1439494—1437536 SP0542 AAK74699.1 NC_003028.3I:515940—516059 SP0441 AAK74602.1 NC_003028.3I:414869—415057 SP0350 AAK74523.1 NC_003028.3|:323990—324625 SP0014 AAK74207.1 NC_003028.3I:14450—14929 SP1965 AAK76032.1 028.3I:c1873279—1873073 SP0117 AAK74303.1 NC_003028.3I:118423—120657 SP0981 AAK75102.1 028.3I:927115—928056 SP2229 AAK76277.1 NC_003028.3I:c2148627—2147602 SP2136 AAK76194.1 NC_003028.3I:c2048521—2046656 SP1179 AAK75288.1 NC_003028.3I:1116230—1118389 SP1174 AAK75283.1 NC_003028.3I:c1110717—1108258 SP2216 AAK76264.1 NC_003028.3I:c2136445—2135267 SP1393 AAK75491.1 NC_003028.3I:1316756—1318027 SP1384 AAK75482.1 NC_003028.3I:c1309464—1308967 SP2032 AAK76097.1 NC_003028.3|:c1939994—1938321 Typically, the polypeptides t in compounds of the invention are immunogenic, either alone or as a t, Which includes polypeptides fused to another polypeptide or mixed With or complexed to an adjuvant. Variants also include sequences With less than 100% sequence identity, as bed herein. In certain embodiments, an antigen of Table 1 or 2 is provided as a full length polypeptide. In addition, one may use fragments, precursors and analogs that have an appropriate immunogenicity.
These polypeptides may be immunogenic in mammals, for example mice, guinea pigs, or humans. An immunogenic polypeptide is lly one capable of g a icant immune response in an assay or in a subject. The immune response may be innate, humoral, cell- mediated, or mucosal (combining elements of innate, humoral and cell-mediated immunity). For instance, an immunogenic polypeptide may se the amount of lL-17 produced by T cells.
The lL-17 assay described in Examples 1-4 is an example of an assay that may be used to identify an genic polypeptide. Alternatively or additionally, an immunogenic polypeptide may(i) induce production of antibodies, e. g., neutralizing antibodies, that bind to the -21_ polypeptide and/or the whole ia, (ii) induce THl7 immunity, (iii) activate the CD4+ T cell response, for example by increasing CD4+ T cells and/or sing localization of CD4+ T cells to the site of infection or ction, (iv) activate the CD8+ CTL response, for example by sing CD8+ T cells and/or increasing localization of CD8+ T cells to the site of infection or reinfection, (V) induce THl 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.
In certain embodiments, polypeptides have less than 20%, 30%, 40%, 50%, 60% or 70% ty 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 e insulin, proliferating cell nuclear n, rome P450, and myelin basic protein.
The present invention also provides an immunogenic composition comprising a pharmaceutically acceptable carrier, a polypeptide having an amino acid sequence comprising SEQ ID NO: 265, 266, or 268 or an immunogenic fragment thereof, and one or more polypeptides having amino acid sequences comprising any of SEQ ID NOS: 1-23 and SPl574, SPl655, SP2106, SP1473, SP0605, SPll77, SP0335, SP0906, SP1828, , , SPll28, SP1836, SP1865, SP0904, SP0765, SP1634, SP0418, SP1923, SPl3l3, SP0775, SP0314, SP0912, SP0159, SP0910, SP2148, SPl4l2, SP0372, SP1304, SP2002, , SP1988, SP0484, , SP1527, SP0542, SP0441, SP0350, SP0014, SP1965, SP0117, SP0981, , SP2136, SP1179, SP1174, SP2216, SP1393, SP064l.l, SP1384, and SP2032, or an immunogenic fragment thereof.
In some embodiments, the vaccine formulation comprises at least two polypeptides, each polypeptide ing to a different group of (i)-(vii): (i) SEQ ID NO: 1 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 2-5 and 14-17 or an immunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 and 18-19 or an immunogenic nt thereof, (iv) SEQ ID NO: 8 or an immunogenic fragment thereof, (v) one of SEQ ID NOS: 9-10 and 20-21 or an immunogenic fragment thereof, (vi) one of SEQ ID NOS: 11-13, 267, and 269-270 or an immunogenic fragment thereof, and (vii) one of SEQ ID NOS: 265-266 and 268 or an immunogenic fragment thereof. Examples of such combinations are listed below. Additional ations may be made by replacing one of the sequences below with the corresponding -22_ consensus sequence, e.g., one of SEQ ID NOS: 14-21 or 268-270. In some embodiments, one of the polypeptides is one of SEQ ID NOS: 265-266 and 268 or an immunogenic fragment thereof.
In some embodiments, the vaccine formulation further comprises a pneumolysoid. In some ments, the vaccine formulation further comprises Cpr or a derivative thereof. In some embodiments, the vaccine formulation further comprises PspA or a derivative f sing all or a fragment of the proline-rich region of PspA.
SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: \OOO\]O\Ul-I>U~)l\) SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: SEQ ID NO: 1 and SEQ ID NO: l—tl—tl—tl—t WNl—‘O SEQ ID NO: 1 and SEQ ID NO: 265 SEQ ID NO: 1 and SEQ ID NO: 266 SEQ ID NO: 1 and SEQ ID NO: 267 SEQ ID NO: 2 and SEQ ID NO: SEQ ID NO: 2 and SEQ ID NO: SEQ ID NO: 2 and SEQ ID NO: SEQ ID NO: 2 and SEQ ID NO: \OOO\10\ SEQ ID NO: 2 and SEQ ID NO: 10 SEQ ID NO: 2 and SEQ ID NO: ll SEQ ID NO: 2 and SEQ ID NO: l2 SEQ ID NO: 2 and SEQ ID NO: 13 -23_ SEQ ID NO: 2 and SEQ ID NO: 265 SEQ ID NO: 2 and SEQ ID NO: 266 SEQ ID NO: 2 and SEQ ID NO: 267 SEQ ID NO: 3 and SEQ ID NO: SEQ ID NO: 3 and SEQ ID NO: SEQ ID NO: 3 and SEQ ID NO: SEQ ID NO: 3 and SEQ ID NO: 0\ SEQ ID NO: 3 and SEQ ID NO: 10 SEQ ID NO: 3 and SEQ ID NO: 1 1 SEQ ID NO: 3 and SEQ ID NO: 12 SEQ ID NO: 3 and SEQ ID NO: 13 SEQ ID NO: 3 and SEQ ID NO: 265 SEQ ID NO: 3 and SEQ ID NO: 266 SEQ ID NO: 3 and SEQ ID NO: 267 SEQ ID NO: 4 and SEQ ID NO: SEQ ID NO: 4 and SEQ ID NO: SEQ ID NO: 4 and SEQ ID NO: SEQ ID NO: 4 and SEQ ID NO: \OOO\10\ SEQ ID NO: 4 and SEQ ID NO: 10 SEQ ID NO: 4 and SEQ ID NO: 1 1 SEQ ID NO: 4 and SEQ ID NO: 12 SEQ ID NO: 4 and SEQ ID NO: 13 SEQ ID NO: 4 and SEQ ID NO: 265 SEQ ID NO: 4 and SEQ ID NO: 266 SEQ ID NO: 4 and SEQ ID NO: 267 -24_ SEQ ID NO: 5 and SEQ ID NO: SEQ ID NO: 5 and SEQ ID NO: SEQ ID NO: 5 and SEQ ID NO: SEQ ID NO: 5 and SEQ ID NO: 0\ SEQ ID NO: 5 and SEQ ID NO: 10 SEQ ID NO: 5 and SEQ ID NO: 1 1 SEQ ID NO: 5 and SEQ ID NO: 12 SEQ ID NO: 5 and SEQ ID NO: 13 SEQ ID NO: 5 and SEQ ID NO: 265 SEQ ID NO: 5 and SEQ ID NO: 266 SEQ ID NO: 5 and SEQ ID NO: 267 SEQ ID NO: 6 and SEQ ID NO: SEQ ID NO: 6 and SEQ ID NO: SEQ ID NO: 6 and SEQ ID NO: 10 SEQ ID NO: 6 and SEQ ID NO: 1 1 SEQ ID NO: 6 and SEQ ID NO: 12 SEQ ID NO: 6 and SEQ ID NO: 13 SEQ ID NO: 6 and SEQ ID NO: 265 SEQ ID NO: 6 and SEQ ID NO: 266 SEQ ID NO: 6 and SEQ ID NO: 267 SEQ ID NO: 7 and SEQ ID NO: SEQ ID NO: 7 and SEQ ID NO: SEQ ID NO: 7 and SEQ ID NO: 10 SEQ ID NO: 7 and SEQ ID NO: 11 SEQ ID NO: 7 and SEQ ID NO: 12 SEQ ID NO: 7 and SEQ ID NO: 13 SEQ ID NO: 7 and SEQ ID NO: 265 -25_ SEQ ID NO: 7 and SEQ ID NO: 266 SEQ ID NO: 7 and SEQ ID NO: 267 SEQ ID NO: 8 and SEQ ID NO: SEQ ID NO: 8 and SEQ ID NO: 10 SEQ ID NO: 8 and SEQ ID NO: 1 1 SEQ ID NO: 8 and SEQ ID NO: 12 SEQ ID NO: 8 and SEQ ID NO: 13 SEQ ID NO: 8 and SEQ ID NO: 265 SEQ ID NO: 8 and SEQ ID NO: 266 SEQ ID NO: 8 and SEQ ID NO: 267 SEQ ID NO: 9 and SEQ ID NO: 1 1 SEQ ID NO: 9 and SEQ ID NO: 12 SEQ ID NO: 9 and SEQ ID NO: 13 SEQ ID NO: 9 and SEQ ID NO: 265 SEQ ID NO: 9 and SEQ ID NO: 266 SEQ ID NO: 9 and SEQ ID NO: 267 SEQ ID NO:10 and SEQ ID NO: 11 SEQ ID NO: 10 and SEQ ID NO: 12 SEQ ID NO: 10 and SEQ ID NO: 13 SEQ ID NO: 10 and SEQ ID NO: 265 SEQ ID NO: 10 and SEQ ID NO: 266 SEQ ID NO: 10 and SEQ ID NO: 267 SEQ ID NO: 11 and SEQ ID NO: 265 SEQ ID NO: 11 and SEQ ID NO: 266 -26— SEQ ID NO: 12 and SEQ ID NO: 265 SEQ ID NO: 12 and SEQ ID NO: 266 SEQ ID NO: 13 and SEQ ID NO: 265 SEQ ID NO: 13 and SEQ ID NO: 266 In certain ments, the vaccine formulation comprises at least three different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 1-13, 265, 266, and 267, or an immunogenic fragment thereof, each polypeptide belonging to a different group of (i)-(Vii): (i) SEQ ID NO: 1 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 2-5 or an immunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 or an immunogenic fragment thereof, (iV) SEQ ID NO: 8 or an immunogenic fragment thereof, (V) one of SEQ ID NOS: 9-10 or an immunogenic fragment thereof, (Vi) one of SEQ ID NO: 11-13 and 267, or an immunogenic fragment thereof, and (Vii) one of SEQ ID NOS: 6 or an immunogenic fragment thereof. Examples of such combinations are listed below. Additional combinations may be made by replacing one of the ces below With the corresponding consensus sequence, e.g., one of SEQ ID NOS: 14-21 or 268-270. In some embodiments, one of the polypeptides is one of SEQ ID NOS: 265-266 and 268 or an immunogenic fragment thereof. In some embodiments, the Vaccine formulation r comprises a pneumolysoid. In some embodiments, the Vaccine formulation further comprises Cpr or a deriVatiVe thereof. In some embodiments, the Vaccine ation further comprises PspA or a deriVatiVe f comprising all or a nt of the proline-rich region of PspA.
SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: \OOO\10\ SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10 SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 13 -27_ SEQ ID NO ; 1, SEQ ID NO: 2, and SEQ ID NO: 265 SEQ ID NO ; 1, SEQ ID NO: 2, and SEQ ID NO: 266 SEQ ID NO ; 1, SEQ ID NO: 2, and SEQ ID NO: 267 SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: O\ SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 10 SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 267 SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: \OOO\10\ SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 10 SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 267 SEQ ID NO ; 1, SEQ ID NO: 5; and SEQ ID NO: SEQ ID NO ; 1, SEQ ID NO: 5; and SEQ ID NO: SEQ ID NO ; 1, SEQ ID NO: 5; and SEQ ID NO: -28— SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 10 SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 267 SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 10 SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 267 SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 10 SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 267 SEQ ID NO:1, SEQ ID NO: 8; and SEQ ID NO: SEQ ID NO:1, SEQ ID NO: 8; and SEQ ID NO: 10 -29_ SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 267 SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 267 SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 1, SEQ ID NO: 267, and SEQ ID NO: 265 -30_ SEQ ID NO:1, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 8 SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 9 SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 10 SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 11 SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 12 SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 13 SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 267 SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 8 SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 9 SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 10 SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 11 SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 12 SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 13 SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 267 SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 9 SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 10 SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 11 SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 12 SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 13 SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 267 -31_ SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 1 1 SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 1 2 SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 1 3 SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 267 SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 2, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 2, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 2, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 8 SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 9 SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 1 0 -32_ SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 11 SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 12 SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 13 SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 267 SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 8 SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 9 SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 10 SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 11 SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 12 SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 13 SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 267 SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 9 SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 10 SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 11 SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 12 SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 13 SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 8, and SEQ ID NO: 267 SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 11 SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 12 SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 13 SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 266 -33_ SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 267 SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 3, SEQ ID NO: 10, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 3, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 3, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 3, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 8 SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 9 SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 10 SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 11 SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 12 SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 13 SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 267 -34_ SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 8 SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 9 SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 10 SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 11 SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 12 SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 13 SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 267 SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 9 SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 10 SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 11 SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 12 SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 13 SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 267 SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 11 SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 12 SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 13 SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 267 SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 265 -35_ SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 4, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 4, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 4, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 10 SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 11 SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 12 SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 13 SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 267 SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 10 SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 11 SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 12 -36— SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 13 SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 267 SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 9 SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 10 SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 11 SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 12 SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 13 SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 267 SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 11 SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 12 SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 13 SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 267 SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 266 -37_ SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 5, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 5, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 9 SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 10 SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 11 SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 12 SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 13 SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 265 SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 266 SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 267 SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 11 SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 12 SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 13 SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 266 SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 267 SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 265 SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 266 -3g_ SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 6, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 6, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 6, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 9 SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 10 SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 11 SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 12 SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 13 SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 265 SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 266 SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 267 SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 11 SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 12 SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 13 SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 266 SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 267 -39_ SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 265 SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 7, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 7, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 7, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 7, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 11 SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 12 SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 13 SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 265 SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 266 SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 267 SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 11 SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 12 SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 13 SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 265 -40_ SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 8, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 8, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 265 SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 266 SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 267 SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 265 SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 9, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 9, SEQ ID NO: 267, and SEQ ID NO: 266 SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 265 _ 41 _ SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 266 SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 265 SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 266 SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 265 SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 266 SEQ ID NO: 10, SEQ ID NO: 267, and SEQ ID NO: 265 SEQ ID NO: 10, SEQ ID NO: 267, and SEQ ID NO: 266 In some embodiments, the vaccine formulation comprises at least two different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21, 268, 269 and 270, or an immunogenic fragment thereof. In certain such embodiments, the vaccine formulation comprises at least two polypeptides, each polypeptide belonging to a different group of (i)-(v): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragment f; (iii) one of SEQ ID NOS: 20-21 or an immunogenic nt thereof, (iv) one of SEQ ID NO: 268 or an immunogenic fragment thereof, and (v) one of SEQ ID NOS: 269-279 or an immunogenic nt thereof. Examples of such combinations are listed below. The ations below specify consensus sequences.
However, additional combinations may be made by replacing one of the consensus sequences with the corresponding non-consensus sequence, e.g., one of SEQ ID NOS: 1-13 or 266-267. In some embodiments, one of the polypeptides is SEQ ID NO: 268 or an immunogenic fragment f. In some embodiments, the vaccine formulation further comprises a pneumolysoid. In some embodiments, the vaccine formulation r comprises Cpr or a derivative thereof. In some embodiments, the vaccine ation further comprises PspA or a derivative thereof comprising all or a fragment of the proline-rich region of PspA.
SEQ ID NO: 14 and SEQ ID NO: 18 SEQ ID NO: 14 and SEQ ID NO: 19 SEQ ID NO: 14 and SEQ ID NO: 20 SEQ ID NO: 14 and SEQ ID NO: 21 -42_ SEQ ID NO: 14 and SEQ ID NO: 268 SEQ ID NO: 14 and SEQ ID NO: 269 SEQ ID NO: 14 and SEQ ID NO: 270 SEQ ID NO: 15 and SEQ ID NO: 18 SEQ ID NO: 15 and SEQ ID NO: 19 SEQ ID NO: 15 and SEQ ID NO: 20 SEQ ID NO: 15 and SEQ ID NO: 21 SEQ ID NO: 15 and SEQ ID NO: 268 SEQ ID NO: 15 and SEQ ID NO: 269 SEQ ID NO: 15 and SEQ ID NO: 270 SEQ ID NO: 16 and SEQ ID NO: 18 SEQ ID NO: 16 and SEQ ID NO: 19 SEQ ID NO: 16 and SEQ ID NO: 20 SEQ ID NO: 16 and SEQ ID NO: 21 SEQ ID NO: 16 and SEQ ID NO: 268 SEQ ID NO: 16 and SEQ ID NO: 269 SEQ ID NO: 16 and SEQ ID NO: 270 SEQ ID NO: 17 and SEQ ID NO: 18 SEQ ID NO: 17 and SEQ ID NO: 19 SEQ ID NO: 17 and SEQ ID NO: 20 SEQ ID NO: 17 and SEQ ID NO: 21 SEQ ID NO: 17 and SEQ ID NO: 268 SEQ ID NO: 17 and SEQ ID NO: 269 SEQ ID NO: 17 and SEQ ID NO: 270 -43_ SEQ ID NO: 18 and SEQ ID NO: 20 SEQ ID NO: 18 and SEQ ID NO: 21 SEQ ID NO: 18 and SEQ ID NO: 268 SEQ ID NO: 18 and SEQ ID NO: 269 SEQ ID NO: 18 and SEQ ID NO: 270 SEQ ID NO: 19 and SEQ ID NO: 20 SEQ ID NO: 19 and SEQ ID NO: 21 SEQ ID NO: 19 and SEQ ID NO: 268 SEQ ID NO: 19 and SEQ ID NO: 269 SEQ ID NO: 19 and SEQ ID NO: 270 SEQ ID No: 20 and SEQ ID NO: 268 SEQ ID No: 20 and SEQ ID NO: 269 SEQ ID No: 20 and SEQ ID NO: 270 SEQ ID No: 21 and SEQ ID NO: 268 SEQ ID No: 21 and SEQ ID NO: 269 SEQ ID No: 21 and SEQ ID NO: 270 SEQ ID NO: 268 and SEQ ID NO: 269 SEQ ID NO: 268 and SEQ ID NO: 270 In some embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: 14- 21 and 268-270 wherein from 1-20 amino acid residues are removed from the inus, C- terminus, or both.
In some embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising any of SEQ ID NOS: 14-17. In some embodiments, the vaccine -44_ formulation ses a polypeptide having an amino acid sequence comprising either of SEQ ID NOS: 18-19. In some embodiments, the vaccine formulation comprises a polypeptide having an amino acid sequence comprising either of SEQ ID NOS: 20-21. In some embodiments, the vaccine formulation ses a polypeptide having an amino acid sequence comprising any of SEQ ID NOS: 268—270.
In some s, a vaccine formulation comprising one or more of SEQ ID NOS: 14-21, 268, 269 and 270 further comprises a polypeptide having an amino acid sequence comprising any of SEQ ID NOS: 1-13, 265, 266 and 267.
In certain ments, the e formulation comprises at least three different polypeptides having an amino acid sequence comprising any of SEQ ID NOS: 14-21, 268, 269 and 270, or an immunogenic fragment thereof. In certain such embodiments, the vaccine formulation comprises three of ): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragment f, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragment thereof; and (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragment thereof, (iv) one of SEQ ID NO: 268 or an immunogenic fragment thereof, and (v) one of SEQ ID NOS: 269-270 or an immunogenic fragment thereof. Examples of such combinations are listed below. The combinations below specify consensus ces. However, additional combinations may be made by ing one of the consensus sequences with the corresponding non-consensus sequence, e.g., one of SEQ ID NOS: 1-13 or 266-267. In some embodiments, one of the polypeptides is SEQ ID NO: 268 or an immunogenic fragment thereof. In some embodiments, the vaccine ation further comprises a pneumolysoid. In some embodiments, the vaccine formulation further comprises Cpr or a derivative thereof. In some embodiments, the vaccine formulation further comprises PspA or a derivative thereof comprising all or a fragment of the proline-rich region of PspA.
SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 20 SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 21 SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 268 SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 269 SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 270 SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 20 SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 21 -45_ SEQ ID NO; 14, SEQ ID NO : 19, and SEQ ID NO: 268 SEQ ID NO; 14, SEQ ID NO: 19, and SEQ ID NO: 269 SEQ ID NO; 14, SEQ ID NO : 19, and SEQ ID NO: 270 SEQ ID NO: 14, SEQ ID NO: 268, and SEQ ID NO: 269 SEQ ID NO: 14, SEQ ID NO: 268, and SEQ ID NO: 270 SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 20 SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21 SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 268 SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 269 SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 270 SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 20 SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 21 SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 268 SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 269 SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 270 SEQ ID NO: 15, SEQ ID NO: 268, and SEQ ID NO: 269 SEQ ID NO: 15, SEQ ID NO: 268, and SEQ ID NO: 270 SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 21 SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 268 SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 269 SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 270 SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20 SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21 SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 268 -46— SEQ ID NO: 16, SEQ ID NO : 19, and SEQ ID NO: 269 SEQ ID NO: 16, SEQ ID NO : 19, and SEQ ID NO: 270 SEQ ID NO: 16, SEQ ID NO: 268, and SEQ ID NO: 269 SEQ ID NO: 16, SEQ ID NO: 268, and SEQ ID NO: 270 SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 20 SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 21 SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 268 SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 269 SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 270 SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 20 SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21 SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 268 SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 269 SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 270 SEQ ID NO: 17, SEQ ID NO: 268, and SEQ ID NO: 269 SEQ ID NO: 17, SEQ ID NO: 268, and SEQ ID NO: 270 SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 268 SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 269 SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 270 SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 268 SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 269 SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 270 SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 268 -47_ SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 269 SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 270 SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 268 SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 269 SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 270 SEQ ID NO: 20, SEQ ID NO: 268, and SEQ ID NO: 269 SEQ ID NO: 20, SEQ ID NO: 268, and SEQ ID NO: 270 SEQ ID NO: 21, SEQ ID NO: 268, and SEQ ID NO: 269 SEQ ID NO: 21, SEQ ID NO: 268, and SEQ ID NO: 270 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 fied by computer algorithms. Some such algorithms, like riX (produced by EinaX), use a ational matrix approach. Other computational tools for fying antigenic epitopes e PEPVAC (Promiscuous EPitope-based VACcine, hosted by Dana Farber Cancer Institute on the world wide web at X.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.ul</MHCPred), and Immune e 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. Peptides identified by the tools described above include the following: .48_ SP2108 SP0148 SP1634 SP0882 SP0314 Fragments Fragments Fragments Fragments Fragments (SEQ ID NOS 34— (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS 57, tively, in 58—82, 83—109, 1 10—130, 131—169, order of respectively, in respectively, respectively, in respectively, in appearance) order of in order of order of order of appearance) appearance) appearance) appearance) AIIDGPWKA ALGLVAAGV PQV HLDNLVLKV AFL VMMAPYDRV ELTGYEIEV MLEIPAHQI DLIAGRVHL SLADYTYKV SIAGINYAK AVNNLSYTK KNFFAHHPK ILLPKDYEK FLLLGAFYL VWDPAKNML TYLPAEADI KVILAGHSK EYQDQIGCL VLIDGLSQL QPLPNISQM RYNMAVNNL SFDNLVSTL YFHDGQNVF ILASLGFLL GSL DFQQIMVRL YYDLPLNEL NPDISRMIV GLSQLLPVI APAVIESLV EHTDNPTIL YFDLFFGTI IPWSENLPD FLLNHYMTV FYYTYGLLA APIAQNPNV ALEYIHHLF QFGGKGVEY MLIPNVDRA SKYAFAGE LPSDQQPYV LPLNELDIL DQI KLEEMAKQV TEGAGNLI YVYPLLAQG IGM VYFHDGQN VLKRGVYTI LADWTNFYY QGLDNLKVI DPELQKQFA MEVVKPFI KVIAGLLRK NKD KYLYAAPI AVYTFDAPG YLKMKEHKL TLNYEHMNK KEAGVKVTL GELTGYEI QSLTPEERE KLSPDQRIF NIGYFFFKK KSTAVLGTV NPNVLVVKK AIYAASQI RIFIYVGTE KYTDVIEKF GAKTDDTTK KLSKQFFGD QI FIDETYRTK KYDDSVSTI SQKFVDFLV GSPRPFIYE LLDLAPQVP DTDRSYPVV TFNQMIKEL QAFKDAKVN AVNNLSYTK WQIEDKHFV YIDSSLCYY DYPETQSVF AVIESLVMY KIFDKIGVE QLL TQFIGLEYQ TPRAINNTL DAKTAANDA MVRLSDGQF LYFDLFFGT KDTDRSYPV APLLVNGEL YGVATIPTL AQG SINDLASLK LIA YIDHTNVAY KTAAIIDGP VVQATTSAK SINDLASLK NVFNSKESF YGY KAYEKEAGV TLEKLSKQF YYDLPLNEL FLLNHYMTV -49_ AGNGAYVFG VAAGVLAAC QKVILAGHS FYLYNGDLS AWVIPQAVK IEL GTDDSIIGW KSFAPLLV NMAVNNLSY TYLSFDNLV DETVVRTV FGTILDAGI YIDHTNVAY NQITAVYTF MLKDKIAFL KLELFYETG KIAFLGSNI SVPRTSYLS FGFGLSLFS STIRSIEQV FRKTTDNPF TVVRTVRDS STIRSIEQV DGLSQLLPV FGFGLSLFS KLVDQGEGF SP0024 Fragments SP1072 Fragments SP0641 Fragments SP1912 Fragments (SEQ ID NOS 170— (SEQ ID NOS 194— (SEQ ID NOS 228— (SEQ ID NOS 0, 193, respectively, 227, respectively, in 264, respectively, in respectively, in order of in order of order of appearance) order of appearance) appearance) appearance) AIVTCMDSR GIEVEKPLY AAYAPNEVV KMWMAGLALLGIGSL AQTFENEPF AEAHLLYRM AGDLRGKII LALATKKVA MAGLALLGIGSLALA GQL ALLNQDNMR DEIANEVWY WMAGLALLGIGSLAL DDVIISGAI APPERNYLY DNYLIYGDL GLALLGIGSLALATK FENEPFQEY AQNSYIHIL DQKEHPEKF LALLGIGSLALATKK FMQANQAYV AVASMGTAL DSLTDRLKL FSDMGEIATLYVQVY ISQQQMGTR AYLLTKTRI KFV KAKKMWMAGLALLGI KPKTRVAIV DAAKFYHAI EGQGRNRKL ALLGIGSLALATKKVAK LHGQLNLPL DTALEELER EIKGAGDLR KMWMAGLALLGIG LGL EEYQGVPFI EPIAEGQYF SDMGEIATLYVQVYE DMGEIATLYVQVYES LPLKPKTRV EFLEKIAPL EVSELKPHR AGLALLGIGSLALAT MGTREIVVL EFQVLYDLL GAFFDKSKI MGEIATLYVQVYESS MQLLIESPL EHVEHLKRL GLI KKMWMAGLALLGIGS VAL ELSEVEMTR GEVEKNLEV GMKAKKMWMAGLALL QFMQANQAY ESPLVLNDY IHFESVEEM MKAKKMWMAGLALLG QLNLPLKPK GEKTPSFNV IMFIVGIFL HFSDMGEIATLYVQV QQMGTREIV GLCPFHGEK IPGTLNKGI MNGMKAKKMWMAGLA REIVVLHHT IGDMPVQIV TFK MWMAGLALLGIGSLA SPLIPDDVI ITMPVTKQL ISDKGGFNW DHFSDMGEIATLYVQ SRLHVAQAL KALLNQDNM IVSEEDFIL RDHFSDMGEIATLYV NGMKAKKMWMAGLAL TEDMIRSLV KRLTKKLVL KEIGVEEAI VDVSDQDFL LTKTRISPI KIVVKDFAR VSDQDFLPF LVLVYDGDK KKINFQPSL VTEDMIRSL MRAEAHLLY KLKFVYIGK NGPEDLAYL KVYYGNNYK QTEEVERAW KYWQAIRAL SEIYLMEGF RDF SPHQALYDM MRFKKEDLK EEI NESVVDNYL VEMTRNKAL NEVWYAGAA VLYDLLGQY NINDIVDGL VPFIEAVQI QYLLKDNII QDL SPRQQGAGL YLMEGFMDV SRSKTLGGY TAAVILAAY WTELPAMGY -51_ Thus, in some aspects, this application provides an immunogenic fragment of an antigen described herein. The fragments, in some instances, are close in size to the full-length ptide or the polypeptide of Table l or 2. For example, they may lack at most one, two, three, four, five, ten, twenty, or thirty amino acids from one or both i. In certain ments, 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 virus sion 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) ably 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.
In some embodiments, the fragment is a truncated fragment of any of SEQ ID NOS: l-2l or 265-270, having from l-5, l-lO, or l-20 amino acid es removed from the N-terminus, C—terminus, or both. In some such embodiments, the same number of residues is d from the N—terminus and the C-terminus, while in other ments, a different number of residues is removed from the N—terminus compared to the C—terminus.
In certain aspects, this application provides genic polypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to a polypeptide of Table l or 2. In certain embodiments, the e formulation comprises at least two different polypeptides having an amino acid sequence comprising a sequence at least 90%, 95%, 98%, or 99% cal to any of SEQ ID NOS: l-2l or 265-270, or an immunogenic fragment f.
In some embodiments, one or more, e.g., two, three, four, or more polypeptides from Table l or 2 or immunogenic fragments or variants thereof are provided 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. -52_ In some embodiments, rather than being in a simple physical mixture, two, three, four, or more polypeptides from Table l or 2 or immunogenic fragments or variants thereof are covalently bound to each other, e.g. as a fusion protein. In some embodiments, the vaccine formulation contains substantially no other S. niae polypeptides other than polypeptides having an amino acid sequence comprising any of SEQ ID NOS: l-23 or 265-270. In some embodiments, the e formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides of Table 1. In some embodiments, the vaccine formulation contains substantially no other S. pneumoniae polypeptides other than polypeptides of Tables 1 and/or 2.
In certain embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid ce comprising any of SEQ ID NO: l-23 or 265-270. In certain such embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence ting of any of SEQ ID NO: l-23 or 265-270. In some embodiments, vaccine formulations or immunogenic compositions contain substantially no other S. pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising (or consisting of) any of the amino acid sequences of the ptides of Tables 1 and/or 2. Substantially, in this context, refers to less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2, or even less than 1% of the other S. niae polypeptides.
In certain embodiments, the e composition s a THl7 cell response at least l.5-fold greater than that induced by a control unrelated antigen (such as the HSV-2 protein ICP47 With the gene name USl2) after contacting THl7 cells. In some embodiments, the vaccine formulation inhibits infection by S. pneumoniae in an uninfected subject. In n embodiments, the vaccine formulation reduces occurrence or duration of S. pneumoniae aryngeal colonization in an individual infected by S. pneumoniae. In some embodiments, the e formulation inhibits development of sepsis in an dual infected by S. pneumoniae. In some embodiments, the vaccine formulation inhibits development of pneumonia, meningitis, otitis media, sinusitis or infection of other sites or organs With S. pneumoniae. -53_ In certain embodiments, this application provides nucleic acids encoding one or more of the ptides described above, such as DNA, RNA, or an analog thereof. The ying DNA sequences for the polypeptides bed 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 e 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.
In certain embodiments, this application provides nucleic acids (such as 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 l or 2, or a variant or n of said gene. In n embodiments, the nucleic acid is 600—2000, 800—1800, 1000-1600, 1200-1400 nucleotides in length. In some embodiments, the c 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 and 2, or immunogenic fragments thereof.
In some embodiments, the e or genic composition may comprise fusion proteins and/or fusion DNA constructs. The polypeptides described herein may be used t cation. In certain embodiments, when r 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, pneumolysin, keyhole limpet hemocyanin or the like.
In certain embodiments, the vaccine formulation comprises at least one lipidated polypeptide. Conjugation to the lipid moiety may be direct or ct (e.g., via a linker). The lipid moiety may be synthetic or naturally produced. In certain embodiments, a polypeptide from Table l or 2 may be chemically conjugated to a lipid moiety. In certain embodiments, a construct may comprise a gene or polypeptide from Table l or 2, 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: 274. A lipidation sequence may be N-terminal or inal to the protein, and may be embedded in a signal or other sequence, or in a fusion protein. Exemplary tion sequences include the signal sequence of SP2108 (SEQ ID NO: 275) and the signal sequence of the E. coli gene RlpB (SEQ ID NO: 276). A signal sequence may be, for example, -54_ an E. coli or S. pneumoniae signal sequence. Exemplary E. coli signal sequences include the mlpA signal sequence (Lin, J.J. et al., “An ichia coli mutant With an amino acid tion 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), 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, RA. et al. “Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli” Plasmid. 2003 Jan;49( 1): 18-29.) E. coli signal sequences that direct lipidation are also bed 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: 276) us S. pneumoniae signal sequences are known in the art. One such signal sequence is SEQ ID NO: 275.
In other embodiments, a construct may comprise a gene or protein from Table 1 or 2, 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 c acid. For instance, a cation tag may be a e, oligopeptide, or polypeptide that may be used in affinity purification. Examples include His, GST, TAP, FLAG, myc, HA, MBP, VSV-G, doxin, V5, avidin, streptavidin, BCCP, Calmodulin, Nus, S tags, lipoprotein D, and B galactosidase. Particular exemplary His tags include HHHHHH (SEQ ID NO: 32) and MSYYHHHHHH (SEQ ID NO: 33). In other ments, 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 n is short. This, in some ces, 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 or 2. -55_ B. genic compositions The present disclosure also provides pharmaceutical compositions containing immunogenic polypeptides or polynucleotides encoding these immunogenic polypeptides together with a pharmaceutical carrier. Antigens from S. pneumoniae were identified by screening immune cells from mice ed with S. pneumoniae, or from y human donors.
The human donors had presumably been exposed to S. pneumoniae at some point during their lifetimes, because S. pneumoniae is a very common disease and colonizing pathogen. Briefly, a library of S. pneumoniae antigens was expressed in bacteria and mixed with antigen presenting cells (APCs). The APCs, in turn, presented S. pneumoniae-derived polypeptides to lymphocytes that had been isolated from mice or from human donors. cyte responses were assayed for reactivity to S. pneumoniae. Human donors, as well as mice immunized with S. pneumoniae, produced lymphocytes specific to S. niae antigens. Thus, the present disclosure contemplates compositions of the S. niae antigens that elicit a strong immune response in immunized or infected mice or humans for counteracting infection by S. pneumoniae.
Tables 1 and 2 list the protein sequence and corresponding nucleotide sequence for S. pneumoniae antigens identified according to the screening methods described . The antigens were identified in screens of mouse and human T cells. In the screens of mouse T cells, the identified antigens were subjected to at least two rounds of screening: a genome-wide round to identify pools of 4 antigens that elicited an immune response, followed by a deconvolution round to individually test and identify single antigens that elicited an immune response from a pool fied in the -wide round. In contrast, in the screens of human T cells, two ent sets of antigen pools were created, such that a polypeptide was combined with different polypeptides between the first and second pools. Consequently, it is possible to determine which polypeptides are antigens by identifying which polypeptides are in ve pools in both the first and second sets. Table 1 lists antigens (and variants thereof) that were identified by one of the above screening methods, and were uently subjected to further g in the mouse models described in es 5-12. Thus, compositions ing to this disclosure may include one or two or more of the genes listed in Table l or 2, or the corresponding gene products.
An immunogenic composition may also comprise portions of said Streptococcus polypeptides, for example deletion mutants, truncation mutants, oligonucleotides, and peptide -56_ 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 ine the immunogenicity of the full-length protein. In some ments, 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 n (e.g., polypeptides of Tables 1 and 2). The peptide fragments may be, for e, linear, ar, or branched.
Some embodiments of the vaccine formulations and immunogenic compositions described herein include an immunogenic ptide (e.g., a polypeptide of Table l or 2) that contains a membrane translocating sequence (MTS), to facilitate introduction of the polypeptide into the ian 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 0L-synuclein, clein, or y-synuclein, the third helix of the Antennapedia homeodomain, SN50, integrin B3 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, 680 and 6,248,558.
In certain embodiments, an antigen (e. g., a ptide of Table l or 2) is covalently bound to another le. This may, for example, increase the half-life, solubility, bioabailability, or immunogenicity of the antigen. Molecules that may be covalently bound to the antigen include a carbohydrate, biotin, 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 , branched, or with comb or star geometries. In some embodiments, the naturally produced form of a protein is covalently bound to a moeity that stimulates the immune system. An example of such a moeity is a lipid . 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.
C. Antibodies specific to the proteins of Tables 1 and 2 Another aspect disclosed herein is an antibody preparation generated against an antigenic composition (e.g., one of the proteins listed in Table l or 2 or an immunogenic fragment -57_ thereof). For instance, this disclosure provides combinations of two, three, four, or five antibodies each izing a different protein of Table l or 2. Any of a variety of antibodies are included. Such antibodies include, e.g., polyclonal, monoclonal, recombinant, humanized or partially humanized, single chain, Fab, and fragments thereof, etc. The antibodies can be of any isotype, e.g., IgG, s IgG isotypes such as IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, etc.; and they can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like. In some embodiments, Fab molecules are expressed and assembled in a genetically transformed host like E. coli. A lambda vector system is available thus to express a population of Fab‘s with a ial diversity equal to or exceeding that of subject generating the predecessor dy. See Huse et al. (1989), Science 246, 1275-81.
D. Components of a vaccine or genic composition comprising S. pneumoniae antigens 0r antibodies izing the same In certain embodiments, the vaccine or immunogenic composition comprises an antigen and one or more of the following: an adjuvant, stabilizer, buffer, surfactant, controlled release component, salt, preservative, and/or an antibody specific to said n. 1. Adjuvants 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 al., Curr. HIV Res. 1309-20, 2003). In many vaccine formulations, the adjuvant provides a signal to the immune system so that it tes a se to the antigen, and the antigen is required for driving the specificity of the response to the pathogen. Vaccine ry 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 d from pathogens and can represent pathogen associated lar patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system.
Alternatively, adjuvants may be fied as organic and inorganic. Inorganic adjuvants e alum salts such as aluminum phosphate, ous aluminum hydroxyphosphate -58_ sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules ing macromolecules. An e 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 THl cells or TH2 cells. In other embodiments, the adjuvant induces the tion of B cells. In yet other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually ive; in some cases, an adjuvant activates more than one type of cell.
In certain embodiments, the adjuvant induces the activation of TH17 cells. It may e 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, mental 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 e more than 2-fold, or more than 10-fold more IL-17 than the cells of population (2). IL-17 may be ed, 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 5-8). One form of labile toxin is produced by Intercell. Mutant tes 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 s lacking ADP-ribosyltransferase activity. Particular detoxified mutant derivatives of labile toxin include LTK7 (Douce et al., “Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants” PNAS Vol. 92, pp. 648, February 1995) and LTK63 (Williams et al., e Imprinting by the Modified abile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease” The l 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 -59_ Sep;67(9):4400-6), and LTR72 (“Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of ichia coli heat-labile enterotoxin with partial knockout of ADP- ribosyltransferase activity.” J Exp Med. 1998 Apr 6;187(7): 1 123-32).
In some embodiments, the adjuvant comprises a VLP (virus-like particle). One such adjuvant platform, Alphavirus ons, induces the activation of THl7 cells using irus and is produced by Alphavax. In certain embodiments of the Alphavirus replicon system, alphavirus may be engineered to s an antigen of interest, a ne of interest (for e, IL-17 or a ne 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.
Certain classes of adjuvants activate toll-like receptors (TLRs) in order to activate a THl7 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 nt that activates TLRs comprises Monophosphoryl Lipid A (MPL). ionally, MPL has been produced as a detoxified lipopolysaccharide (LPS) endotoxin ed from gram ve 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 tic TLR agonists (in particular, TLR-4 agonists) are disclosed in Evans JT et al. cement of antigen-specific immunity via the TLR-4 ligands MPL adjuvant and 29.” Expert Rev es 2003 Apr;2(2):2l9-29.
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 -60_ dioctadecyldimethylammonium bromide (DDA). Another TLR-activating adjuvant is R848 (resiquimod).
In some ments, the nt 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 nt ty and With acceptable toxicity.
Typical ons 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 olipids such as phosphatidyl choline). In certain embodiments, an ISCOM is assembled together With a polypeptide or nucleic acid of interest. However, different n fractions may be used in different ratios. In addition, the different n 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 t, "substantially" refers to less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or even 1%. Such nts may comprise fraction A and fraction C mixed into a ratio of 70-95 A: 30-5 C, such as 70A:30Cto75A:5C,75A:5Ct080A:20C,80A:20Ct085A:15C, 85A:l5Cto 90A: 10C,90A: 10Cto95A:5C,or95A:5Cto99A:1C.
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.
Adjuvants may be covalently bound to antigens. In some embodiments, the adjuvant may comprise a protein Which induces inflammatory responses through activation of antigen- presenting cells (APCs). In some ments, 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(11), pp 954). In certain embodiments, a polypeptide described herein is presented in the context of the trivalent conjugate system, sing a fusion protein of S. niae Pneumococcal surface adhesin A (PsaA) With the pneumolysoid -61— 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 (W43 3F, D385N, and C428G) which render the molecule nontoxic but do not ere with its TLRmediated atory properties. Conjugation of a ccharide to the fusion of a polypeptide to the TLRagonist PdT results in greatly enhances immunological response to the polypeptide. In some embodiments, one or more ptides bed 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.
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 t (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 ofa vaccine or genic composition In addition to the antigens and the adjuvants described above, a vaccine formulation or immunogenic composition may include one or more additional components.
In certain embodiments, the e ation or genic 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 n, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L—histidine, alanine, valine, e, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
In certain ments, the vaccine formulation or immunogenic composition includes one or more s such as a mixture of sodium bicarbonate and ascorbic acid. In some _ 62 _ embodiments, the vaccine formulation may be administered in saline, such as phosphate buffered saline (PBS), or distilled water.
In certain embodiments, the vaccine ation or immunogenic ition includes one or more surfactants such as polysorbate 80 (Tween 80), Triton X-100, Polyethylene glycol tert-octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-( l l ,3,3-Tetramethylbutyl)phenyl- polyethylene glycol (TRITON X-lOO); Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN 20); and 4-(l,l,3,3-Tetramethylbutyl)phenol polymer With formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic or nonionic.
In certain embodiments, the vaccine ation or immunogenic composition includes one or more salts such as sodium de, ammonium de, calcium chloride, or potassium 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 e vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl l, and/or sorbic acid.
In certain ments, the vaccine ation or immunogenic composition is a controlled release formulation.
E. DNA vaccines In certain aspects, the vaccine comprises one or more of the nucleic acids disclosed herein or corresponding to the polypeptides described herein. When a nucleic acid vaccine is administered to a patient, the corresponding gene product (such as a desired antigen) is produced in the t’s body. In some ments, nucleic acid vaccine vectors that include optimized recombinant polynucleotides can be delivered to a mammal (including humans) to induce a therapeutic or prophylactic immune response. The nucleic acid may be, for example, DNA, RNA, or a synthetic nucleic acid. The nucleic acid may be single stranded or double stranded.
Nucleic acid vaccine vectors (e.g., adenoviruses, liposomes, papillomaviruses, retroviruses, etc.) can be administered directly to the mammal for uction of cells in vivo.
The c acid vaccines can be formulated as pharmaceutical compositions for administration in any suitable manner, including parenteral administration. Plasmid vectors are typically more -63_ efficient for gene transfer to muscle tissue. The potential to deliver DNA vectors to mucosal surfaces by oral administration has also been reported (PLGA ulated Rotavirus and Hepatitis B) and DNA plasmids have been ed for direct introduction of genes into other tissues. DNA es have been introduced into animals primarily by intramuscular injection, by gene gun delivery, or by electroporation. After being introduced, the plasmids are generally maintained episomally without replication. Expression of the encoded proteins has been shown to persist for extended time periods, ing stimulation of B and T cells.
In ining the effective amount of the vector to be administered in the treatment or prophylaxis of an infection or other condition, the ian evaluates vector toxicities, progression of the disease, and the tion of anti-vector antibodies, if any. Often, the dose equivalent of a naked nucleic acid from a vector is from about 1 u g to 1 mg for a typical 70 kilogram patient, and doses of vectors used to deliver the nucleic acid are ated to yield an equivalent amount of therapeutic nucleic acid. Administration can be accomplished via single or divided doses. The toxicity and eutic efficacy of the nucleic acid vaccine s can be determined using standard pharmaceutical procedures in cell cultures or experimental animals.
A nucleic acid e can contain DNA, RNA, a modified nucleic acid, or a ation thereof. In some embodiments, the vaccine comprises one or more cloning or expression vectors; for instance, the vaccine may comprise a plurality of expression vectors each capable of autonomous expression of a nucleotide coding region in a mammalian cell to produce at least one immunogenic polypeptide. An expression vector often includes a eukaryotic promoter sequence, such as the tide sequence of a strong eukaryotic promoter, operably linked to one or more coding regions. The compositions and methods herein may involve the use of any particular eukaryotic promoter, and a wide variety are known; such as a CMV or RSV promoter. The promoter can be heterologous with respect to the host cell. The promoter used may be a constitutive promoter.
A vector useful in the present compositions and methods can be circular or linear, single- stranded or double stranded and can be a plasmid, cosmid, or episome. In a le embodiment, each nucleotide coding region is on a separate vector; however, it is to be understood that one or more coding regions can be present on a single vector, and these coding regions can be under the control of a single or multiple promoters. -64— us plasmids may be used for the production of nucleic acid vaccines. Suitable embodiments of the nucleic acid vaccine employ ucts using the plasmids VR1012 (Vical Inc., San Diego Calif), pCMVI.UBF3/2 (S. Johnston, University of Texas) or pcDNA3.1 (InVitrogen Corporation, Carlsbad, Calif.) as the vector. In addition, the vector construct can contain stimulatory sequences (188), such as unmethylated deG motifs, that stimulate the animal‘s immune system. The nucleic acid vaccine can also encode a fusion product containing the immunogenic polypeptide. Plasmid DNA can also be delivered using attenuated bacteria as delivery system, a method that is suitable for DNA vaccines that are administered orally. Bacteria are transformed with an independently replicating plasmid, which becomes released into the host cell cytoplasm following the death of the attenuated bacterium in the host cell.
DNA vaccines, including the DNA encoding the desired antigen, can be uced into a host cell in any suitable form including, the fragment alone, a linearized d, a circular plasmid, a d e of ation, an episome, RNA, etc. Preferably, the gene is contained in a plasmid. In certain embodiments, the plasmid is an expression vector. Individual expression vectors capable of expressing the c material can be produced using standard recombinant techniques. See e.g., Maniatis et al., 1985 Molecular Cloning: A Laboratory Manual or DNA Cloning, Vol. I and II (D. N. Glover, ed., 1985) for general cloning methods.
Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterially, intraoccularly and oral as well as lly, transdermally, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, al, buccal and sublingual tissue. Typical routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. c constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, "microproj ectile bombardment gene guns", or other physical methods such as electroporation (”EP"), "hydrodynamic method", or ultrasound. DNA vaccines can be delivered by any method that can be used to deliver DNA as long as the DNA is expressed and the desired antigen is made in the cell.
In some ments, a DNA vaccine is delivered via known ection reagents such as cationic mes, fluorocarbon emulsion, ate, tubules, gold particles, biodegradable -65_ microspheres, or cationic polymers. Cochleate ry es are stable phospholipid m itants consisting of phosphatidyl serine, cholesterol, and calcium; this nontoxic and noninflammatory transfection reagent can be present in a digestive system. Biodegradable microspheres comprise polymers such as poly(lactide-co-glycolide), a polyester that can be used in ing apsules of DNA for transfection. Lipid-based microtubes often consist of a lipid of spirally wound two layers packed with their edges joined to each other. When a tubule is used, the nucleic acid can be arranged in the central hollow part thereof for delivery and controlled release into the body of an .
In some embodiments, DNA vaccine is delivered to mucosal surfaces via microspheres.
Bioadhesive microspheres can be prepared using different techniques and can be tailored to adhere to any mucosal tissue including those found in eye, nasal cavity, urinary tract, colon and gastrointestinal tract, offering the possibilities of localized as well as systemic controlled release of vaccines. Application of bioadhesive microspheres to ic mucosal tissues can also be used for localized vaccine action. In some embodiments, an alternative approach for mucosal vaccine delivery is the direct administration to l surfaces of a plasmid DNA expression vector which encodes the gene for a ic protein antigen.
The DNA plasmid vaccines according to the present invention are formulated according to the mode of administration to be used. In some embodiments where DNA d vaccines are injectable compositions, they are sterile, and/or pyrogen free and/or particulate free. In some embodiments, an isotonic formulation is preferably used. Generally, additives for icity can include sodium chloride, se, mannitol, ol and lactose. In some embodiments, isotonic solutions such as phosphate buffered saline are preferred. In some embodiments, stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. In some embodiments, a stabilizing agent that allows the formulation to be stable at room or ambient temperature for extended periods of time, such as LGS or other polycations or polyanions is added to the formulation.
In some embodiments, the DNA vaccine may further comprises a pharmacologically acceptable carrier or diluent. Suitable carriers for the vaccine are well known to those d in the art and include but are not limited to proteins, sugars, etc. Such carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. es of non-aqueous carriers are -66_ propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer‘s dextrose and sodium de, lactated Ringer‘s or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, olyte replenishers such as those based on Ringer‘s se, and the like. Preservatives and crobials include antioxidants, chelating agents, inert gases and the like. Preferred preservatives include formalin, thimerosal, neomycin, polymyxin B and ericin B.
An ative approach to delivering the nucleic acid to an animal involves the use of a viral or bacterial vector. Examples of suitable viral vectors include adenovirus, polio virus, pox s such as vaccinia, canary pox, and fowl pox, herpes viruses, including catfish herpes virus, adenovirus-associated vector, and retroviruses. ary bacterial vectors include attenuated forms of Salmonella, Shigella, Edwardsiella ri, Yersinia ruckerii, and Listeria togenes. In some embodiments, the nucleic acid is a vector, such as a plasmid, that is capable of gous expression of the nucleotide sequence encoding the immunogenic polypeptide.
F. Use of Vaccines The S. pneumoniae vaccines described herein may be used for prophylactic and/or therapeutic treatment of S. pneumoniae. Accordingly, this application es 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 or sequelae, such as sepsis. 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 In prophylactic embodiments, the vaccine is administered to a t to induce an immune se that can help protect against the establishment of S. pneumoniae, for example by ting against colonization, the first and necessary step in disease. Thus, in some aspects, the method inhibits infection by S. niae in a non-colonized or uninfected subject. In -67— another aspect, the method may reduce the duration of colonization in an individual who is already colonized.
In some embodiments, the vaccine itions of the ion 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 ty of symptoms or sequelae is at least 25%, 40%, 50%, 60%, 70%, 80% or even 90%. In ular embodiments, vaccinated individuals may display no symptoms or ae upon contact with S. niae, do not become colonized by S. pneumoniae, or both. Protective immunity is lly achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal ty is primarily the result of secretory IgA (sIGA) antibodies on mucosal surfaces of the atory, gastrointestinal, and genitourinary tracts. The sIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, that result in sIGA production by B lymphocytes on mucosa-lined tissues of the body. Humoral immunity is lly 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 THl or THl7 cells.
Essentially any individual has a certain risk of becoming infected with S. pneumoniae.
However, certain sub-populations have an increased risk of infection. In some embodiments, a vaccine formulation as described herein (e. g., a composition comprising one or more polypeptides from Table l or 2, or nucleic acids encoding the polypeptides, or antibodies reactive with the polypeptides) is administered to patients that are immunocompromised.
An compromising ion arising from a medical treatment is likely to expose the individual in question to a higher risk of infection with S. pneumoniae. It is possible to treat an infection prophylactically in an individual having the immunocompromised condition before or during treatments known to compromise immune on. By lactically treating with an nic composition (e.g., two or more antigens from Table l or 2, or nucleic acids encoding the antigens), or with dies reactive to two or more antigens from Table l or 2, before or during a treatment known to compromise immune function, it is possible to prevent a -68_ 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 S. niae infection e. g., following a treatment g 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 ts 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 ated with immunosuppression.
In another embodiment, at least one dose of the pneumococcal antigen composition is given to adults in the following groups at increased risk of pneumococcal disease or its complications: all s 65 years of age; adults with asplenia, splenic dysfunction or sickle-cell e; adults with the following conditions: c cardiorespiratory disease, cirrhosis, alcoholism, chronic renal disease, nephrotic syndrome, diabetes mellitus, chronic cerebrospinal fluid leak, HIV infection, AIDS and other ions associated with immunosuppression (Hodgkin‘s disease, lymphoma, multiple myeloma, immunosuppression for organ transplantation), individuals with cochlear implants; individuals with erm health ms such as heart disease and lung disease, as well as individuals who are taking any drug or ent that lowers the body‘s resistance to infection, such as long-term ds, certain cancer drugs, radiation therapy; Alaskan natives and certain Native American populations. 2. Therapeutic use In therapeutic applications, the vaccine may be administered to a patient suffering from S. 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 ae bin an infected dual. In some embodiments, treating the patient refers to reducing the duration of symptoms or ae, or reducing the intensity of symptoms or sequelae. In some ments, 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%. -69_ In eutic embodiments, the vaccine is administered to an individual post-infection.
The vaccine may be administered shortly after infection, e.g. before ms or sequelae manifest, or may be administered during or after manifestation of ms or sequelae.
A therapeutic S. pneumoniae vaccine 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 tis, otitis media (ear infection), itis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brain abscess.
Sepsis is a rare but life-threatening complication of S. niae 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(1):2-8. 3. Assaying vaccination efficacy The efficacy of vaccination with the 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-l7A) by stimulating T cells derived from the subject after vaccination.
The IL-l7 levels may be compared to IL-17 levels in the same subject before vaccination.
Increased IL-l7 (e.g., IL-l7A) , such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or ld or more increase, would indicate an increased se 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, d, d or lOO-fold or more increase, would indicate an increased response to the vaccine. In addition, one may measure THl7 cell activation, where increased THl7 cell activation, such as a 1.5 fold, , 5-fold, 10-fold, 20-fold, 50-fold or lOO-fold or more increase, ates with an increased se to the vaccine. One may also measure levels of an antibody ic 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 lOO-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 e IL-l7 levels and the levels of vaccine-specific antibody. Alternatively, one may follow epidemiological s such as incidence of, severity -70_ of, or duration of pneumococcal infection in vaccinated individuals ed 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 the test vaccine 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 zation or the reduction in lethality in vaccinated animals. es 5-8 and 10 show the efficacy of polypeptides of Table l in inhibiting S. pneumoniae nasal zation following intranasal challenge in the mouse model. Examples 11 and 12 show the efficacy of polypeptides of Table l in protecting against sepsis and death following infection with S. pneumoniae via tion in the mouse model.
G. Use of Immunogenic Compositions 1. Defense against S. pneumoniae infection The immunogenic compositions of the present disclosure are designed to elicit an immune response t S. pneumoniae. Compositions described herein (e.g., ones comprising one or more ptides of Table l or 2, or nucleic acids encoding the polypeptides) 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 THl-biased CD4+ T cell response, a iased CD4+ T cell response and/or a CD8+ T cell response. In some embodiments, the composition stimulates an antibody response. In some embodiments, the composition stimulates a THl-biased CD4+ T cell response, THl7-biased CD4+ T cell response and/or a CD8+ T cell response, and an antibody response.
In certain ments, the composition (e.g., one comprising one or more polypeptides of Table l or 2, or nucleic acids ng the polypeptides, or antibodies ve with the peptides) es a cytokine or nucleotide coding region ng a cytokine such as IL-l7, to e additional stimulation to the immune system of the mammal. In certain embodiments, the composition comprises a cytokine such as IL-l7. -71_ While not Wishing to be bound by theory, in some embodiments a THl7 cell response is desirable in mounting an immune response to the compositions sed herein, e.g., ones comprising one or more polypeptides of Table l or 2. In certain embodiments, an active THl7 response is beneficial in clearing a coccal infection. For instance, mice lacking the IL- l7A receptor show decreased Whole cell e-based protection from a pneumococcal challenge (Lu et al., “Interleukin-17A mediates acquired immunity to pneumococcal colonization.” PLoS Pathog. 2008 Sep l9;4(9)).
Thus, herein is provided a method of increasing IL-17 production by stering the compositions described herein (e.g., ones comprising one or more polypeptides of Table l or 2) to a subject. Furthermore, this application provides a method of activating THl7 cells by administering said compositions to a subject. In certain ments, increased IL-l7A 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 coccal killing is independent of antibodies and complement.
However, specific antibody production and complement activation may be useful additional mechanisms that contribute to ng of a coccal infection.
Immunogenic compositions containing immunogenic polypeptides or cleotides encoding immunogenic polypeptides together With a pharmaceutical carrier are also provided.
In some instances, the genic composition comprises one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13, 265, 266 and 267, such as one or more nucleic acids selected from SEQ ID Nos. 24-31, 271, 272 and 273. In some embodiments these nucleic acids are expressed in the immunized individual, producing the encoded S. pneumoniae antigens, and the S. pneumoniae ns so produced can produce an immunostimulatory effect in the immunized individual.
Such a nucleic acid-containing stimulatory composition may comprise, for e, 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: 1-13, 265, 266 and 267. Such a composition may also comprise a bacterial plasmid vector into Which is inserted a promoter (sometimes a strong viral er), one or more nucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13, 265, 266 and 267, and a enylation/transcriptional termination sequence. In some instances, the nucleic acid is DNA. -72_ H. Diagnostic uses This application provides, inter alia, a rapid, inexpensive, sensitive, and specific method for detection of S. pneumoniae in patients. In this respect it should be useful to all hospitals and physicians examining and treating patients with or at risk for S. pneumoniae ion. Detection kits can be simple enough to be set up in any local hospital laboratory, and the antibodies and antigen-binding portions thereof can readily be made available to all hospitals treating patients with or at risk for S. niae infection. As used herein, “patient” refers to an individual (such as a human) that either has an S. pneumoniae infection or has the potential to contract an S. pneumoniae infection. A patient may be an individual (such as a human) that has an S. pneumoniae ion, has the potential to contract an S. pneumoniae infection, who has recovered from S. pneumoniae infection, and/or an individual whose infection status is unknown.
In some embodiments, one may perform a diagnostic assay using two or more antibodies, each of which binds one of the antigens of Table l or 2 to detect S. pneumoniae in an individual.
In some embodiment, one of the antigens is SEQ ID NO: 265, 266, or 268. The instant disclosure also provides a method of phenotyping biological samples from patients suspected of having a S. pneumoniae ion: (a) obtaining a biological sample from a patient; (b) ting the sample with two or more S. pneumoniae fic antibodies or antigen-binding portions thereof under conditions that allow for binding of the antibody or n-binding portion to an e of S. pneumoniae; where binding indicates the presence of S. pneumoniae in the sample. In some embodiments, the binding to the biological sample is compared to binding of the same antibody to a negative l tissue, wherein if the biological sample shows the presence of S. pneumoniae as compared to the negative control tissue, the patient is identified as likely having a S. pneumoniae infection. In some cases, binding of one antibody indicates the presence of S. niae; in other cases, the g of two or more antibodies indicates the presence of S. pneumoniae. The aforementioned test may be appropriately adjusted to detect other bacterial infections, for ce by using an antibody reactive a homolog (from another bacterial species) of one of the proteins bed in Table 1. In some embodiments, the antibodies raised against a S. pneumoniae protein in Table l or 2 will also bind the homolog in another Streptococcus species, especially if the homologs have a high percentage sequence identity. _ 73 _ Alternatively, one may use an antigen of Table l or 2 (such as SEQ ID NO: 265, 266, or 268) to detect anti-S. pneumoniae dies in an individual. The instant disclosure also provides a method of phenotyping biological samples from patients suspected of having a S. pneumoniae infection: (a) obtaining a biological sample from a patient; (b) ting the sample with two or more S. pneumoniae -specific antigens selected from Table l or 2 or portions thereof under ions that allow for binding of the n (or portion f) to any host antibodies present in the sample; where binding indicates the presence of . pneumoniae antibodies in the sample. In some embodiments, the binding to the biological sample is compared to g of the same antigen to a negative control tissue, wherein if the biological sample shows the presence of anti-S. pneumoniae dies as compared to the negative control tissue, the patient is fied as likely either (1) having a S. niae infection, or (2) having had a S. pneumoniae infection in the past. In some cases, detecting one antibody indicates a current or past infection with S. pneumoniae; in other cases, detecting two or more dies indicates a current or past infection with S. pneumoniae. The aforementioned test may be appropriately adjusted to detect other bacterial infections, for instance by using a homolog (from r bacterial species (e.g., a Streptococcal species) of the proteins described in Table 1.
In some embodiments, the immune cell response of a mammalian cell may be quantified ex vivo. A method for such quantification comprises administering the compositions herein disclosed to a mammalian T cell ex vivo, and quantifying the change in ne tion of the mammalian T cell in response to the composition. In these methods, the cytokine may be, for example, IL-l7.
The binding of an S. pneumoniae antibody to an antigen (e. g., a polypeptide of Table l or 2, such as SEQ ID NO: 265, 266, or 268) may be measured using any appropriate method. Such methods include ELISA (enzyme-linked immunosorbent assay), Western blotting, competition assay, and spot-blot. The detection step may be, for ce, chemiluminescent, fluorescent, or colorimetric. One suitable method for measuring antibody-protein binding is the Luminex xMAP system, where peptides are bound to a dye-containing microsphere. Certain systems, including the xMAP system, are amenable to measuring several different markers in multiplex, and could be used to measure levels of antibodies at once. In some embodiments, other systems are used to assay a plurality of markers in multiplex. For example, profiling may be performed using any of the following systems: antigen microarrays, bead microarrays, nanobarcodes -74_ particle technology, d proteins from cDNA expression libraries, protein in situ array, protein arrays of living transformants, universal n array, lab-on-a-chip microfluidics, and peptides on pins. Another type of clinical assay is a chemiluminescent assay to detect antibody binding. In some such assays, ing the VITROS Eci anti-HCV assay, antibodies are bound to a solid-phase support made up of microparticles in liquid suspension, and a surface fluorometer is used to quantify the enzymatic generation of a fluorescent product.
In some embodiments, if the biological sample shows the presence of S. pneumoniae (e. g., by detecting one or more polypeptide of Table l or 2, such as SEQ ID NO: 265, 266, or 268, or an antibody that binds one of said polypeptides), one may administer a therapeutically effective amount of the compositions and therapies described herein to the patient. The biological sample may comprise, for e, blood, semen, urine, vaginal fluid, mucus, saliva, feces, urine, cerebrospinal fluid, or a tissue sample. In some embodiments, the biological sample is an organ intended for lantation. In certain embodiments, before the detection step, the ical sample is subject to culture conditions that promote the growth of S. pneumoniae.
The diagnostic tests herein (e.g., those that detect a polypeptide of Table l or 2, such as SEQ ID NO: 265, 266, or 268, or an antibody that binds one of said polypeptides) may be used to detect S. pneumoniae in a variety of samples, ing samples taken from patients and s obtained from other sources. For example, the diagnostic tests may be used to detect S. pneumoniae in food, drink, or ients for food and drink; on objects such as medical instruments, medical devices such as cochlear implants and pacemakers, shoes, ng, ure including hospital furniture, and drapes including hospital drapes; or in samples taken from the environment such as plant samples. In some embodiments, the tests herein may be performed on samples taken from s such as agricultural animals (cows, pigs, ns, goats, horses and the like), companion animals (dogs, cats, birds, and the like), or wild animals.
In certain embodiments, the tests herein may be performed on samples taken from cell cultures such as cultures of human cells that produce a eutic protein, cultures of bacteria intended to produce a useful biological molecule, or cultures of cells grown for research purposes.
This disclosure also provides a method of determining the location of a S. pneumoniae ion in a patient comprising: (a) administering a pharmaceutical composition comprising a labeled S. pneumoniae antibody or antigen-binding portion thereof to the patient, and (b) detecting the label, wherein binding indicates a S. pneumoniae infection in a particular location -75_ in the patient. Such a diagnostic may also comprise comparing the levels of binding in the patient to a control. In certain embodiments, the method further comprises, if the patient has a S. pneumoniae infection, treating the infection by administering a therapeutically ive amount of a S. pneumoniae -binding antibody or antigen-binding portion thereof to the patient. In certain ments, the method further comprises, if the patient has a S. niae infection, treating the infection by administering a therapeutically effective amount of a S. pneumoniae protein of Table l or 2, or genic portion thereof, to the patient. The method may further comprise determining the location and/or volume of the S. pneumoniae in the patient. This method may be used to evaluate the spread of S. pneumoniae in the patient and ine whether a localized therapy is appropriate.
In some embodiments, the anti-S. pneumoniae dies or T cells described herein may be used to make a sis of the course of infection. In some embodiments, the anti-S. pneumoniae antibodies or T cells herein may be detected in a sample taken from a patient. If antibodies or T cells are t at normal levels, it would te that the patient has raised an immune response against anti-S. pneumoniae. If antibodies or T cells are absent, or present at reduced , it would indicate that the patient is failing to raise a ient response against anti-S. pneumoniae, and a more aggressive ent would be recommended. In some embodiments, antibodies or T cells present at reduced levels refers to antibodies that are present at less than 50%, 20%, 10%, 5%, 2%, or 1% the level of antibodies or T cells typical in a patient with a normal immune system. Antibodies may be detected by ty for any of the antigens described herein (e.g., those in Table 1 and/or 2), for example using ELISA. T cells may be detected by ex vivo responses for any of the antigens described herein (e.g., those in Table 1 and/or 2), for example using ELISA or ELISPOT assays.
In some embodiments, detection of specific S. pneumoniae antigens (e. g., those in Table 1 and/or 2, such as SEQ ID NO: 265, 266, or 268) may be used to predict the progress and symptoms of S. niae infection in a patient. It will be understood by one of skill in the art that the methods herein are not limited to detection of S. pneumoniae. Other embodiments include the detection of related bacteria including bacteria with proteins homologous to the proteins described in Table l or 2. Such related bacteria include, for e, other strains of Streptococcus. —76— I. Doses and Routes of Administration 1. forms, s, and timing The amount of antigen in each vaccine or immunogenic composition dose is ed as an effective amount, which induces a lactic or eutic response, as described above, in either a single dose or over multiple doses. ably, the dose is without significant e 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 pg of each protein, in some instances 2-100 pg, for instance 4-40 pg. In some aspects, the e formulation comprises 1-1000 pg of the polypeptide and 1-250 pg of the nt. In some embodiments, the appropriate amount of antigen to be delivered will depend on the age, weight, and health (e.g. immunocompromised status) of a subject. When present, typically an adjuvant will be present in amounts from 1 pg — 250 pg per dose, for example 50-150 pg, 75-125 pg or 100 pg.
In some embodiments, only one dose of the vaccine is administered to achieve the s described above. In other ments, 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 , 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. 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 ments, a dosage form is provided as a dose of lyophilized composition and at least one separate sterile container of diluent.
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.
In eutic applications, compositions are administered to a patient suffering from a disease in an amount sufficient to treat the patient. Therapeutic applications of a composition -77_ described herein e ng transmissibility, slowing disease progression, reducing bacterial viability or replication, or ting 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.
In prophylactic embodiments, compositions are administered to a human or other mammal to induce an immune response that can t the establishment of an infectious e or other condition. In some embodiments, a composition may partially block the bacterium from ishing an infection.
In some embodiments, the compositions are administered in combination with antibiotics.
This co-administration is ularly appropriate when the pharmaceutical composition is administered to a patient who has recently been exposed (or is suspected of having been recently exposed) to S. pneumoniae. Many otics are used to treat pneumococcal infections, including penicillin, amoxicillin, amoxicillin/clavulanate, cefuroxime, cefotaxime, ceftriaxone, and ycin. The riate 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 otic choices” Am J Med. 1999 May 3;106(5A):19S—25S). 2. Routes ofadministration The e formulations and pharmaceutical compositions herein can be delivered by administration to an individual, typically by systemic administration (e. g., intravenous, intraperitoneal, intramuscular, ermal, subcutaneous, subdermal, transdermal, intracranial, intranasal, mucosal, anal, vaginal, oral, buccal route or they can be inhaled) or they can be administered by topical application. In some embodiments, the route of administration is intramuscular. In other embodiments, the route of stration is subcutaneous. In yet other embodiments, the route of administration is mucosal. In certain embodiments, the route of administration is transdermal or intradermal 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 S. pneumoniae vaccines comprising toxins (e.g. a toxin or labile toxin); in other embodiments, the administration is intranasal.
Vaccines formulated with Alphavirus replicons may be administered, for example, by the —78— uscular or the subcutaneous route. Vaccines sing 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 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 ition is suitable for administration to a man 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 ments, 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, ins, and/or inactive protein nts. 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 als for vaccines, such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in the vaccine may be e, 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)/l<g of product for an intrathecal able composition; 5 EU/kg of product for a non-intrathecal inj ectable composition, and 0.25-0.5 EU/mL for sterile water.
In certain embodiments, the preparation comprises less than 50%, 20%, 10%, or 5% (by dry weight) contaminating protein. In certain embodiments, the desired molecule is present in the ntial e of other biological olecules, such as other proteins cularly 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 t reconstituted mixture). In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of -79_ biological macromolecules of the same type present (but water, buffers, and other small les, 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 ns were expressed, ve 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 ments, 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 ) 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).
It is preferred that the vaccine or genic composition has low or no toxicity, within a reasonable risk-benefit ratio. In certain embodiments, the vaccine or immunogenic composition ses ingredients at concentrations that are less than LD50 measurements for the animal being ated. LD50 measurements may be obtained in mice or other mental model systems, and extrapolated to humans and other animals. Methods for estimating the LD50 of compounds in humans and other animals are nown 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/l<g, 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 num toxin.
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 e, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non- aqueous, isotonic sterile ion solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended -80_ recipient, and aqueous and non-aqueous sterile suspensions that can e suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed ners, such as ampoules and vials.
Injection ons and suspensions can be prepared from sterile powders, granules, and s of the kind previously described. In the case of adoptive transfer of therapeutic T cells, the cells can be administered intravenously or parenterally.
Formulations suitable for oral stration can consist of (a) liquid solutions, such as an effective amount of the polypeptides or packaged nucleic acids suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a ermined 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, ol, 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 rs. e 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 n 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 ulated, e.g., in liposomes, or in a formulation that es for slow release of the active ingredient.
The ns, 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. l formulations can be placed into pressurized acceptable propellants, such as rodifluoromethane, propane, nitrogen, and the like. Aerosol formulations can be delivered orally or nasally.
Suitable formulations for vaginal or rectal administration e, for example, itories, 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 -81— combination of the polypeptides or packaged nucleic acids With a base, including, for example, liquid triglycerides, polyethylene glycols, and in hydrocarbons.
J. ation and Storage of Vaccine Formulations and Immunogenic Compositions The S. pneumoniae vaccines and immunogenic compositions described herein may be produced using a variety of techniques. For example, a polypeptide 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 s 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 enylation ce. In some embodiments, the promoter is inducible or repressible. Other regulatory sequences may provide for secretion or ion of the ptide 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 hromosomal plasmid, or may be ated into the host . 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.
Once a recombinant cell line has been produced, a polypeptide may be isolated from it.
The isolation may be accomplished, for example, by affinity purification ques or by physical separation techniques (e. g., a size column).
In a further aspect of the present disclosure, there is provided a method of manufacture comprising mixing one or more ptides or an immunogenic fragment or variant thereof With a carrier and/or an adjuvant.
In some embodiments, antigens for inclusion the vaccine formulations and immunogenic compositions may be produced in cell culture. One method ses providing one or more expression vectors and cloning nucleotides encoding one or more polypeptides selected from polypeptides having an amino acid sequence of Table l or 2, such as SEQ ID NO: 265, 266, or 268, then expressing and isolating the polypeptides. -82— The immunogenic polypeptides described herein, and nucleic acid compositions that express the ptides, can be packaged in packs, dispenser devices, and kits for administering nucleic acid compositions to a mammal. For e, packs or dispenser devices that contain one or more unit dosage forms are provided. Typically, instructions for administration of the compounds will be provided with the packaging, along with a suitable tion on the label that the compound is suitable for ent of an indicated condition, such as those disclosed herein.
V. Examples e 1. Antigen identification and pooled murine screens Each open reading frame predicted in the S. pneumoniae TIGR4 genome was cloned into an expression vector comprising a tag that is able to be presented by the major histocompatibility complex (MHC). Each construct was then expressed in E. coli, and full-length expression validated by a surrogate assay that fies the tag in the t of MHC. The screen is described in more detail in International Application . In order to facilitate screening the large library, the library was pooled such that four induced library clones were present in each well. In order to screen T cells from mice immunized against S. pneumoniae, an aliquot of the pooled library was added to peritoneal-derived macrophages. The macrophages were allowed to bind the tagged S. pneumoniae antigens via the MHC. After 2 hr at 37°C, the macrophages were washed with PBS. The macrophages were then fixed with 1% paraformaldehyde for 15 min and washed extensively with PBS. 105 T cells were added to each well in 200 uL of RP-lO media. The T cells had previously been isolated from mice that had been zed 2 times with killed S. pneumoniae bacteria with cholera toxin adjuvant. The assay plates were incubated for 72 hrs at 37°C. The amount of IL-17 in the supernatant of each well was determined through the use of an IL-17 ELISA assay. The threshold for a positive result was set at two standard deviations above the mean of all samples.
Example 2. Deconvolution 0f the positive murine pools A secondary screen was used to determine which antigen(s) out of the four clones in each well induced the positive response observed in the pooled screen described in Example 1. All the clones in each positive pool were pulsed individually onto neal macrophages in duplicate wells. T cells isolated from zed mice from the same genetic background as the initial screen were used to screen the pulsed hages using the IL-17 assay described in -83_ Example 1. Individual antigens that induced an e se in the duplicate wells greater than two standard deviations above the mean of negative control samples were considered positive responses. The library plasmids present in these positive clones were sequenced to confirm the identity of the antigen. The antigens SP1574, SP1655, SP2106, SP0148, SP1473, SP0605, SP1177, SP0335, , SP1828, SP2157, SP1229, SP1128, , SP1865, SP0904, SP0882, SP0765, SP1634, SP0418, SP1923, SP1313, SP0775, SP0314, SP0912, SP0159, SP0910, SP2148, SP1412, SP0372, SP1304, SP2002, SP0612, SP1988, , SP0847, SP1527, SP0542, SP0441, SP0350, SP0014, SP1965, SP0117, and SP2108 were confirmed using this method.
Example 3. Antigen identification and pooled human s CD4+ T cells and CD14+ monocytes were isolated from peripheral blood acquired from human donors. The monocytes were differentiated into tic cells by culturing them in GM- CSF and IL-4 containing media, ially as described in Tedder TF and Jansen PJ (1997 “Isolation and generation of human dendritic cells.” t Protocols in logy Supp 23: 7.32.1-7.32.16). After five days in culture, the dendritic cells were seeded into 384 well .
The CD4+ T cells were non-specifically expanded in culture to ensure sufficient quantities.
Each open reading frame ted in the S. pneumoniae TIGR4 genome was cloned into an expression vector comprising a tag that is able to be presented by the major ompatibility complex (MHC). Each construct was then expressed in E. coli, and full-length expression validated by a surrogate assay that fies the tag in the context of MHC. In order to facilitate screening the large library, the library was pooled such that four induced library clones were present in each well. In order to screen the human T cells, an aliquot of the pooled library was added to the seeded dendritic cells in 384-well plates. After 2 hr at 37°C, the tic cells were fixed with 1% paraformaldehyde for 15 min and washed extensively with phosphate buffer and lysine buffer. 40,000 of the CD4+ T cells in 70 uL of RP-10 media were added to each well of a 384-well plate. The assay plates were incubated for 3 days at 37°C. The amount of IL-17 in the supernatant of each well was determined through the use of an IL-17 ELISA assay. In different iterations of the screen, the threshold for a positive result was set at two standard deviations above the mean of all samples, two standard deviations above the mean of negative controls, or 1.78 times the median absolution deviation of the data set. Positive pools were then deconvoluted as described in Example 4. .84_ Example 4. Deconvolution of the ve human pools For all antigens, deconvolution was performed by comparing the results of two pool screens. In this method, two different sets of pools were prepared, so that a polypeptide was with three different polypeptides between the first and second pools. Consequently, it is possible to determine which polypeptides are antigens by identifying which polypeptides are in positive pools in both the first and second sets. In this deconvolution method, a pool was identified as positive if it was at least 1.78 times the median absolution deviation of the data set.
An antigen was identified as a positive hit if it was positive in at least two ed secondary screens. The antigens SP2108, SP0641, SP1393, SP0024, .1, SP1072, SP1384 and SP2032 were identified using the above approach.
Example 5 , SP0148 and SP1634 polypeptides The SP2108 polypeptide (SEQ ID NO: 9), SP0148 polypeptide (SEQ ID NO: 7) and SP1634 polypeptide (see Table 2) were formulated as vaccine compositions using 4 pg of the polypeptide in ation with 1 pg a toxin adjuvant (CT). For combinations, 4 pg of each polypeptide was used. The compositions were administered intranasally to 6 mice three times, one week apart. The subjects 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 ptides 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-l7 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 SP0882 and SP0314 polypeptides -85_ This example used the same protocols as Example 5, except that only two doses of the vaccine composition were administered. In these experiments, the SP0882 polypeptide (SEQ ID NO: 2) and SP0314 polypeptides (see Table 2) were tested in parallel with two of the three polypeptides tested in Example 5. Results of the IL-l7 immunogenicity assay are shown in FIGS. 4 and 5. Results of the colonization assay are shown in , SP0641N, and SP0024 polypeptides This example used a protocol similar to that of Example 5, except that two doses of the vaccine compositions were administered, one week apart. Vaccine compositions comprised the polypeptides SP1072 (SEQ ID NO: 8), SP0641N (SEQ ID NO: 13) or SP0024 (SEQ ID NO: 1), and cholera toxin adjuvant (CT). Four weeks after the last immunization, the mice were challenged intranasally with live type 6B S. pneumoniae. One week later the bacterial burden was ed in each mouse by plating a nasal lavage on selective media and counting resultant CFU. The number of CFU isolated from each mouse is plotted for each immunized cohort. The results of this colonization assay are shown in Statistically icant results are ted in the figure (* = e < 0.05).
Example 8 SP0148, SP0314, SP0882, and SP2108 polypeptides tested in the BALB/c mouse To determine whether similar immune responses were seen across different mouse genotypes, vaccine compositions were stered to BALB/c mice. Vaccine compositions sed the polypeptides SP0148 (SEQ ID NO: 2), SP0314 (see Table 2), SP0882 (SEQ ID NO: 2) or SP2108 (SEQ ID NO: 9), and cholera toxin adjuvant (CT). Using a protocol similar to that of Example 5, the mice were immunized, challenged intranasally with S. pneumoniae, and the number of CFU was ed. The results of this colonization experiment are shown in Example 9 SP1912, SP2108 and SP0148 ptides: IL-17A immunogenicity assay The polypeptides SPl9l2 (SEQ ID NO: 265), SP2108 (SEQ ID NO: 9) or SP0148 (SEQ ID NO: 7) were formulated as vaccine compositions with cholera toxin adjuvant (CT). The vaccine compositions were administered to mice two times, one week apart. The positive l 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 SPl9l2). Three weeks after -86_ the last immunization, peripheral blood was collected from the retroorbital sinus and evaluated in a whole blood assay. Briefly, the heparizined whole blood was d 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-l7A levels measured by ELISA. Results of the IL-l7A immunogenicity assay are shown in Each symbol in the graph represents responses from individual mice, and the line indicates the median response of the group.
Example 10 SP1912, SP2108 and SP0148 ptides: colonization assay Animals were immunized with vaccine formulations comprising the polypeptides SP1912 (SEQ ID NO: 265), SP2108 (SEQ ID NO: 9) or SP0148 (SEQ ID NO: 7) and cholera toxin adjuvant (CT) as described in Example 9, and then challenged intranasally with 107 live type 6B S. pneumoniae four weeks after the last immunization (and one week after retroorbital blood collection). Seven days after challenge, s were euthanized and the nasopharyngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Results are shown in 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. ("‘*"< = e <0.05).
Example 11 SP1912 polypeptide: aspiration challenge (sepsis assay) Polypeptide SP1912 was evaluated for its ability to t mice from sepsis. Groups of ten mice were subcutaneously immunized three times, two weeks apart with e compositions comprising either the SP1912 polypeptide (SEQ ID NO: 265) or pneumolysoid (PdT) adsorbed to alum. The positive control was killed, unencapsulated whole cell S. pneumoniae + alum (WCB), and the negative l was alum alone. Three weeks after the final immunization, blood was collected for evaluation of IL-l7A response and antibody levels, and then one week later, the mice underwent tion nge with 107 live strain 0603 (type 6B) S. pneumoniae.
Animals were monitored for survival for eight days. Results of the aspiration challenge are shown in as survival curves for each immunized group.
Example 12 Pneumolysoid PdT, SP0148 and SP0641N polypeptides: tion challenge (sepsis assay) .87_ Polypeptide SP0148 was evaluated for its ability to protect mice from sepsis when zed singly or in combination with SP0641N and/or pneumolysoid (PdT). Groups of ten mice were subcutaneously zed three times, two weeks apart with vaccine compositions comprising polypeptide SP0148 (SEQ ID NO: 7), singly or in combination with polypeptide SP0641N (SEQ ID NO: 13) and/or PdT, adsorbed to alum. The ve 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 ted 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 12 as survival curves for each immunized group. e 13 SP1912, SP2108 and SP0148 polypeptides: colonization assay onal studies were performed essentially as described in Example 10, for a total of four separate studies. Briefly, animals were immunized with vaccine formulations sing the ptides SP1912 (SEQ ID NO: 265), SP2108 (SEQ ID NO: 9), SP0148 (SEQ ID NO: 7), or onally SP2108 plus , and cholera toxin adjuvant (CT) as described in Example 9.
Control animals were immunized with killed, unencapsulated whole cell S. pneumoniae plus CT (WCB), or CT alone. Immunized animals were challenged intranasally with 107 live type 6B S. pneumoniae four weeks after the last zation. Seven days after challenge, animals were euthanized and the nasopharyngeal cavities lavaged and ed on permissive media to evaluate the S. pneumoniae titers. Pooled results of four studies are shown in 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 14 SP1912 and SP0148 polypeptides: IL-17A immunogenicity assay Groups of ten mice were subcutaneously immunized twice, two weeks apart with vaccine compositions comprising either SP1912 polypeptide (SEQ ID NO: 265), SP0148 polypeptide (SEQ ID NO: 7), 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 -88_ and evaluated for IL-l7A 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 ted and IL-l7A levels measured by ELISA. Results of the IL-l7A immunogenicity assay are shown in . Each symbol in the graph represents responses from individual mice, and the line indicates the median response of the group. e 15 SP1912 and SP0148 polypeptides: colonization assay Animals were aneously immunized three times, two weeks apart with vaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 7) at different doses plus and minus SP1912 (SEQ ID NO: 265), 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 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 are shown in 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 zation out of the number of animals in the group is indicated at the top of the figure.
Example 16 SP1912, SP0148, and SP2108 polypeptides: colonization assay In two separate studies, s were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 7) and SP0148 plus SP1912 (SEQ ID NO: 265), or onally with SP2108 (SEQ ID NO: 9), 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 asally with 107 live type 6B S. pneumoniae four weeks after the last immunization. Seven days after challenge, s were euthanized and the aryngeal cavities lavaged and cultured on permissive media to evaluate the S. pneumoniae titers. Pooled results of the two s are shown in as the colony forming units of bacteria (CFU) per . Each symbol represents a titer from an dual mouse response, and the horizontal line represents the median of the group. The number of animals protected from colonization out -89_ of the number of animals in the group and corresponding percentage of animals protected from colonization are indicated at the top of the . 05, >“*p<0.01, >“""“p<0.001 Dunn’s le Comparison Test compared to Alum control) e 17 Pneumolysoid L460D, PspA derivative PR+NPB, SP1912, SP0148, and SP2108 polypeptides: colonization assay Animals were subcutaneously immunized three times, two weeks apart with vaccine formulations comprising the polypeptides SP0148 (SEQ ID NO: 7), SP2108 (SEQ ID NO: 9), SP0148 plus SP2108, and SP0148 plus SP2108 in combination with SP1912 (SEQ ID NO: 265) or known S. pneumoniae antigens L460D plus PR+NPD, adsorbed to alum. Two separate studies were conducted. Control animals were immunized with alum alone. zed 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. niae . Results of the second study are shown in 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 ted from colonization in the four studies described in Examples 16 and 17. # not % not colonized/ colonized total —---——- ——————— —---——- —--——-- -90_ —----3/10 —---——- 0148 + 2108 + L46OD + 10 0 40V PR+N PB ------ e 18 PspA, SP0148 and SP2108 passive antibody transfer and aspiration challenge (sepsis assay) Groups of ten mice were injected with monoclonal antibodies specific for PspA, heat-inactivated rabbit sera ic 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 (WCB). One day after injection, the mice underwent tion challenge with 106 live S. pneumoniae type WU-2 . Animals were monitored for survival for eight days. The data are shown in as survival curves for each immunized group. shows the percent of animals protected from sepsis in the studies described in Examples 12 and 18, as well as two additional studies.
SEQUENCES SEQ ID NO: 1 SP0024 >gi|l497l488|gb|AAK74215.l| conserved hypothetical protein Streptococcus paeu oniae TIGR4 FMQANQAYVALHGQ.N.PLKPKTRVAIVTCMDSRLHVAQALGJALGDAHILRNAGGRVTEDMIRSLVISQQ Q GTRVIVV.HHiDCGAQitnNnPthYLKnnLGVDVSDQDtLPtQDInnSVRnDMQLLI?SPLIPDDVIISGAIYN V DTGS TVVEL SEQ ID NO: 2 SP0882 >gi|l4972356|gb|AAK75009.l| conserved hypothetical oro:eia (Streptococcus pneumoniae TIGR4) YLKMK?HK.KVPYTGK?RRVRIL.PKDY?KDTDRSYPVVYFHDGQ VF SKESFIGHSWKIIPAIKRNPDI SRMIVVAIDNDGMGRMNLYAAW<tQLSPIPGQQbGGKGVnYAnbVMnVVKPtID; YR KADCQHTAMIGSSLGGNI TQFIGLEYQDQIGCJGVFSSANWL{QLAt RYbLCQKLSPDQRItIYVGinnADD D< KQAYIDSSLCYY HDLIAGGVHLDNLVJKVQSGAI{SnIPWSnNLPDCLRFFA?KW -91_ SEQ ID NO: 3 M QSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDI S RMIVVAIDNDGMGRMNEYAAW<bQESPIPGQQtGGKGVnYAntVMnVVKPtI SEQ ID NO: 4 SP0882 with exogenous signal sequence MSSKF KSAAVLG"ATLASL..VACMNQSYFYLKMKEHK.KVPYTGKERRVRIL.PKDYEKDTDRSYPVVYFHDGQA A VF SKESFIGHSWKIIPAIKR PDISRMIVVAIDNDGMGRMNEYAAW<tQESPIPGQQbGGKGVnYAntVMnVVKPt IDL‘ YR "AMIGSSEGGNITQFIGLEYQDQIGCEGVFSSANWL{QEAt RYtECQKLSPDQRItIYVGl..A 4 ADD D< LMDGNIKQAYIDSSECYYHDLIAGGVHLDNLVEKVQSGAI{SnIPWSnNLPDCLRFFAEKWA SEQ ID NO: 5 SP0882N with ous signal sequence MSSKF KSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQN VFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAW<bQESPIPGQQtGGKGVnYAntVMnVVKPt SEQ ID NO: 6 SP0148 lac<ing signal sequence MCSGGAKKnGnAASKKnIIVAlNGSPKPtIY A.nNGnLlGYnInVVRAIt(DSDKYDVKtnKanSGVtAGLDADRYN MAV N.SYlKnRAnKY.YAAPIAQNPNVLVV<<DDSSIKSEDDIGGKSlEVVQAllSAKQL A HlDNPlILNY TKADFQQI VRLSDGQFDYKIFD<IGVETVI< QG.DNLKVIELPSDQQPYVYPLLAQGQD _‘J .KSFVDKRIKELYKD GanK-SKthGDlYLPAnADIK.A SEQ ID NO: 7 SPOl48 including signal sequence (277 amino acids witq N—:ermiqal E) M<KIV<YSSLAALALVAAGVLAACSGGAKKnGnAASKKnIIVAlNGSPKPtIY A.nNGnLlGYnInVVRAIt<DSDKY DVKt A .<anSGVtAGLDADRYNMAV nRAnKY.YAAPIAQNPNVLVV<(DDSSIKSEDDIGGKSTEVVQAT lSAKQ.nAYNAnHlDNPlILNYlKADbQQI VRLSDGQFDYKIFD<IGVETVI< QG-DNLKVIELPSDQQPYVYPL LAQGQDE.KSFVDKRIKnLYKDGanK.SKQttGDlYLPAnADIK.A SEQ I) O: 8 SPlO72 >gi|l4972547|gb|AAK75185.l| DNA primase Streptococcus pneumoniae TIGR4 -92_ Z<U A10< H H W ANIVEVIGDVISLQKAGRNY.G.CPtHGn<lPSbNVV.DKQtYiCtGCGRSGDVbKtI.L‘ ,_<: 10 G)< RVGInVnKP.YSnQKSASPHQA.YDMHEDAA<FY{AIEMlllMG.nARNY.YQRG.A DnV-(HbWI .APPE Y-YQR-SDQYR A .nDL.DSGLFYLSDA QFVDTF { RI FPE" DQGKVIAFSGRIWQKTDSQTS<Y<NS H.YiMDRAKRSSG<ASnIYLMnGtMDVIAAY aCUG) H L‘J 3:CW (/3 GlA-SRnHVnHL<R. K<LV.VYDG <AGQAATL<A .DEIGDMPVQIVSMPDNLDPDEY.QKNGPED.AY..l< RISPIEbYIHQYKPnNSnN.QAQIEFL E<IAPLIVQE<SIAAQNSYIHILADSEAStDYlQInQIVNn S RQVQRQ R EGISRPTPIT PVTKQESAIMRAEAH ..YRMMESP-V . DYRLRnDbAbAantQVLYD..GQYGN.PPnV.AnQ nnVnRAWYQVLAQD.PAnISPQnLSnV E TRNKALL Q D RIKK<VQEASHVGDlDlA.nn.nRLISQ<RRME SEQ ID NO: 9 SP2108 including signal sequence >gi|l497362 O|gb|AAK76l67.l| mal:ose/mal:odextrin ABC transporter, maltose/maltodextri1—binding n (S:reotococcus pneu oniae TIGR4) MSSKFMKSAAVLGTATLASELLVACGSKTAD(PADSGSSnVKnLlVYVDnGYKSYI A .nVAKAYnKnAGVKVlEKlGD ALGGLDKLSEDNQSG VPDVMMAPYDRVGSLGSDGQ-SEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIES.VMYY NKDLVKDAP<TFADLENLA<DSKYAtAGEDG<A lAbEADWleYYlYGLEAGNGAYVFGQNGKDA<DIGLANDGSIV GINYAKSWYE<WPKGA QD EGAG LIQTQFQEGKTAAIIDGPWKAQAFKDA(VNYGVATIPTLPNGKEYAAFGGGKA WVIPQAVKN-nASQKtVDt.VAanQKV.YD< NnIPA lnARSYAnGK DnLllAVIKQtKNlQPLPNISQ SAVW DPAKNMLFDAVSGQKDAK"AANDAVILI<ElIKQ<bG L‘ SEQ ID NO: 10 SP2108 lacking signal sequence AD<PADSGSSnVKnLlVYVDnGYKSYI .nVAKAYnKnAGVKVl.KlGDALGGLDKLS.DNQSG VPDVMMAA PYDRVGSLGSDGQ-SEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIES.VMYYNKDLVKDAP<TFADLENLA<DSK DG< lAbEADWthYYlYGLEAGNGAYVFGQNGKDA<DIGLANDGSIVGINYAKSWYE(WPKGA QD EGAG LIQTQFQEGKTAAIIDGPWKAQAFKDA<VNYGVATIPTLPNGKEYAAFGGGKAWVIPQAVKN.EASQKFVDFEVAT EQQKV-YD< N nIPA lnARSYAnGK VIKQtKNlQPLPNISQ SAVWDPAKNMLFDAVSGQKDAK"AAND AVlLI<ElIKQ<tGE SEQ ID NO: 11 MSGTS ATPIVAASTVLIRPK.KnM.nRPVL(NLKGDDKIDETSL"KIALQNTARP MDATSWKEKSQYFASPRQQG AGEI VANALRNEVVATFKNTDS<GEVNSYGSISL<EIKGD<KYb IKEHNlS RPE tKVSASAITTDS-TDR-K.
DnlY<DnKSPDG<QIVPnIHPnKVKGANITFEHDTFTIGANSSFDLNAVINVG L‘JAK KNKanStIanSVnnMnA.
NS G<<INFQPS-SMP- GFAGNWNiEPILD<WAW S(TEGGYDDDGKP<IPG"LNKGIGGEHGID<F PAGV IQ R<D<NTTSLDQNP..tAbA NnGI APSSSGSKIANIYPEDSNGNPQDAQLnRG. RSA««G.ISIVNT NKnG.A QRDLKVISR _‘J {FIRGIL S<SNDAKGIKSS<LKVWGD.KWDG.IYNPRGR A.nNAPnSKD QDPATKIRGQF -93__ «PIAnGQYtYKt(YRLIKDYPWQVSYIPV(IDNTAPKIVSVDFSNP EKIKLITKDTYHKVKDQYK LiLtARDQKdHA IANnVWYAGAALVNnDG nKNLnViYAG nGQGRNRKLDK EIKGAGDLRGKIIEVIALDGSSNFT (IHRI(FANQADEKGMISYYJVDPDQDSS (YQ SEQ ID NO: SPO64l >gi|l49721_.7lgblAAK7479l.l| seri 1e protease, sub :ilase fa ily [St otococcus pneumoniae TIGR4] MKKSTVJSLT'"AAVIJAAYAPN LVVLADISSSnDALNIS DKnKVAnNK. IHSA LISQDEK A (iAVIKnKnV IDNN .nNSN (SQGDY DStVNKNi nNPKKnDKVVYIA ntK «SG nKAIK.LSSL T<V.Y TYDRIF GSAI TTP IKQInGISSV A.RAQKVQPM NiAR<nIGV .nAIDY-KSINAPFGK DGRG VIS I DTG"DYRHKAM RbK .KG D< YWLSD (IPHAF YY HG) (HNVDVVSVSSGFTGTG-VGGKITVEKYDDGRDYF {GM IAGIJA GND LQDIKNb DGIAP AQIFSYKMYS DAGSGbAGDA i bHAIADA .(YWQAI RALR<AGIPMVVATG YATSASSSSWD-VA Ni-KMTDTG VTRTAAH UAIAVASAKNQTV A IGGESFKYR NIGAFFD<SKITTN L‘J DGT<APSKJKFVYIG <GQDQD-IG D-RGKIAV RIYTK JKNAFK<AMDKGARAIMVVN" VNYY RD WT?LPA A nGi <8QVtSISGDDGV<LW MINPD<KT <(RNNK.DtKDKLnQYYPID 38F SN< <nIDthAP ..YK nDIIVPAGSTSWGPRI ..LKPDVSAPGKNIKSTLNVINGKSTYGY SGTS A" PIVAASTVLI RPK .RPVL<NLKG DDKID-TS-"(IA-QNTARP DATSW EKSQYFASPRQQGAGJI VA ALRNEVVATF (NT SYGSISL EIKGD<KYb I <4{NIS RP. tKVSASAITT .iY<Dn< SPDG<QIVPnIHP A.(VKGA ITF EHDTFTIGA SSFDL EAK KbV fiSbIHb A FQPS-SMP. ?PILD<WAW S<TJGGY D DDGKP<IPG' GIHGIDA TTSLDQNPn nGI APSSSGSKIANIYPJDS G PQDAQLnRG PSP-VLRSA.. G) 10 N D-KVISRHu ,_L‘ “21 H (SNDAKGIKSS <LKVWGD-(WDG .IYNPRGR..A nSKD A .PIAnGQY R4T<D (IDNTAP (IVSVDFS PEKI<LIT<DTY DQYK DQK. .(bDnIA YAG nGQGR RKJDKDG TIYL‘J IKGAG (IIH .DGSSNFT {RI<FA (YQK nIA nS<tK -G G <?GS-<KDIIGV. .nSI «KSStiI DR IST I niGKR nnYDY<YDD (GNIIAYD 3G1 DnI (SKIYGVJSPS<DG i (SI IKAi<YDbiSK MitDLYANI DIV JAFAG JFVKDN (AEIKI a P «LG .GDLS< KPD L K LSGKIYS _*.i (QQY RKGYA .KVTTY PG <T) .? GVYS< DIAKIQ<ANPN n iIYADSRNV EDGRSTQSV DGF IIRYQVFTF DKGEAI D D .tGKDD nYiG. VnAIKnDGSMLtIDiKPV PSKS (IYVR L*.i FYLRGK I D < nSVVDNY-IYG "RDF I<LNV <DGDIMDWGM<DY<A TD JQTGYS D4 A<AVGViYQFJYDNVKP L‘JVNIDP <GNTSIEYADG<SVVFNINDKR NGtDGnIQnQHIYI DI<QI I D<TJNI<IVV<DbARNiiV<ntIL nVSnL<PiRViViIQNGK LMSSIIVS.nDtI.PVY .(GYQbDG GK<DAGYVINLSK <IT WW H .(KnnnNKPibDVSKK (DNPQV HSQLN. A .nHSQKS DSTKDVTATVL DKNNISSKST' (LPKTGTASGAQTLLAAGIMFIVGIFLG. (NQ SEQ ID NO: -94_ SPO641N MVVLADTSSS?DALNISDKnKVAnNKn(in IHSA LISQDtKLKKIAVIKnKnVVSKNPVIDNNISNnnAKIKnnN SN<SQGDY DSbVNKNi«NPKKnDKVVYIAntK3KnSGnKAIKnLSSL< T<V.YTYDRIFNGSAIETTPDNLD<IK QInGISSVnRAQKVQPM NiAR<nIGVnnAIDY.KSINAPFGK FDGRG VIS IDTG"DYRHKAMRIDDDAKAS R tK<nD-KG DKNYWLSD<IPHAF YY GGKITVEKYDDGRDYFDPHGMiIAGI.AGND nQDIKNt GIDGIAP AQ IFSYKMYSDAGSGtAGDLI tHAILDSI(HNVDVVSVSSGFTGTGLVGE(YWQAIRALR(AGIPMVVATGNYATSAS VA DTGNVTRTAAHEDAIAVASAKNQTVEFD(VNIGGESFKYRNIGAFFD(SKITTNEDGTKAPS KJKFVYIG<GQDQDLIG.D.RGKIAV DRIYTKDLKNAFK(AMDKGARAIMVVNTVNYY RD WT?LPA GYnADnG T<SQVFSISGDDGVKLW KTEV<RNNKnDtKDKLnQYYPIDMESFNSNKPNVGD«KnIDtKbAPDiDKn.Y KEDIIVPAGSTSWGPRIDLJLKPDVSAPGKNIKSTLNVINGKSTYG SEQ ID NO: 14 SP0882 consensus M QSYFYLKMKEHKLKVPYTGK?RRVRILLPKDY?KDTDRSYPVVYFHDGQNVFNSKESF I Y IGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMN LYAAWKtQ LSPIPGQQbGGKGV A.YAn Y H m m bVM ID;iYRiKADCQHTAMIGSSLGGNITQFIGLIL‘ ._<: 10 DQIGCLGVFSSANWLHQ LAbNRYbLCQKLSPDQRIbIYVGi A .nADDiDKILMDGNIKQAYIDSSLCYYHDLIAGGVH H R LDNLVLKVQSGAIHS nIPWS nNLPDCLRFFA71 KW S3Q ID NO: 15 SP0882N consensus M QSYFYLKMKEHKLKVPYTGK?RRVRILLPKDY QKDTDRSYPVVYFHDGQNVFNSKE U) “21 I Y IGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMN LYAAWKtQ LSPIPGQQbGGKGV A .YAn Y H m m FVM EVVKPFI -95__ SEQ ID NO: 16 SP0882 consensus with exogenous signal sequence MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDY_‘J T T V I KDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNL‘J Y Y H YAAWKFQESPIPGQQtGGKGV A.YAnbVMnVVKPtIDElYRlKADCQHTAMIGSSLGGNIT L‘J L‘J QFIGLL‘J ,_<:10 DQIGCLGVFSSANWLHQEAtNRYtECQKLSPDQRIbIYVGl A DKlLM EK I H DGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSnIPWSnNLPDCLRFFAEKWA SEQ ID NO: 17 SP0882N consensus with exogenous signal sequence MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDY_‘J T T V I KDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNL‘J Y Y H YAAWKFQESPIPGQQtGGKGV A.YAnbVMnVVKPbI L‘J L‘J SEQ ID NO: 18 SPOl48 consensus lacking signal sequence KK A.GnAASKKnIIVAlNGSPKPtIY A.nNGnLlGYnInVVRAItKDSDKYDVKFL‘J Q SRN N X KTEWSGVFAGLDADRYNMAVNNLSYlK.é «KYLYAAPIAQNPNVLVVKK DDSSIKSLDD I m -96__ IGGKSlLVVQAllSAKQLnAYNAnHlDNPlILNYlKADLQQIMVRLSDGQFDYKIFDKIG VETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKnLYKDGlL«KLSKQ Y S tbGDlYLPAnADIK(.A V SEQ ID NO: 19 SPOl48 consensus including signal sequence M<KIVKYSSLAALALVAAGVLAACSGGAKK A.GnAASKKnIIVAlNGSPKPtIY A.nNGnLl G L Q S R N GYnInVVRAItKDSDKYDVKt A .KlnWSGVtAGLDADRYNMAVNNLSYIK LYAAPA N X I IAQNPNVLVVKKDDSSIKSLDDIGGKSlLVVQAllSAKQLnAYNAnHlDNPlILNYIKAD L*.i RLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKA F Y S SFVDKRIKnLYKDGanKLSKthGDlYLPAnADIK(.A V SEQ ID NO: 20 SP2108 consensus lacking signal sequence MCGSKTADKPADSGSSnVKnLlVYVDnGYKSYI A .nVAKAYnK«AGVKVILKIGDALGGLD A I KLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGA I X T PAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAbAGLDGKIlAbLADWleYYlYGL -97_ LAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQL‘J G P A X H GKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVIPQAVKNLEASQK FVDFLVAT?QQKVLYDKiNnIPANinARSYAnGKNDnLiiAVIKQbKNiQPLPNISQMSA S A S VWDPAKNMLFDAVSGQKDAKTAANDAViLIKLiIKQKtG L‘ SEQ ID NO: 21 SP2108 consensus including signal sequence MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSnVKnLiVYVDnGYKSYI A .nVA T T V A KAYnKnAGVKViLKiGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLS I I X DGAKTDDTTKSLVTAANGKVYGAPAVI3SLVMYYNKDLVKDAPKTFADLENLAKDSKYAF AGE FLADWTNFYYTYGLLAGNGAYVFGQNGKDAKD I GLANDGS IVGINYAKSWY A G P A X EKWPKGMQDTEGAGNLIQTQFQEGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKL‘J ,_<: AAFGGGKAWVIPQAVKNLLASQKbVDtLVAinQQKVLYDKiNnIPANinARSYAnGKND.A A S A LTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQK FGIL‘J -98__ SEQ ID NO: 22 SPI634 >gi|l4973lZ4|gb|AA (757l4.l| etical protei 1 SP_1634 Streptococcus pneumoniae TIGR4 YJKDVAYDSYY D .PLN «T. DILI .InIiYLSbDNLVSI .PQ R.LD .APQVPR DPTMLTSKNR .QL .D?LAQH KJSHFINDIDPn-—u .QKQFAA TY RVSLD"YLIVFRGTD DSIIGWK nDbHLiY KnIPAQKHA-RY-K FFAH HPKQKVIJAG {SKGGNLAIYAASQI?QS .Q QI"AVYTFDAPG-HQH_‘ .TQTAGYQRI DRSKIFIPQGSIIG ML31 PAHQIIVQSTALGGIAQ {DTFSWQIE 3K {FVQLDKTNSDSQQVDiit(LWVAIVPD naLQLYbDthGiI .DAGISS I DLASL(ALEYIHHLFVQAQSLIPn nR<J LL IDIRYQAW( R SEQ ID 23 SPO3I4 >gi|l497l788|gb|AAK7449l.l| hyanur01idase Streptococcus oqeumoniae TIGR4 QTKTKKLIVSLSSLV .SGFL .NHYM IGAnni iiNiIQQSQKEVQYQQ RDI (N-VnNGDbGQIL DGSSPWT GSKAQGWSAWVDQKNSA DAS RVI LA<DGAI ISS{n( .RAALHRMVPIEAKK (YKJ RF(I(TDNKIGIA(V RIInn SGKDKRLWNSAIISGIK DWQ ILA DYSP iLDVDKI( .n.bY niGiGiVSbKDIn .V< .VA DQ- S «DSQID (Q .nnKI D LPIGK(HVFS4ADYTYKVF_‘4 P DVASV (NGIL nPT. (nGi iNVIVS(DG( QVKKIP .KI .ASV(DAYTDRL 33W GIIA GNQYY DS(N'QQMA (LNQn .nG (VA DSDS SISSQADRIY .Wn(tS YKISANLIAI Y ((Lnn A (QVINPSSRYYQDE TVVRTVRDS EWM {KHVY SE (SI_‘4 VG WWDYEIGTP RAIN i-S-MKanSDn nIK (YIDVIL(bVPDPL{bR(i D NPFKA .GGN .VDMGRVKVIAG.. 3Q RSI nQVt (-VDQG?GFYQ DGSYI DHT VAYTGAYG V.I4 DGLS QLJPVIQ(" ( PI DKDKMQT Y LV .MD SRGRSISRA SEGHVAAVEVL RGIHRIAD SnGn QC .QS .V(" IVQS DSYYDVF ( (D ISLMQS DAGVASVPRPSYLSAt KMD (iA Y ALKGbiG-S- RI .NY* .H (n KRGWYTS 3G FY. G DLSiYSDGYWPTVNPY(MPGTTET DAKRA DSDTG(V4PSAFVGTS A ATATMDFT WNQTLTAH (SWF JKDKIAFJGS IQ TSTDTAATTIDQRK .?SGNPY (VYVNDKnASLI flQfl Pn QSVt-nStDS (KNIGYFFFK (SSIS S(ALQ (GAWKDIN nGQSDKnVnN*ILIISQAH (QN RDSYGYM.JIP RA"FNQMI(n-nSSLI nNN niLQSVY DA (QGVWGIVKYDDSVSTISNQFQVL( RGVYII RKnGDnY(IAYY Pn SAPDQ th( .nQAAQPQVQNSK<.K* .(Sn nnKNHSDQKNLPQTG?GQSILAS .GFLLLGAFYLF RRGKNN S3Q ID NO: 24 SP0882N DNA GAA CAA CC AC iiiA C AAAAA ACACAAAC CAAGGiiCC A ACAGG AAGGAGCGCCGTGT ACG Cl C CC AAAGAI A GAGAAAGATACAGACCGI CC A CCiG G AIACI CAIGACGGGCAAA AA AGCAAAGAG C CA GGACAI CA GGAAGA A CCCAGCTATCAAACGAAATCCGGATA'"C GA GiCGiiGC A GAIGG Al GGGGCGGA GAAIGAG A GCGGC GGAAGIICCAAGA AiCCCAGGGCAGCAG GG GGiAAGGGiG GGAGiA GC GAGII G CAiGGAGGiGGiCAAGCC'"T -99__ SEQ ID NO: 25 SP0882 with exogenous signal sequence (nucleotides) A G CA ClAAAl A GAAGAGCGC GCGG AAC GC ACAC lGClAGCllGC ll GGlAGCl GCA GAA CAA CC AC 1 A C AAAAA GAAAGAACACAAAC CAAGGllCC A ACAGG AAGGAGCGCCG"GTAC C C CC AAAGAl A GAGAAAGA"ACAGACCGl CC A CC G G AlACl CA GACGGGCAAAA" AA AGCAAAGAG C CA GGACAl CA GGAAGA A CCCAGCTATCAAACGAAATCCGGATA"CAG GA GlCG l GC A GACAAlGA GG AlGGGGCGGA GAAlGAG A GCGGC GGAAGllCCAAGAA" CCCAGGGCAGCAG GG GGlAAGGGlG GGAG A GC GAG G CA GGAGGlGGlCAAGCC l GAGACC A CG ACAAAAGCAGAC'"GCCAGCAlACGGC A GA GG CC CAC CAAlA AC CCAG A CGG l GGAAlACCAAGACCAAAl GG GC GGGCGl CAlC "GGCTCCACCAAG AAGCC lAACCGC A l lCGAGlGCCAGAAAC A CGCC GACCAGCGCA C ClA GlAGGAACAGAAGAA GCAGA GA ACAGACAAGACCl GA GGAlGGCAA A CAAACAAGCC A A CGlCGC l GC A lGAll GA AGCAGGGGGAGlACAlClGGAlAA C GlGClAAAAG CAG GCCAlCCA AG C CTTGG"CAGAAAATCTACCAGA lG ClGAGAl l GCAGAAAAA GG AA SEQ ID NO: 26 SP0882N with exogenous signal ce (nucleotides) A G CA ClAAAl A GAAGAGCGC GCGG GCllGGAAC GC ACAC lGClAGCllGC ll l GCA GAA CAA CC AC l A C AAAAA GAAAGAACACAAAC CAAGGllCC A ACAGG AAGGAGCGCCG'"GTAC C C CC AAAGAl A GAGAAAGA"ACAGACCGl CC A CC G G AlACl CA GACGGGCAAAA' AA AGCAAAGAG C CA GGACAl CA GGAAGA A CCCAGCTATCAAACGAAATCCGGATA'"CAG lCGCA GA GlCGllGC A GACAAlGAlGG AlGGGGCGGA GAAlGAG A GCGGC GGAAGllCCAAGAA' CC AlCCCAGGGCAGCAG GG GGlAAGGGlG GGAGl A GC GAGll G CAlGGAGGlGGlCAAGCC ll A'"C SEQ ID NO: 27 SPOl48 lacking signal ce (nucleotide S) A"GTGCTCAGGGGGTGCTAAGAAAGAAGGAGAAGCAGC'"AGCAAGAAAGAAA CA CG lGCAACCAA'"GGATCACC AAAGCCA lAlC A GAAGAAAATGGCGAATTGACTGGTTACGAGA GAAG CGl CGCGClA C l C"GACAAA AlGA G CAAGl lGAAAAGACAGAA GG CAGGlG C GC GG C GACGC GA CGllACAA A GGC G CAACAA C llAGC ACAC AAAGAACG"GCGGAGAAA ACC C A GCCGCACCAA GCCCAAAA"CC AAlG CC lGlCG GAAGAAAGAlGAC ClAG A CAAG ClClCGA GA A CGG GGAAAA'"CGACGGAAG'"CG CAAGCCACTACA"CAGCTAAGCAG AGAAGCA ACAA GClGAACACACGGACAACCCAAC AlCCllAAC Al AC"AAGGCAGAC CCAACAAA CAl GG ACG lGAGCGA"GGACAA llGAC AlAAGAlll lGA AAAA CGG G lGAAACAGTGA"CAAGAACCAAGG lGGACAACl GAAAG AlCGAACl CCAAGCGACCAACAACCG'"ACG l ACCCACllC GC CAGGG CAAGA GAG GAAA CG l G AGACAAACGCATCAAAGAACTT'"ATAAAGAT GGAACTCTTGAAAAAT'"GTCTAAACAA CllCGGAGACAC A ClACCGGCAGAAGCTGATATTAAAGAGTAA —100— SEQ ID NO: 28 SPOl48 including signal sequence (nucleotides) ATGAAAAAAAlCG lAAAlAC CAlClC GCAGCCC lGC Cl G GClGCAGG G GC lGCGGC llGClCAGG GGGTGCTAAGAAAGAAGGAGAAGCAGC'"AGCAAGAAAGAAA CA CG lGCAACCAA GGA CACCAAAGCCA lA lC A GAAGAAAA'"GGCGAAl GAClGG ACGAGA GAAG CGl CGCGClA C AAAGAl C GACAAA"AT GA G CAAGl lGAAAAGACAGAA GG CAGGlG C GC GG C GACGC GA AA A GGC G CAA CAA CllAGC ACAC AAAGAACG"GCGGAGAAA ACC C A GCCGCACCAA GCCCAAAA CC AAlG CC lG AGAAAGAlGAC ClAG A CAAG ClClCGA GA A CGG GGAAAA"CGACGGAAG CG CAAGCCACT ACA"CAGCTAAGCAG AGAAGCA ACAA GClGAACACACGGACAACCCAAC AlCCllAAC AlAC AAGGCAGA C CCAACAAA CAlGG ACG lGAGCGA"GGACAA llGAC AlAAGAlll lGA AAAA CGG G lGAAACAG TGA"CAAGAACCAAGG lGGACAACl GAAAG AlCGAACl CCAAGCGACCAACAACCG ACG l ACCCACTT C GC CAGGG CAAGA GAG GAAA CG lG AGACAAACGCATCAAAGAACTT"ATAAAGATGGAACTCTTGA GTCTAAACAA Cl lCGGAGACAC A ClACCGGCAGAAGCTGATATTAAAGAGTAA SEQ ID NO: 29 SP2108 lacking signal sequence (nucleotides) A"GTGCGGAAGCAAAAClGC GAlAAGCC GC GAl ClGGl CAlC GAAG CAAAGAAC CAC G A A GlAGA CGAGGGA"ATAAGAGClAlA GAAGAGG GClAAAGCl A GAAAAAGAAGC GGAGlAAAAG CAC C AAAA C GGlGA GClClAGGAGGlC GAlAAAC l ClC lGACAACCAA C GG AA GlCCC GAlGl A GA GGCl CCATACGACCGlG AGG AGCC lGGl C GACGGACAAC CAGAAG GAAA GAGCGA GG GC AGA CGACACAAClAAA ClC G AACAGC GC AAlGG AAAG ACGG GC CC GCCGl A CGAG CAC GllA G AClACAACAAAGAC GGlGAAAGATGCTCCAAAAACA GClGACl GGAAAACC"TGCTAAAGATAGCAAA ACGCA lCGClGGlGAAGA GG AAAAC AC GCC lCClAGClGAC GGACAAACllC AC AlACAlA GGAC C GCCGGlAACGGlGC ACG Cl lGGCCAAAACGGTAAAGACGC'"AAAGACA CGG Cl GCAAACGACGGT" ClA CG AGGlAlCAAClACGClAAA C l GG ACGAAAAA GGCC AAAGGlAlGCAAGATACAGAAGGTGC"GGA AAC lAA CCAAAClCAA CCAAGAAGG"AAAACAGClGC A CA CGACGGACC GGAAAGCTCAAGCC lAA AGA GC AAAGlAAAC ACGGAG lA CCCAACTC'"TCCAAATGGAAAAGAAlA GC GCA CGG GGlG GlAAAGC lGGGlCA CC CAAGCCGTTAAGAACCl GAAGC C CAAAAA llG AGAC CC GllGCAACl GAACAACAAAAAGlA AlAlGA AAGAC AACGAAA"CCCAGC GAGGClCGl CA ACGC GAAGGlAA AAACGATGAG'"TGACAACAGClG AlCAAACAG'"TCAAGAACAC'"CAACCAC"GCCAAACA ClC CAAAlGlClG CAGl lGGGA CCAGCGAAAAAlA GCl C llGA GC GlAAGlGGlCAAAAAGATGCTAAAACAGCTGCTAACGAT GClG AACAl GAlCAAAGAAACAATCAAACAAAAAl lGGlGAA AA SEQ ID NO: 3O M (nucleotides) A"GTCAGGAAC AG AC CCAA CG GGCAGCllC AClG lGAl AAAT"AAAGGAAATGCT TGAAAGACClG A GAAAAATC'"TAAGGGAGATGACAAAA AGAlC ACAAG CllACAAAAATTGCCCTACAAA A"ACTGCGCGACC GAlGCAAC lC lGGAAAGAAAAAAG CAAlAC lGCAlCACC AGACAACAGGGA —101— GCAGGCC AA 1AA G GGCCAAiGC '"GAAGTTGTAGCAACTT'"CAAAAACAC GAiiC AAAGGi GG AAAC CA Al GG CCA C C AAAGAAA"AAAAGG'"GATAAAAAA AC ACAA CAAGC CACAA CA"CAAACAGACC GACi AAAG CAGCA CAGCGA AAC ACAGA C C AACi GACAGA AAAAC GA"GAAACATATAAAGATGAAAAA C CCAGAiGG AAGCAAA iG CCAGAAA GAAAAAGTCAAAGG AGCAAAiAiCACA GAGCA GA AC CAC A AAAi C AGC iGA GAAiGCGG AiAAA G TTGGAGAGGCCAAAAACAAAAAiAAA GiAGAA CAi iAi CA GAG CAG GGAAGAAA"GGAAGCTC"A AACTCCAACGGGAAGAAAA AAAC CCAACCi C i G CGA GCC C AA GGGA GC iGGGAA GGAACCA AA CCiiGAiAAA GGGC GGGAAGAAGGG CAAGA CAAAAACAC"GGGAGG AiGA GA GAiGG A AACCGAAAAT'"CCAGGAACCi AAA AAGGGAA GG GGAGAACA GG A AGA AAA GCAGGAG AA"AGAAAAGATAAAAA"ACAACA CCC GGA CAAAAiCCAGAA Al GC CAAiAACGAAGGGA" CAACGCiCCA CAiCAAGiGG i GC AACA iAiCCi iAGA CAAA GGAAAiCC CAAGA GC AACTTGAAAGAGGATTAACACC C CCAC iG A iAAGAAG'"GCAGAAGAAGGA GA iCAA AGiAAA AATAAAGAGGGAGAAAATCAAAGAGACi AAAAG CAi CGAGAGAACAC A AGAGGAAii iAAA AAGCAATGATGCAAAGGGAAiCAAA CA C AAAC AAAAG i GGGGiGAC GAAG GGGA GGACiCA C ATCCTAGAGGTAGAGAAGAAAATGCACCAGAAAG AAGGA AA CAAGA CC GC AC AAGA AAGAGG CAA i GAACCGAT"GCGGAAGGiCAAiAi CiAiAAAi AAA A AGA iAAC AAAGAi ACCCA GGCAGG iCC A A iCCiG AAAAA iGAiAACACCGCCCCiAAGA G CGGi GAi CAAA CCiGAAAAAAiiAAG GA "TACAAAGGA AC i AiCA AAGG AAAAGA CAG AiAAGAAiGAAACGC A GCGAGAGATCAAAAAGAACA" CC"GAAAAA iGACGAGA'"TGCGAACGAAG i GG A GCiGGCGCCGC C G iAAiGAAGAiGGAGAGG GA AAAAAA C GAAG AAC ACGCAGGTGAGGG'"CAAGGAAGAAA'"AGAAAAC GA AAAGACGGAAAiACCA i AiGAAA AAAGG GCGGGAGAT"TAAGGGGAAAAA CAiiGAAG CAi GCA AGAiGG iC AGCAAi iCACA AAGA iCA AGAA AAA iGC AAiCAGGCiGAiGAAAAGGGGAiGA iiCC Al AiC AG AGAiCC GA CA AGAi CA CiAAA Al CAA SEQ ID NO: 31 SPO641N (nucleotides) AiGGiAGiC AGCAGACACA CiAGC C GAAGAiGCiiiAAACA C CiGAiAAAGAAAAAGTAGCAGAAAATAA AGAGAAACA GAAAAiAiCCA AGiGC A GGAAACT'"CACAGGAi i AAAGAGAAGAAAACAGCAGTCA"TAAGG AAAAAGAAG G iAG AAAAAiCCi G GA AGACAA'"AACACTAGCAATGAAGAAGCAAAAA"CAAAGAAGAAAA" CCAA AAA CCCAAGGAGAT"ATACGGAC CA iG GAAiAAAAACACAGAAAA'"CCCAAAAAAGAAGA AAAG GiC A A GC GAA i AAAGA AAAGAATC'"GGAGAAAAAGCAATCAAGGAAC A CCAG CiiAAGAA ACAA AAG A AiAC A GA AGAA i ii AACGG AG GCCAiAGAAACAAC CCAGA AAC i AA AAA CAAA"AGAAGGiA CA CGGi GAAAGGGCACAAAAAG'"CCAACCCAiGA GAA CAiGCCAGAAAGGAAA GG AG GAGGAAGCiA GA ACC AAAG CiAiCAA GC CCG iiGGGAAAAAi iGAiGG AGAGGiA GG CA CAAAiAiCGA AC GGAACAGAi A AGACAiAAGGC A GAGAA CGA GA GAiGCCAAAGCCTCAA"GAGA AAAAAAGAAGAC AAAAGGCAC AAi Al GG GAGiGA AAAA CCCiCAiGCGi CAA A 1A AA GG GGCAAAATCACTGTAGAAAAA AiGA GA GGAAGGGAiiA i GACCCACA CAiA GCAG GGA iC iGCiGGAAA GAiACiGAACAAGACA'"CAAAAACi AACGGCA AGA GGAA iGCACC AAiGCACAA —102— A i iCiCi ACAAAAIG AI C GACGCAGGA CIGGG iiGCGGGiGAiGAAACAAiG CAiGC A iGAAGA i C A CAAACACAACG GA G iG iiCGG A CAiC GG i ACAGGAACAGGIC G AGGIGAGAAA"ATT GGCAAGCTA"TCGGGCA AAGAAAAGCAGGCA CCAA GG G CGCiACGGGiAAC A GCGACi C GC iCA AGI C CA GGGAI iAGiAGCAAA AAICA C GAAAAIGACCGACACiGGAAA G AACACGAAC"GCAGCACA GAAGA GCGA AGCGG CGCiiC GC AAAAA CAAACAG GAG iiGA AAAG iAACAiAGGiGGAGAAAGT" iAAA ACAGAAA A AGGGGCCI CGAIAAGAG AAAA CACAACAAA GAAGA GGAACAAAAGC CCiAG AAA iAAAAi G A A AIAGGCAAGGGGCAAGACCAAGA iGA AGG GGA C AGGGGCAAAA GCAG AA GGAIAGAA A GA AAAAAAIGCI iAAAAAAGC A GGA GCACGCGCCA"TATGG iG AAAIAC G AAA ACIACAA AGAGAIAAI GGACAGAGCi CCAGC A GGGA CGGA GAAGG AC AAAAGiCAAG G CAA i CAGGAGAIGA GG G AAAGC A GGAACA GAI AAICC GAIAAAAAAAC "GAAGTCAAAAGAAA AA AAAGAAGA i iAAAGA AAA GGAGCAA AC A CCAA GAIA GGAAAG A AT"CCAACAAACCGAA G AGG GACGAAAAAGAGA iGAC iAAGi GCACC GACACAGACAAAGAAC C AI AAAGAAGAiAiCAiCG CCAGCAGGA C ACA C GGGGGCCAAGAA AGAi ACi iAAAACCCGAiG C AGCACCIGGIAAAAAIA AAA CCACGC 1AA G AIIAA GGCAAA CAAC AiGGC SEQ ID NO: 32 HiHHHH SEQ ID NO: 33 MSYYHHHHHH SEQ ID NO: 265 SPl912 GMKAKKMW AGLALLGIGSEALATKKVADDRKLMKIQnnLinIVRDHtSDMGnIAiLYVQVYnSSL«SLVGGVIF EDGRHYItVYnNnDLVYnnnV.
SEQ ID 0: 266 SPl912E MRY-AT..LS.AVLITAGCKKVADDRKLMKIQnnLinIVRDHtSDMGnIAiLYVQVYnSSL«SLVGGVIFEDGRHYT tVYnNnD-VYnnnVL SEQ I) O: 267 DTSSSEDALNISDKnKVAnNKn<in IHSA EiSQDbKEKKIAVIKnKnVVSKNPVIDNNISNnnAKIKnnNSN<SQ GDY DStVNKNinNPKKnDKVVYIAntKDKnSGnKAIKnLSSL<NT<V.YTYDRIFNGSAIETTPDNLD(IKQIEGI QKVQPM NiAR<nIGVnnAIDY.KSINAPFGK VIS IDTG"DYRHKAMRIDDDAKAS RFK<ED LKG"DKNYWLSD<IPHAF YYNGGKITVEKYDDGRDYFDPHGMiIAGI.AGND nQDIKNt GIDGIAP AQIFSYK MYSDAGSGtAGDEI tHAIEDSI(HNVDVVSVSSGFTGTGLVGEKYWQAIRALR(AGIPMVVATGNYATSASSSSWD —103— JKMTDTG VTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKY RNIGAFFD<SKITTN L‘J DGT<APSKJKFV YIGKGQDQD IGLD .RGKIAV DRIYTKDJKNAFK <AMDKGARAIMVVN"VNYY RD WT?LPA GY.a; (SQV FSISGDDGV (LWNMINPD (KT:VKRNNK .DtKDKLA4 nQYYPIDMESFNSN (PNVGDnKnIDbeAPDi nDII VPAGSTSWGPRID. .LKP DVSAPGKNIKSTLNVINGKSTYGYMSGTSMA"PIVAASTVLI RPK-(nM-n (NLK -TS "KIA P MDATSW<EKSQYFASPRQQGAGLINVA ALRNEVVATF<NTDS<G4V SYGSISL <3IKGD<KYt IKniNiS RPL tKVSASAITT DSLTDR-K-D niYKD A <QIVPnIHPn<VKGA ITFEHDT FTIGANSSF DLNAVINVGEAKA KNKtV fiStIan SVn nMnA .NSNGKK I FQPSJSMPL GFAG WNi 3PILD<WAW nnGSRS (iLGGYDD DGKP <IPG"LNKGIGGEHGIDKF PAGVIQNRKD < TTSL SEQ ID NO: 268 SP1912 sus M GMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKIQ A .nLi nIVRDHbSDMG nIAiLYVQVY nSSL nSLVGGVIF H A L I L S LDGRHYibVY.Z nDLVY A A .nVL I SEQ ID NO: 269 SP641N consensus MVVLADTSSS?DALNISDK:KVA————— nNK «KH nNIHSAMLISQDt LKKIAVI <. nVVSKNPVIDNNiSN A.nAK N S VVDK (D N I K 1.L‘ 1I LG A T TK IKn nNSNKSQGDYiDStVNKNi nNPKK nDKVVYIA anDK 1.SG nKAIK «LSSLKNTKVLYTYDRIFNGSAIETTPDN 3- Q H Q Q N G Q NAH SA G RL G LDKIKQI nGISSV nRAQKVQPMMNHARK nIGV A.«AIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDA T I KASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGI APNAQIFSYKMYSDAGSGtAGDLthHAILDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNY ATSASSSSWDLVANNHLKMTDTGNVTRTAAH EDAIAVASAKNQTVIEFDKVNIGGESFKYRNIGAFFDKSKITTNZ UG) Q N -104— TKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGY nADnGiKSQVtSISGDDGVKLWNMINPDKKTEVKRNNKnDtKDKLnQYYPIDMESFNSNKPNVGD.W nIDbKtAPDi DKnLYKnDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYG SEQ ID NO: 270 SP6 41M consensus MSGTSMATPIVAASTVLIRPKLKnMLnRPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQG K T AGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKL DniYKDnKSPDGKQIVPnIHPnKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKtVnStIHt A nAL Y R A NSNGKKINFQPSLSMPLMGFAGNWNH?PILDKWAW A.nGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGV 8 TD K M3 IQNRKDKNTTSLDQNPnLtAtNNnGINAPSSSGSKIANIYPLDS GNPQDAQL?RGLTPSPLVLRSA A.nGLISIVNT R D D Q VH 3 T NKnGnNQRDLKVISRTHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGR A.nNAPnSKDNQDPATKIRGQF K V G QYtYKtKYRLiKDYPWQVSYIPVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQYKN;iLtARDQKAHA PnKtDnIANnVWYAGAALVN A.DGnVnKNLnViYAGnGQGRNRKLDKDGNTIYEIKGAGDLRGKIIEVIALDGSSNFT S A KIHRIKFANQADEKGMISYYLVDPDQDSSKYQ —105— DH K A m SEQ ID NO: 271 SP1912 (nucleotides) uIGGLbiLAIITTT ELAGIC '1‘ '1‘AT SA SEQ ID NO: 272 SP1912L (nucleotides) ATGAGATACCTGGCAACAIiGiiGiiAiCiCiGGCGGiGiiAAiCACCGCCGGGTGCAAAAAAGTTG m7ckc3\\;r‘__.T‘»_v.’-‘».7 mm W‘r firm, :1 r'? r: (“y“) r1401“ “In“ {Wham-‘7 me >‘1‘r .L L k_:!-\!-\\:_v-E\. TC}: bbrs..z-.\\:r.:-.\\:r.L .L \_'r:-\‘__. 1-‘L‘,_'.'.’- RTIGIGGC“C“CCATTTTTCCGACATGGGGG“““mmCCCACCC\ \, 3,93. um \ farms; .L \II'\/\:-'t “.1 ; .T; TIES T :3: 1- 2 {~41 EACC SEQ ID NO: 273 SPO64l.l(nucneotides) GACACA CIAGC C GAAGAIGCIIIAAACA C CiGAIAAAGAAAAAGTAGCAGAAAATAAAGAGAAACA"GAAAA iAiCCA AGIGC A GGAAACT"CACAGGAI i AAAGAGAAGAAAACAGCAGTCA"TAAGGAAAAAGAAG iG iA G AAAAAICCIG GA AGACAA"AACACTAGCAATGAAGAAGCAAAAA"CAAAGAAGAAAA CCAA AAA CCCAA GGAGAT"ATACGGAC CA iG GAAIAAAAACACAGAAAA"CCCAAAAAAGAAGA AAAG GIC A A GC GA A i AAAGA AAAGAATC"GGAGAAAAAGCAATCAAGGAAC A CCAG CIIAAGAA G A AIAC A GA AGAA iii AACGG AG GCCAIAGAAACAAC CCAGA AACI GGACAAAA A"AGAAGGIA CA CGGi GAAAGGGCACAAAAAG"CCAACCCAiGA GAA CAIGCCAGAAAGGAAA GGAG GAGGAAGCTA" GA ACC AAAG CIAICAA GC CCG iiGGGAAAAAi iGAiGG AGAGGIA GG CA CAAAIAICGA A C"GGAACAGAI A AGACAIAAGGC A GAGAA CGA GA GAIGCCAAAGCCICAA GAGA AAAAAAGAAGAC AAAAGGCAC GAIAAAAAI AI GG GAGIGA AAAA CCCICAIGCGI CAA A 1A AA GG GGCAAAAT CAC G AGAAAAA AiGA GA GGAAGGGAIIA i CA GGGA GCAiA GCAGGGA C iGCiGGAA A"GATACTGAACAAGACA"CAAAAAC AACGGCA AGA GGAA GCACC AA A i C Ci ACAAA A G A C GACGCAGGA CiGGG i GCGGGiGA AIG CA GC A GAAGAI C A CAAACACAA CG GA G iG iiCGG A CAiC GG i ACAGGAACAGGIC G AGGiGAGAAA"ATTGGCAAGCTA"TCGGG CA AAGAAAAGCAGGCA CCAA GG G CGCiACGGGiAAC A GCGACi C GC iCAAGi C CA GGGAi "TAGTAGCAAA AAICA C GAAAAIGACCGACACIGGAAA G AAC"GCAGCACA GAAGA GCGA AGC GG CGCiiC GC AAAAA CAAACAG GAG iiGA AAAG iAACAiAGGiGGAGAAAGi iAAA ACAGAAA"A "AGGGGCCi CGAiAAGAG AAAA CACAACAAA GAAGA GGAACAAAAGC CCIAG AAA iAAAAi G A A AiAGGCAAGGGGCAAGACCAAGA iGA AGG GGA C CAAAA GCAG AAiGGAiAGAA A 1"ACAAAGGA AAAAAATGCT"TTAAAAAAGCTA"GGATAAGGGTGCACGCGCCA iAiGGiiG AAAIAC G AA —106— A AClACAAlAGAGAlAAl GGACAGAGCl CCAGC A GGGA AlGAAGCGGAlGAAGGlAC AAAAGlCAAGlG CAA llCAGGAGAlGA GGlG AAAGC A GGAACA GAl AAlCC AAAACTGAAGTCAAAAGAAA AA AAAGAAGA l lAAAGA AAA GGAGCAA AC A CCAA GAlA GGAAAG AAl CCAACAAACCGA A G AGG GACGAAAAAGAGA lGAC lAAGl GCACC GACACAGACAAAGAAC C AlAAAGAAGATA"CATC G CCAGCAGGA C ACA C GGGGGCCAAGAA AGAl ACl AAAACCCGAlG CAGCACCTGGTAAAAA A AAA CCACGC lAA G Al AA GGCAAA CAAC AlGGC A A GlCAGGAAC AG AlGGCGAC"CCAA CG llC AClG GA AAAT"AAAGGAAA GC lGAAAGACC G A GAAAAATC"TAAG GGAGATGACAAAA AGAlC ACAAG CllACAAAAA GCCClACAAAA"ACTGCGCGACC A GAlGGA GCAAC lC lGGAAAGAAAAAAG CAA AC GCAlCACC AGACAACAGGGAGCAGGCC AA AA G GGCCAA GC "GAGAAA GAAGl GlAGCAAC l CAAAAACAC GA C AAAGGl GG AAAC CA AlGG CCA C C AAAGAAA"AAAAGG"GATAAAAAA AC ACAA CAAGC CACAA ACA CAAACAGACC GACl AAAG CAGCA CAGCGA AAC ACAGA C C AAClGACAGA AAAAC GA GAAACATATAAAGATGAAAAA C C CAGATGG"AAGCAAA lG CCAGAAA CACCCAGAAAAAGTCAAAGGAGCAAAlAlCACA GAGCA GA AC CAC A AGGCGCAAAl C AGC lGA GAAlGCGG AlAAA GllGGAGAGGCCAAAAACAAAAAlAAA GlAGAA CAl lAl CA GAG CAG GGAAGAAA"GGAAGCTC"AAACTCCAACGGGAAGAAAA AAAC CC AACCl C l G CGA GCC C AA GGGA GClGGGAA GGAACCACGAACCAA CCllGAlAAA GGGC GG GAAGAAGGG CAAGA CAAAAACAC"GGGAGG AlGA GA GAlGG AAACCGAAAATTCCAGGAACCTTAAA"AA GGGAA GG GGAGAACAlGG AlAGAlAAA AAlCCAGCAGGAG"TATACAAAA"AGAAAAGATAAAAATACAA CATCCC"G SEQ ID NO: 274 Canonical lipobox motif [.IVMFESTAGPC]—[LVIAMFTG]—[IVMSTAGCP]—[AGS]—C SEQ ID NO: 275 SP2108 signal sequence MSSKFMKSAAVLGTATLASLLLVAC SEQ ID NO: 276 m coli RlpB signal ce RYLATLLLSLAVLITAG[C] SEQ ID NO: 301 I munogenic PspA/PspC ptides including the —coil structure (PR + NPB) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPI.DnIADnYQGKLlVA KLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQ.KEFLDANLAGSGSG HMHHHHHHSSGLVPRGSGMKElAAAKbERQiMDSPDLGTDDDDKAMAD.KKAVNE - 107-— "U k"UCU .nPnNPAPAPKPAPAPQP EKPAPAPAPKP EKSADQQA.A «DYARRS.nnYNRLlQQ1 IO "U "U WCUI<PAPAPVPKRL‘J P (TGWGQENGMWCRQACGRT RAPPPPPLR GC SEQ ID NO: 302 H munogenic PspA/PspC polypeptides i 1g the coiled—coil structure (PR only) MSDKIIHLTDDSFDTDVL (A DGAILVDFWA EWCGPCKMIAPI .DnIAD nYQGKLlVA KENIDQNPGTAPKYGIRGIPTLL.FKNG EVAATKVGALSKGQ .KEFLDANLAGSGSG H HHHHHHSSGLVPEGSG KEIAAAKbE EQ {MDSPDLGTDDDDKAMAD .KKAVNE PETPAPAPAPAPAPAPTP EAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKP APAPAPAPKP nKPAnKPAPAPKPLIPKIGW (QENGMWCRQACGRTRAPPPPPLRSG SEQ ID NO: 303 I unogenic Ps oA/PspC polypeptides lacki 1g the coiled—coil structure (PR + NPB) U ((AVNn «KPAnnPnNPAPAPKPAPAPQREKPAPAPAP<PEKSADQQA A .nDYARR SnnnYNR.lQQQPP<AEKPAPAPVPKP EQPAPAPKTGWGQENGMW SEQ ID 0: 304 I munogenic spC poly oeptides lacking :he coiled—coil ure (PR only) D.KKAVNnPnlPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAP (PAPAPKPAPAPKPA PAPKPAPAP(PAPAPAPAPKPnKPAn<PAPAPKP ElPKlGWKQ LNGMW SEQ ID 0: 305 I munogenic spC polypeptides lacking :1e coiled—coil structure (PR + NPB) MAKKAnLnKanKPAnnPnNPAPAPQPEKSADQQA<.nDYARRSA A .nYNRLlQQQPPKA SEQ I) O: 306 40 on—proliqe Bloc: (NPB) E<SADQQAnnDYARRSnnnYNRLlQQQ SEQ ID NO: 307 Non—oroline Block (NPB) DQQAnnDYARRSnnnYNRLlQQQ 50 SEQ ID NO: 308 oliqe Block ( PB) MEKSADQQAnnDYARRSnnnYNRLlQQQ SEQ ID NO: 309 —108— Amino—terminal boundary to the ion D .KKAVNE_‘ s3Q ID O: 310 Carboxy—terminal boundary to the PR—region (K/G)TGW(K/G)Q ENGMW —109—

Claims (56)

The claims defining the invention are as follows:
1. A vaccine formulation sing a pharmaceutically acceptable carrier and a first polypeptide comprising the amino acid sequence of SEQ ID NO: 265, 266, or 268, or an 5 immunogenic fragment or homologue thereof, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 9, 10, 20, or 21, or an immunogenic fragment or homologue thereof.
2. The vaccine formulation of claim 1, further comprising a third polypeptide comprising 10 the amino acid sequence of SEQ ID NO: 6, 7, 18, or 19, or an immunogenic fragment or gue thereof.
3. A vaccine formulation comprising a pharmaceutically acceptable r and a first polypeptide sing the amino acid of SEQ ID NOS: 265, 266, or 268, or an genic 15 fragment or homologue thereof, and a second polypeptide comprising the amino acid sequence of SEQ ID NOS: 6, 7, 18, or 19, or an immunogenic fragment or homologue thereof.
4. The vaccine formulation of claim 1 or 3, wherein the first polypeptide consists of the amino acid sequence of SEQ ID NO: 265.
5. The e formulation of claim 1 or 3, wherein the first polypeptide consists of the amino acid sequence of SEQ ID NO: 266.
6. The vaccine formulation of claim 1 or 3, wherein the first polypeptide consists of the 25 amino acid sequence of SEQ ID NO: 268.
7. The vaccine ation of claim 1, n the second polypeptide consists of the amino acid sequence of SEQ ID NO: 9. 30
8. The vaccine formulation of claim 2, wherein the third polypeptide consists of the amino acid sequence of SEQ ID NO: 6.
9. The vaccine formulation of claim 2, wherein the third polypeptide consists of the amino acid sequence of SEQ ID NO: 7. 5
10. The vaccine formulation of claim 3, wherein the second ptide consists of the amino acid sequence of SEQ ID NO: 6.
11. The vaccine formulation of claim 3, wherein the second polypeptide consists of the amino acid sequence of SEQ ID NO: 7.
12. The vaccine formulation of any one of claims 1-11, wherein the immunogenic fragment of the first ptide is a truncated fragment of the first polypeptide having from 1-20 amino acid residues removed from the N-terminus, C-terminus, or both. 15
13. The vaccine formulation of any one of claims 1-11, wherein the genic fragment of the second polypeptide is a truncated fragment of the second polypeptide having from 1-20 amino acid residues removed from the N-terminus, inus, or both.
14. The vaccine ation of any one of claims 2, 8, and 9, wherein the immunogenic 20 fragment of the third polypeptide is a truncated fragment of the third polypeptide having from 1- 20 amino acid residues removed from the N-terminus, C-terminus, or both.
15. The vaccine formulation of any of claims 1-14, which ns no other S. pneumoniae polypeptides.
16. The vaccine formulation of claim 1, further comprising one or more polypeptides having an amino acid sequence comprising SEQ ID NOS: 22 or 23 or an immunogenic fragment or homologue f. 30
17. The vaccine formulation of any of claims 1-16, wherein one or more polypeptides are ated to an immunogenic carrier.
18. The vaccine formulation of any of claims 1-17, which comprises at least one lipidated polypeptide.
19. A vaccine formulation according to claim 3, wherein the second polypeptide comprises 5 the amino acid sequence of SEQ ID NO: 6, or an immunogenic fragment or homologue thereof, and further comprising a third polypeptide comprising the amino acid sequence of SEQ ID NO: 7, or an immunogenic nt or homologue f.
20. The vaccine formulation of claim 19, wherein the second polypeptide is lipidated.
21. The vaccine formulation of claim 19 or 20, further sing a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 9, or an immunogenic fragment or homologue thereof, and/or a fifth ptide comprising the amino acid sequence of SEQ ID NO: 10, or an immunogenic fragment or homologue thereof.
22. The vaccine formulation of claim 21, wherein the fifth polypeptide is lipidated.
23. A vaccine ation according to claim 1, wherein the second ptide comprises the amino acid sequence of SEQ ID NO: 9, or an immunogenic fragment or homologue thereof, 20 and further comprising a third polypeptide comprising the amino acid sequence of SEQ ID NO: 10, or an immunogenic fragment or homologue thereof.
24. The e formulation of claim 23, wherein the third polypeptide is lipidated.
25 25. The e formulation of any of claims 1-24, further comprising an adjuvant.
26. The vaccine formulation of claim 25 wherein the adjuvant is an agonist of a toll-like receptor (TLR).
27. The e formulation of claim 25, wherein the nt is alum.
28. The vaccine formulation of claim 25, wherein the vaccine formulation comprises 1-1000 µg of each polypeptide and 1-250 µg of the adjuvant.
29. The vaccine formulation of any of claims 1-28, which induces a TH17 cell response at 10 least 1.5-fold r than that induced by a control unrelated antigen after contacting TH17 cells.
30. The vaccine formulation of any of claims 1-28, wherein the e formulation inhibits infection by S. pneumoniae in an uninfected subject. 15
31. The vaccine formulation of any of claims 1-28, wherein the vaccine formulation inhibits S. niae colonization in a subject.
32. The vaccine formulation of any of claims 1-28, wherein the vaccine formulation ts one or more S. pneumoniae symptoms in a subject.
33. The vaccine formulation of any one of claims 1-28, wherein the vaccine formulation inhibits S. pneumoniae-induced sepsis in the subject.
34. The vaccine formulation of any one of claims 1-28, n the vaccine formulation 25 reduces the duration of S. pneumoniae colonization in a subject.
35. The vaccine ation of any one of claims 1-28, wherein the vaccine formulation elicits a cell-mediated immune response to S. pneumoniae in a subject. 30
36. The vaccine formulation of any one of claims 1-28, wherein the vaccine formulation elicits a T cell response to S. niae in a subject.
37. The vaccine ation of any one of claims 1-28, wherein the vaccine formulation elicits a CD4+ T cell response to S. pneumoniae in a subject. 5
38. The e formulation of any one of claims 1-28, wherein the vaccine formulation elicits a TH17 cell response to S. pneumoniae in a subject.
39. The vaccine formulation of any one of claims 1-28, wherein the vaccine formulation induces an increased level of IL-17 in a t.
40. The vaccine formulation of any one of claims 1-28, wherein the vaccine ation elicits a TH1 cell response to S. pneumoniae in a t.
41. The e formulation of any one of claims 1-28, n the vaccine formulation 15 elicits a CD8+ T cell response to S. pneumoniae in a subject.
42. The vaccine formulation of any one of claims 1-28, wherein the vaccine formulation elicits a CD8+ Cytotoxic T Lymphocyte (CTL) response to S. pneumoniae in a subject. 20
43. The vaccine formulation of any one of claims 1-28, wherein the vaccine formulation s an innate immune response to S. pneumoniae in a subject.
44. The vaccine formulation of any one of claims 1-28, wherein the vaccine formulation inhibits development of otitis media in a subject.
45. The vaccine ation of any one of claims 1-28, wherein the vaccine formulation reduces intensity and/or severity of otitis media in a subject.
46. Use of a vaccine formulation according to any of claims 1-28 in the manufacture of a 30 medicament for treating a subject suffering from or susceptible to S. pneumoniae infection.
47. The use of claim 46, wherein the medicament inhibits infection by S. pneumoniae in an uninfected subject.
48. The use of claim 46, wherein the medicament inhibits S. pneumoniae colonization in the 5 subject.
49. The use of claim 46, wherein the medicament inhibits S. niae symptoms in the 10
50. The use of claim 46, wherein the medicament inhibits S. pneumoniae-induced sepsis in the subject.
51. The use of claim 46, wherein the medicament inhibits development of otitis media in the
52. The use of claim 46, wherein the medicament s intensity and/or severity of otitis media in the subject.
53. The use of claim 46, wherein the medicament treats a subject with one dose.
54. The use of claim 46, n the medicament treats a subject within three doses.
55. The use of claim 46, wherein the subject is a human. 25
56. The vaccine formulation of any one of claims 1, 3, 19 or 23, substantially as hereinbefore described with reference to any of the Examples and/or
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US201161434818P 2011-01-20 2011-01-20
US61/434,818 2011-01-20
PCT/US2012/022128 WO2012100234A1 (en) 2011-01-20 2012-01-20 Vaccines and compositions against streptococcus pneumoniae

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