HK1192166A - Vaccines and compositions against streptococcus pneumoniae - Google Patents

Description

Vaccines and compositions against Streptococcus Pneumoniae (Streptococcus Pneumoniae)

RELATED APPLICATIONS

This application claims benefit of filing date of U.S. provisional application No. 61/434,818 filed on 20/1/2011. The entire teachings of the referenced application are expressly incorporated herein by reference.

Government support

This work was performed with government support under license number AI066013 awarded by the National Institutes of Health. Accordingly, the U.S. government has certain rights in this invention.

I. Background of the invention

Pneumococcal disease has been the leading cause of disease and death in the united states and even globally. Millions of cases of pneumonia, meningitis, bacteremia, and otitis media each year are caused by the pathogenic streptococcus pneumoniae infection. Streptococcus pneumoniae is a gram-positive bacterium with pods that colonize the nasopharynx of approximately 5% -10% of healthy adults and 20% -40% of healthy children. When streptococcus pneumoniae is carried into the eustachian tube, sinuses, lungs, blood stream, meninges, joint spaces, bones, and peritoneal cavity, normal colonization becomes infectious. Streptococcus pneumoniae has several virulence factors that enable the organism to evade the immune system. Examples include polysaccharide capsules, which prevent phagocytosis by host immune cells; proteases, which inhibit complement mediated opsonization; and proteins, which cause host cell lysis. In the polysaccharide capsule, the presence of complex polysaccharides forms the basis for the classification of pneumococci into different serotypes. To date, 93 serotypes of streptococcus pneumoniae have been identified.

Different pharmaceutical compositions have been used to exploit the immune response against streptococcus pneumoniae infection. The multivalent pneumococcal vaccine PPV-23 was developed to prevent pneumonia and other invasive diseases caused by streptococcus pneumoniae in adult and elderly populations. The vaccine contains Capsular Polysaccharides (CPs) from 23 serotypes of streptococcus pneumoniae. As T cell independent antigens, these CPs induce only a transient antibody response, requiring repeated dosing, which increases the risk of immune tolerance. Antibodies raised against streptococcus pneumoniae, called anti-capsular antibodies, are thought to have a protective effect in adults and immunocompetent individuals. However, children and immunocompromised individuals, including the elderly, under the age of 2 years do not respond well to T cell-independent antigens and, therefore, do not receive optimal protection from PPV-23. Another streptococcus pneumoniae vaccine (Prevnar) includes bacterial polysaccharides from 7 streptococcus pneumoniae strains conjugated to diphtheria toxoid protein. This vaccine induces B and T cell responses. However, because it only provides protection against 7 pneumococcal serotypes, serotype replacement can render the lung useless. Serotype replacement has been demonstrated in several clinical trials and epidemiological studies, requiring the development of different formulations of these vaccines. An example is the recently introduced Pepper 13 against 13 pneumococcal serotypes (Prevnar 13). In addition, both of these Pepper's preparations are expensive to manufacture, greatly limiting their availability in the developing world. PPV-23, consisting of 23 purified but unbound polysaccharides, has a broader coverage but does not provide protection for the population at the highest risk of pneumococcal disease, i.e. children under the age of 2 years.

Thus, there remains a need to design pharmaceutical compositions that are more effective than current strategy proposals. In particular, these compositions need to incorporate novel or specific antigens to elicit an immune response against streptococcus pneumoniae.

Disclosure of the invention

Streptococcus pneumoniae is a major health problem, especially in very young, elderly or immunocompromised patients. Although DNA and protein sequence information of streptococcus pneumoniae has been known for some time and researchers have long attempted to produce vaccines against streptococcus pneumoniae, the main problem is how to identify protective polypeptides from about 2100 genes in the streptococcus pneumoniae genome. The present application presents the results of a whole genome screen designed to identify the most immunogenic proteins in the streptococcus pneumoniae genome. Several hits generated by this screen have shown protection against colonization by streptococcus pneumoniae, and in some cases, colonization and streptococcus pneumoniae-induced sepsis, in a mouse model. Thus, the present disclosure provides, among other things, certain highly effective vaccines against streptococcus pneumoniae. These vaccines may be used for therapeutic or prophylactic use. The disclosure also provides specific antigens and methods of using these antigens to elicit an immune response against streptococcus pneumoniae.

In certain aspects, the disclosure provides a vaccine formulation comprising a pharmaceutically acceptable carrier and a peptide having a sequence comprising SEQ ID NO:265 or 268 (or consisting thereof), or an immunogenic fragment thereof. In some embodiments, the polypeptide comprises an exogenous signal sequence. For example, the polypeptide can have a 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 sequence comprising SEQ id no:1-23, 267 and 269-270 or an immunogenic fragment thereof. In certain embodiments, the vaccine formulation further comprises a second polypeptide having an amino acid sequence comprising SEQ id no:1-23, 267 and 269-270 or an immunogenic fragment thereof.

In certain embodiments, the first and the second polypeptide belong to different groups of (i) - (vi): (i) SEQ ID NO:1 or an immunogenic fragment thereof, (ii) SEQ ID NO:2-5 and 14-17 or an immunogenic fragment thereof, (iii) SEQ ID NO:6-7 and 18-19 or an immunogenic fragment thereof, (iv) one of SEQ ID NOs: 8 or an immunogenic fragment thereof, (v) SEQ ID NO:9-10 and 20-21 or an immunogenic fragment thereof, and (vi) one of SEQ ID NOs: 11-13, 267 and 269-270 or an immunogenic fragment thereof.

In some such embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO:9 or SEQ ID NO: 10. In some embodiments, the vaccine formulation comprises a polypeptide consisting of SEQ ID NO:6 or 7 and a polypeptide consisting of SEQ ID NO:9 or 10.

In any of this aspect or embodiments, the vaccine formulation may comprise SEQ ID NO: 265. 266 or 268 which is a truncated fragment having from 1 to 20 amino acid residues removed from the N-terminus, C-terminus or both. In some embodiments, the vaccine formulation is substantially free of other than having a polypeptide comprising SEQ ID NO: 1-21 and 265-270, or a polypeptide other than the amino acid sequence of any one of seq id no.

In certain embodiments, the vaccine formulation comprises one or more polypeptides, which or which have a sequence comprising SEQ ID NO:22 or 23 or an immunogenic fragment thereof.

In another aspect, the invention provides vaccine formulations comprising known streptococcus pneumoniae antigens, such as pneumocolyloid, choline binding protein a (cbpa) or pneumococcal surface protein 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 a sequence comprising SEQ ID NO:1-23 and 265-270 or an immunogenic fragment thereof, (ii) a pneumocolyloid, and (iii) a pharmaceutically acceptable carrier. Another exemplary vaccine formulation comprises: (i) a polypeptide having a sequence comprising SEQ id no:1-23 and 265-270 or an immunogenic fragment thereof, (ii) CbpA or a derivative thereof, and (iii) a pharmaceutically acceptable carrier. Another exemplary vaccine formulation comprises: (i) a polypeptide having a sequence comprising SEQ ID NO: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 SEQ ID NO:2-5, 6,7, 9-13, and 265-267. In some embodiments, the vaccine formulation further comprises a second polypeptide having an amino acid sequence comprising SEQ ID NO:1-23 and 265-270. In some embodiments, the pneumocolyoid is PdT, Pd-A, Pd-B, rPd2, rPd3, Ply8, Δ 6Ply, L460D (see, e.g., US2009/0285846 and L. mitchell (L. mitchell), Protective Immune response to Streptococcus pneumoniae pneumoconiae pneumosoid (Protective Immune Responses to Streptococcus pneumoniae pneumoconiae pneumosoids), ASM2011 conference abstract, 2011) or a variant thereof. In some embodiments, the derivative of PspA comprises all or a fragment of the proline-rich region of PspA.

In certain embodiments, the polypeptide is conjugated to an immunogenic carrier. In some embodiments, the vaccine formulation comprises at least a lipidated polypeptide.

In some embodiments, the vaccine formulation further comprises conjugated streptococcus pneumoniae polysaccharides. These conjugated polysaccharides may be, for example, as described in US patent 5,623,057, US patent 5,371,197, or PCT/US 2011/023526.

In some embodiments, the vaccine formulation further comprises an adjuvant. The adjuvant may be, for example, a toll-like receptor (TLR) agonist. The adjuvant may be, for example, alum. In some embodiments, the vaccine formulation comprises 1-1000 μ g of each polypeptide and 1-250 μ g of the adjuvant.

In certain embodiments, the vaccine formulation induces T_H17 cell response ratio by control group unrelated antigen on T contact_H17T induced after cells_H17 cell responses were at least 1.5 fold greater. In some embodiments, the vaccine formulation inhibits infection by streptococcus pneumoniae in an uninfected subject. In certain embodiments, the vaccine formulation inhibits colonization of streptococcus pneumoniae in an individual. In some embodiments, the vaccine formulation inhibits a symptom or sequelae of streptococcus pneumoniae. For example, the vaccine formulation inhibits streptococcus pneumoniae-induced sepsis.

In certain aspects, the disclosure provides methods for treating a subject suffering from or susceptible to infection by streptococcus pneumoniae, comprising administering an effective amount of any of the vaccine formulations described herein.

In some embodiments, the method inhibits infection by streptococcus pneumoniae in an uninfected subject. In some embodiments, the method inhibits colonization of streptococcus pneumoniae in the subject. In some embodiments, the method inhibits a symptom or sequelae of streptococcus pneumoniae. An exemplary sequelae is sepsis.

In certain embodiments, the method treats the subject with one dose. In other embodiments, the method treats the subject with two or three doses. In some embodiments, the method treats the subject within three doses.

In certain embodiments, the subject is a human.

The present disclosure provides, for example, vaccine formulations comprising a pharmaceutically acceptable carrier and one or more polypeptides having a sequence comprising SEQ ID NO: 1-13, 265, 266, and 267 or an immunogenic fragment thereof.

The disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and at least one polypeptide having a sequence comprising SEQ ID NO: 6. SEQ ID NO:10 or SEQ ID NO:265 or an immunogenic fragment thereof. The 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.

In addition, the present application provides a vaccine formulation comprising a pharmaceutically acceptable carrier and one or more polypeptides having a sequence comprising SEQ ID NO: 14-23, 268, 269 and 270, or an immunogenic fragment thereof.

The present disclosure further provides an immunogenic composition comprising a pharmaceutically acceptable carrier and two or more polypeptides having a sequence comprising 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 a polypeptide sequence comprising SEQ ID NO: 6. SEQ ID NO:10 or SEQ ID NO:265 or an immunogenic fragment thereof. The disclosure further provides a vaccine formulation comprising a pharmaceutically acceptable carrier and two or more polypeptides having a sequence consisting of SEQ ID NO: 7. SEQ ID NO:9 or SEQ ID NO:265 or an immunogenic fragment thereof. The present disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and two or more polypeptides having a polypeptide sequence comprising SEQ ID NOs: 6. SEQ ID NO:9 and SEQ ID NO:265 or an immunogenic fragment thereof. In addition, the disclosure provides a vaccine formulation comprising a pharmaceutically acceptable carrier and two or more polypeptides having a sequence comprising SEQ ID NO: 7. SEQ ID NO:10 and SEQ ID NO:265 or an immunogenic fragment thereof. In some embodiments, the polypeptide comprising SEQ ID NO:265 comprises an exogenous signal sequence.

The disclosure also provides a vaccine formulation comprising a pharmaceutically acceptable carrier and three or more polypeptides having a sequence comprising SEQ ID NOs: 6. SEQ ID NO:10 and SEQ ID NO:265 or an immunogenic fragment thereof. The disclosure further provides vaccine formulations comprising a pharmaceutically acceptable carrier and three or more polypeptides having a sequence consisting of SEQ ID NOs: 7. SEQ ID NO:9 and SEQ ID NO:265 or an immunogenic fragment thereof.

Brief description of the drawings

FIG. 1 shows the concentration of IL-17 produced by blood samples from mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by stimulation with killed, capsular-free, whole cell Streptococcus pneumoniae, as described in example 5. The left panel shows the data in the form of scattered dots, and the right panel shows the mean and standard deviation of each sample. The immunization group "All 3" represents animals immunized with a combination of SP2108, SP0148 and SP 1634.

FIG. 2 shows the concentration of IL-17 produced by blood samples from mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by stimulation with a combination of the three proteins (SP2108, SP0148, and SP1634), as described in example 5.

FIG. 3 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by challenge by intranasal administration of Streptococcus pneumoniae, as described in example 5. 003 represents a control group irrelevant antigen.

FIG. 4 shows the concentration of IL-17 produced by blood samples from mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by stimulation with killed, capsular-free, whole cell Streptococcus pneumoniae, as described in example 6.

FIG. 5 shows the concentration of IL-17 produced by blood samples from mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by stimulation with one or more of the indicated proteins, as described in example 6.

FIG. 6 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by challenge by intranasal administration of Streptococcus pneumoniae, as described in example 6. HSV-2 proteins ICP47 and Ovalbumin (OVA) under the gene name US12 (NP-044543.1, NC-001798.1; shown as 003 in the figure) represent control antigens.

FIG. 7 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by challenge by intranasal administration of Streptococcus pneumoniae, as described in example 7.

FIG. 8 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of BALB/c mice immunized with one or more of the indicated proteins and cholera toxin adjuvant, followed by challenge with intranasal administration of Streptococcus pneumoniae, as described in example 8.

FIG. 9 shows the concentration of IL-17A produced by blood samples from mice immunized with the indicated protein and cholera toxin adjuvant, followed by stimulation with either the immunizing protein (left panel) or killed non-capsulated whole cell Streptococcus pneumoniae (right panel), as described in example 9.

FIG. 10 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with the indicated proteins and cholera toxin adjuvant, followed by challenge with intranasal administration of Streptococcus pneumoniae, as described in example 10.

Figure 11 shows the survival of mice immunized with the indicated proteins and adjuvant alum, followed by an inspiratory challenge with streptococcus pneumoniae, as described in example 11.

Figure 12 shows the survival of mice immunized with the indicated proteins and adjuvant alum, followed by an inspiratory challenge with streptococcus pneumoniae, as described in example 12.

FIG. 13 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with the indicated proteins and cholera toxin adjuvant, followed by challenge with intranasal administration of Streptococcus pneumoniae, as described in example 13.

Figure 14 shows the concentration of IL-17A produced by blood samples from mice immunized with the indicated protein and alum, followed by stimulation with the indicated protein at the top left, as described in example 14.

FIG. 15 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with the indicated proteins and alum or with killed whole cell Streptococcus pneumoniae plus alum Without Capsules (WCV), followed by challenge by intranasal administration of Streptococcus pneumoniae, as described in example 15.

FIG. 16 shows the number of Streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with the indicated proteins and alum or with killed whole cell Streptococcus pneumoniae plus alum Without Capsules (WCV) followed by challenge by intranasal administration of Streptococcus pneumoniae in two pooled studies as described in example 16.

Figure 17 shows the number of streptococcus pneumoniae colonies obtained from nasal washes of mice immunized with the indicated proteins and alum or with killed non-capsulated whole cell streptococcus pneumoniae plus alum (WCB) followed by challenge by intranasal administration of streptococcus pneumoniae as described in example 17.

FIG. 18 shows the survival of mice injected with antibodies or serum specific for the indicated proteins, followed by an inspiratory challenge with S.pneumoniae, as described in example 18.

Figure 19 shows the percentage of animals protected from sepsis in six separate inspiratory challenge studies, two of which are described in more detail in examples 12 and 18.

Detailed description of the invention

A. Specific polypeptides and nucleic acids for use in streptococcus pneumoniae vaccines and immunogenic compositions

The streptococcus pneumoniae vaccines described herein include one or more polypeptides or genes as listed in table 1, or variants or fragments thereof as described below. The vaccine may comprise or consist of a polypeptide comprising or consisting of a sequence of table 1 or a variant or immunogenic fragment thereof. The DNA and protein sequences of each Gene and polypeptide can be found by searching Gene coordinates in the streptococcus pneumoniae TIGR4 genome in the public database Entrez Gene (NCBI NIH website on the world wide web, www.ncbi.nlm.nih.gov/sites/entrezdb ═ Gene), and the sequences specified are also included in the present application.

TABLE 1 immunogenic polypeptides for vaccine formulations

NB: the database sequences incorrectly listed TTG (encoding Leu) at nucleotide positions 541- > 543. As set forth in SEQ ID NO: 28 has a TTC at that codon and encodes Phe. The database sequences further did not include the C-terminal Glu found in some isolates.

Certain polypeptides of table 1 and variants thereof are described in more detail below.

SP1912(SEQ ID NO: 265) and variants thereof

SP1912 is a putative protein with 99 amino acids. Although the protein function is not known with certainty, sequence analysis indicates that it is a putative thioredoxin.

In some embodiments, a vaccine or pharmaceutical composition comprising a streptococcus pneumoniae polypeptide comprises a polypeptide comprising at least 20 contiguous amino acid residues selected from SP 1912. The polypeptide may also be a variant of a fragment of these at least 20 residues. In certain embodiments, the polypeptide comprises no more than 90, 75, 60, 45, or 30 consecutive amino acids of S P1912.

In some embodiments, the compositions and methods herein call for the use of an SP1912 variant that comprises an exogenous lipidated sequence. In some embodiments, the signal sequence directs lipidation. Thus, the lipidation signal may be, for example, the signal sequence of SP2108(SEQ ID NO: 275) or SP0148 or the E.coli (E.coli) signal sequence. An exemplary variant SP1912L comprising the signal sequence of the E.coli gene RlpB (SEQ ID NO: 276) consists of the polypeptide sequence SEQ ID NO: and 266, respectively. SP1912(SEQ ID NO: 265) and SP1912L (SEQ ID NO: 266) may be correspondingly encoded by a sequence according to SEQ ID NO: 271 and 272, but other DNA sequences (including codon optimized sequences) may be used due to the degeneracy of the genetic code.

The consensus sequence illustrating the combination of SP1912 sequences from different serotypes is represented as SEQ ID NO: 268. thus, in certain embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 268 or an amino acid sequence consisting thereof or an immunogenic fragment thereof (e.g., instead of a polypeptide having an amino acid sequence comprising SEQ id no: 265).

SP0024(SEQ ID NO: 1) and variants thereof

SP0024 represents a putative protein of 165 amino acids containing a conserved carbonic anhydrase domain extending from amino acid 27 to amino acid 163. Based on this consensus motif, SP0024 can be a zinc binding protein.

In some embodiments, a vaccine or pharmaceutical composition comprising a streptococcus pneumoniae polypeptide comprises a polypeptide comprising at least 20 contiguous amino acid residues selected from SP 0024. The polypeptide may also be a variant of a fragment of these at least 20 residues. In certain embodiments, the polypeptide comprises no more than 150, 125, or 100 consecutive amino acid residues of SP 0024.

SP0882(SEQ ID NO: 2) and variants thereof

SP0882 is a putative protein with 274 amino acids. The majority of the protein (amino acids 2-270) forms the esterase-like or lipase domain.

In some embodiments, a vaccine or pharmaceutical composition comprising a streptococcus pneumoniae polypeptide comprises a polypeptide comprising at least 20 contiguous amino acid residues selected from SP 0882. The polypeptide may also be a variant of a fragment of these at least 20 residues. In certain embodiments, the polypeptide comprises no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acid residues in SP 0882.

One particular truncated variant, designated SP0882N, consists of the N-terminal 130 amino acids of SP0882 and is shown as SEQ ID NO: 3.SP 0882N contains a region that is very conserved among different serotypes. In certain embodiments, the polypeptide comprising SP0882 or SP0882N, or an immunogenic fragment of either, may further comprise an exogenous signal sequence. In some embodiments, the signal sequence is an escherichia coli or streptococcus pneumoniae signal sequence. The signal sequence may be, for example, the signal sequence of SP 2108. Two exemplary such polypeptides are SEQ ID NOs: 4 and 5.

Variants of the DNA and protein sequences of SP0882 are described, inter alia, in U.S. patent application publication No. 2009/0215149 and International applications WO2002/077021, WO98/18931 and WO 2007/106407. Variants of SP0882N are disclosed in international application WO 2008/146164.

There are sequence differences at the protein level between different streptococcus pneumoniae serotypes, and a consensus sequence illustrating the combination of SP0882 sequences from different streptococcus pneumoniae serotypes is represented as SEQ ID NO: 14-17. Thus, in certain embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 14-17 or an amino acid sequence consisting thereof or an immunogenic fragment thereof (e.g., instead of a polypeptide having an amino acid sequence comprising one of SEQ ID NOs 2-5).

The nucleic acid sequences encoding the different variants of SP0882(SEQ ID NOS: 2-5) are represented as SEQ ID NOs: 24-26, but other DNA sequences (including codon-optimized sequences) may encode these polypeptides due to the degeneracy of the genetic code.

SP0148(SEQ ID NO: 7) and variants thereof

The protein SP0148 was named "ABC transporter, substrate binding protein". Such proteins are typically extracellular proteins that transiently interact with transmembrane protein complexes. Such complexes use the energy generated by ATP hydrolysis to transport specific substrates across cell membranes. SP0148 is a protein of 276 or 277 (depending on the isolate) amino acids containing a conserved PBPb (periplasmic binding protein) domain spanning amino acids 40-246, typical of membrane bound transport complexes. In addition, SP0148 had a bacterial extracellular solute-binding protein family 3 domain which was largely identical in extension to the PBPb domain and extended from amino acids 40 to 244. In some embodiments, the truncated mutant of SP0148 contained in the vaccine or other composition contains or lacks one or more of the domains and motifs.

In some embodiments, a vaccine or pharmaceutical composition comprising a streptococcus pneumoniae polypeptide comprises a polypeptide comprising at least 20 contiguous amino acid residues selected from SP 0148. The polypeptide may also be a variant of a fragment of these at least 20 residues. In certain embodiments, the polypeptide comprises no more than 250, 275, 200, 175, 150, 125, or 100 consecutive amino acids of SP 0148.

Endogenous SP0148 contains a signal sequence directing its secretion and potential lipidation. In some embodiments, the nucleic acid sequence of SEQ ID NO:7 is partially or completely processed by an expression host (e.g., e.coli). In some embodiments, the SP0148 variant (SEQ ID NO: 6) lacking the signal sequence was used. SEQ ID NO:6 consists of the polypeptide 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 screen herein.

Variants of the amino acid sequence and nucleotide sequence of SP0148 can be found in U.S. patent application publication No. 2005/0020813, U.S. patent nos. 7,378,514 and 7,504,110, and european patent application nos. EP1572868 and EP 1855717.

A consensus sequence illustrating the combination of SP0148 sequences from different streptococcus pneumoniae serotypes is shown as SEQ ID NO: 18 and 19. Thus, in certain embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 18-19 or an amino acid sequence consisting thereof or an immunogenic fragment thereof (e.g., instead 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

SP1072, also known as dnaG, is a DNA primer enzyme that catalyzes the formation of RNA primers to allow DNA polymerase to initiate DNA replication. SP1072 is a protein of 586 amino acids that contains several conserved motifs. From the N-terminus, amino acids 2-96 form a zinc finger domain, the DNA primase catalytic core spans amino acids 122-250, and the highly conserved topoisomerase-primase (TORPIM) nucleotidyl transferase/hydrolase domain region extends from amino acids 258 to 330. In some embodiments, the vaccine or other composition comprises a truncated mutant of SP1072 comprising or lacking one or more of the domains and motifs.

In some embodiments, a vaccine or pharmaceutical composition comprising a streptococcus pneumoniae polypeptide comprises a polypeptide comprising at least 20 contiguous amino acid residues selected from SP 1072. The polypeptide may also be a variant of a fragment of these at least 20 residues. In certain embodiments, the polypeptide comprises no more than 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids in SP 1072.

SP2108(SEQ ID NO: 9) and variants thereof

The polypeptide SP2108 is 423 amino acids in length and is also known as MalX, maltose/maltodextrin ABC transporter or maltose/maltodextrin binding protein. The vast majority of the proteins (amino acids 3-423) are classified as MalE (maltose binding periplasmic) domains. In addition, SP2108 contains a signal sequence that directs its secretion and potential lipidation. In some embodiments, the nucleic acid sequence of SEQ ID NO:9 is partially or completely processed by an expression host (e.g., e.coli). In some embodiments, the SP2108 truncation mutant included in the vaccine or other composition includes one or more of the domains and motifs.

In some embodiments, the compositions and methods herein call for the use of SP2108 variants that lack a signal sequence. Such variants consist of the polypeptide sequence SEQ ID NO:10, and can be represented, for example, by a sequence according to SEQ id no: 29, but other DNA sequences (including codon optimized sequences) may be used due to the degeneracy of the genetic code.

In some embodiments, the vaccine or pharmaceutical composition comprising a streptococcus pneumoniae polypeptide comprises a polypeptide comprising at least 20 contiguous amino acid residues selected from SP 2108. The polypeptide may also be a variant of a fragment of these at least 20 residues. In certain embodiments, the polypeptide comprises no more than 400, 350, 300, 250, 200, 150, or 100 consecutive amino acids of SP 2108.

A consensus sequence illustrating the combination of SP2108 sequences from different serotypes is represented as SEQ ID NO: 20 and 21. Thus, in certain embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 20-21 or an amino acid sequence consisting thereof or an immunogenic fragment thereof (e.g., instead of a polypeptide having an amino acid sequence comprising one of SEQ ID NOs 9 or 10).

SP0641(SEQ ID NO: 12) and variants thereof

SP0641, 2144 amino acids in length, also called PrtA, is a cell wall-associated serine protease. Full-length SP0641 contains many conserved motifs: a PA _2 motif extending between amino acids 485 and 597, which can form a protein binding surface; an Fn 3-like domain (amino acids 800-939); and two predicted catalytic domains of the S8C5a type located at amino acids 226-449 and 639-777. In some embodiments, the vaccine or other composition comprises a truncated mutant of SP0641 that comprises or lacks one or more of the domains and motifs.

In some embodiments, a vaccine or pharmaceutical composition comprising a streptococcus pneumoniae polypeptide comprises a polypeptide comprising at least 20 contiguous amino acid residues selected from SP0641. The polypeptide may also be a variant of a fragment of these at least 20 residues. In certain embodiments, the polypeptide comprises no more than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 consecutive amino acids in SP0641.

Certain other truncation mutants of SP0641 may also be used. For example, the polypeptide designated SP0641N (SEQ ID NO: 13) consists of 661 amino acids, which 661 amino acids correspond to amino acids 24-684 near the N-terminus of SP0641. The region of 648 residues captured by the truncated variant SP0641M (SEQ ID NO: 11) was located approximately near SP0641N (and corresponds to amino acids 686 1333 of SP 0641). The polypeptide designated SP0641.1(SEQ ID NO: 267) consists of 978 amino acids, these 978 amino acids corresponding to amino acids 28-1006 of SP0641.

Variants of SP0641 are disclosed, for example, in U.S. patent nos. 7,338,786, 6,573,082 and 7,132,107, and international application WO 00/06738.

SEQ ID NO: 30. 31 and 273 show the DNA sequences of SP0641M (SEQ ID NO: 11), SP0641N (SEQ ID NO: 13) and SP641.1(SEQ ID NO: 267) respectively, but other DNA sequences (including codon-optimized sequences) may encode these SP0641 variants due to the degeneracy of the genetic code.

The consensus sequence illustrating the combination of SP0641N and SP0641M sequences from different streptococcus pneumoniae serotypes is represented as SEQ ID NO: 269 and 270. Thus, in certain embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 269 or 270 or an amino acid sequence consisting thereof or an immunogenic fragment thereof (e.g., instead of a polypeptide having an amino acid sequence comprising one of SEQ ID NO:11 or 13).

Polypeptides homologous to the polypeptides of tables 1 and 2 (e.g., SP1912, S P1912L, SP0024, SP0882N, SP0148 with or without signal sequence, SP1072, SP2108 with or without signal sequence, SP0641M, SP0641N, or SP0641.1) can also be used in the compositions and methods disclosed herein. Individual strains of streptococcus pneumoniae contain many mutations within each other, and some of these mutations result in differences in protein sequence between the different strains. One skilled in the art can readily substitute the amino acid sequence, or a portion thereof, with a homologous amino acid sequence from a different streptococcus pneumoniae strain. In certain aspects, the present application provides immunogenic polypeptides that are at least 90%, 95%, 97%, 98%, 99%, or 99.5% identical to the polypeptides of tables 1 and 2, or immunogenic fragments thereof. These variants of the polypeptides of tables 1 and 2 can be designed using serotype differences.

In some embodiments, the vaccine compositions herein comprise a protein fragment of table 1 or 2 (e.g., a fragment of SP1912, SP1912L, SP0024, SP0882N, OSP148 with or without signal sequence, SP1072, SP2108 with or without signal sequence, SP0641M, SP0641N, or SP 0641.1). In some embodiments, the present application provides truncation mutants that are close in size to a polypeptide of Table 1 or 2 (e.g., one of SEQ ID NOs: 1-13, 265, 266, or 267). For example, they may lack up to one, two, three, four, five, ten or twenty amino acids at one or both termini. Internal deletions of, for example, 1-10, 11-20, 21-30 or 31-40 amino acids are also contemplated.

In certain embodiments, the vaccine formulation comprises one or more polypeptides having a sequence comprising SEQ ID NO: 14-21, 268, 269 and 270 or a polypeptide consisting of an amino acid sequence thereof. In certain embodiments, the fragment is SEQ ID NO: 14-21, 268, 269 and 270, wherein 1-5, 1-10 or 1-20 amino acid residues are removed from the N-terminus, the C-terminus or both. In certain embodiments, the fragment is SEQ ID NO: 14-21, 268, 269 and 270, wherein 1-10 amino acid residues are removed from the N-terminus, the C-terminus or both termini. For example, 10 amino acid residues can be removed from each of the N-and C-termini, thereby producing a protein from which 20 amino acid residues have been removed.

In certain embodiments, the vaccine formulations provided herein comprise or further comprise one or more or two or more known streptococcus pneumoniae antigens. In some cases, these known streptococcus pneumoniae antigens are primarily antibody targets. In some cases, these known streptococcus pneumoniae antigens may provide protection against streptococcus pneumoniae colonization or streptococcus pneumoniae induced sepsis. One suitable class of Streptococcus pneumoniae antigens recognized in the art is pneumocolyxoid. pneumolynoid has homology with the pneumococcal protein Pneumolysin (PLY), but is less toxic than pneumolysin. The pneumocolyloid may be a naturally occurring or engineered pneumolysin derivative. In some embodiments, pneumolyloid has at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with pneumolysin. In some embodiments, pneumolyloid exhibits a toxicity less than 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500, or 1/1000 of pneumolysin toxicity in an assay for one or both of hemolytic activity against erythrocytes and inhibition of polymorphonuclear leukocytes. Both assays are described in Morse F.K. et al ("thiol-activated toxin of Streptococcus pneumoniae Pneumolysin does not require a thiol group for in vitro activity of Pneumolysin"; Infection and immunization, 8 1989; 57 (8): 2547-52) (Saunders F.K.et al ("Pneumolyysin, the thiol-activated toxin of Streptococcus pneumoniae, do not require a thiol group for in vitro activity" Infection Immun.1989 Aug; 57 (8): 2547-52.) exemplary pneumolysosids include a triple mutant of PdT (which is described in Belley A.M. et al (1995); Infection and immunization 63: 1969-74(Berry, A.M.1995) and further purification of the Pneumolysin B.19 and Pneumolyysin by the toxin of Pneumolysaccharide and its binding to Pd-19 (Pneumocyst) and their genetic immunogens are described in Pd-359 J.3519), infection and immunity, 1991, month 7; 59(7): 2297- "Purification and immunization of transgenic microorganisms and their connection to Streptococcus microorganism type 19F" infection Immun.1991Jul; 59 (7): 2297- "304"); rPd2 and rPd3 (Ferela et al, "DNA vaccines based on genetically detoxified pneumolysin derivatives do not protect mice from attack by Streptococcus pneumoniae", "European microbiology Association immunology and medical microbiology (2006) 46: 291-297 (Ferreia et al," DNA vaccines based on genetic testing and microorganisms of genetic testing and infection exchange with Streptococcus pneuma "FEMS Immunol Med Microbiol (2006) 46: 291-297)); ply8, Δ 6Ply, L460D, or a variant thereof. In some embodiments, the pneumolysin has a mutation in the catalytic center, such as at or near amino acid 428 or 433.

Other suitable streptococcus pneumoniae antigens for combination vaccines include pneumococcal surface protein a (pspa); derivatives of PspA, choline binding protein A (CbpA) and derivatives thereof (AD Eurynie et al, "Protection against Streptococcus pneumoniae by immunization with pneumolysin and CbpA"; infection and immunization 10.2001; 69 (10): 5997) -6003(AD Ogunniyi et al, "Protection against infection of Streptococcus pneumoniae by immunization with plasmid and CbpA," infection immunization with plasmid and immunization with pneumolysin and CbpA, "" infection Immun 2001Oct; 69 (10): 5997) -6003); pneumococcal surface adhesin a (psaa); a casein cleaving protease; sortase a (srta); pilus 1RrgA adhesin; ppma; PrtA; PavA; LytA; Stk-PR; PcsB; RrgB and derivatives thereof.

Derivatives of PspA include proline-rich segments with non-proline blocks (PR + NPB, described further below and in denier c.c. et al (2010) Infection and Immunity 78: 2163-72(Daniels, c.c. et al (2010) Infection and Immunity 78: 2163-72)) and related constructs that include all or a fragment of the proline-rich region of PspA (e.g., a region containing one or more of the sequences PAPAP, PKP, PKEPEQ, and PEKP and optionally including a non-proline block). An example of a non-proline block has exemplary sequence EKSADQQAEEDYARRSEEEYNRLTQQQ (SEQ ID NO: 306), which generally lacks proline residues in the additional proline-rich region of the non-frizzled region of PspA. Other examples of non-proline block (NPB) sequences include SEQ ID NO: 307 and 308. PspA and its derivatives may comprise genes that express proline-rich-like structures (i.e., PKP, PKEPEQ, and PEKP) with or without NPB. The amino acid at either end of the NPB marks the boundary of the proline-rich region. In one example, the amino terminal boundary of the PR region is DLKKAVNE (SEQ ID NO: 309) and the carboxy terminal boundary is (K/G) TGW (K/G) QENGMW (SEQ ID NO: 310). Peptides containing NPB are particularly immunogenic, indicating that NPB may be an important epitope. Exemplary immunogenic PspA polypeptide derivatives comprising a coiled coil structure include SEQ ID NO: 301 and 302. Particular embodiments of immunogenic PspA polypeptide derivatives lacking a coiled coil structure have an amino acid sequence shown as seq id NO: 303-305. An immunogenic PspA polypeptide of SEQ ID NO: 301. 303 and 305 contain PR and NPB sequences (PR + NPB). SEQ ID NO: the immunogenic PspA polypeptides of 302 and 304 include only PR sequences (PR only) and lack NPB.

In some cases, another suitable streptococcus pneumoniae antigen has at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to a corresponding wild-type streptococcus pneumoniae protein. The sequences of the above-mentioned polypeptides and the nucleic acids encoding them are known; see, e.g., the streptococcus pneumoniae ATCC700669 whole genome sequence according to genbank accession number FM211187.1 and the polypeptide sequences linked therein.

Other streptococcus pneumoniae antigens for use in combination vaccines include conjugated streptococcus pneumoniae polysaccharides. These conjugated polysaccharides may be, for example, as described in US patent 5,623,057, US patent 5,371,197, or PCT/US 2011/023526.

In addition to those nucleic acids and polypeptides described in table 1 above, the present application also provides immunogenic compositions comprising one or more of the polypeptides or genes listed in table 2, or variants or fragments thereof, as described herein. The DNA and protein sequences of each Gene and protein can be found by searching the locus tags in the public database Entrez Gene as described above.

TABLE 2 immunogenic proteins identified in human and mouse screens

Typically, the polypeptides present in the compounds of the invention are immunogenic, either as such or in variant form, variants including a polypeptide fused to another polypeptide or mixed or complexed with an adjuvant. As described herein, variants also include sequences having less than 100% sequence identity. In certain embodiments, the antigens in table 1 or 2 are provided as full length polypeptides. In addition, fragments, precursors and analogues with appropriate immunogenicity may be used.

These polypeptides may be immunogenic in mammals, for example, in mice, guinea pigs, or humans. Immunogenic polypeptides are typically those that are capable of eliciting a significant 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 example, the immunogenic polypeptide can increase the amount of IL-17 produced by T cells. The IL-17 assay described in examples 1-4 is an example of an assay that can be used to identify immunogenic polypeptides. Alternatively or additionally, the immunogenic polypeptide may (i) induce the production of antibodies, e.g. neutralizing antibodies, that bind to the polypeptide and/or the whole bacterium, (ii) induce T_H17 immunization, (iii) activation of CD4⁺T cell response, e.g. by increasing CD4⁺T cells and/or increase CD4⁺(iii) localization of T cells to sites of infection or reinfection, (iv) activation of CD8⁺CTL responses, e.g. by increasing CD8⁺T cells and/or increase CD8⁺Localization of T cells to sites of infection or re-infection, (v) induction of T_H1 immunization and/or (vi) activation of innate immunity. In some embodiments, the immunogenic polypeptide causes the production of a detectable amount of antibodies specific for that antigen.

In certain embodiments, the polypeptides have less than 20%, 30%, 40%, 50%, 60% or 70% identity to human autoantigens and/or gut commensals (e.g., certain Bacteroides (Bacteroides), Clostridium (Clostridium), Clostridium (Fusobacterium), Eubacterium (Eubacterium), Ruminococcus (Ruminococcus), Peptococcus (Peptococcus), Peptostreptococcus (Peptostreptococcus), Bifidobacterium (Bifidobacterium), Escherichia (Escherichia), and Lactobacillus (Lactobacillus) species). Examples of human autoantigens include insulin, proliferating cell nuclear antigen, cytochrome P450, and myelin basic protein.

The invention also provides an immunogenic composition comprising a pharmaceutically acceptable carrier having an amino acid sequence comprising SEQ ID NO: 265. 266 or 268, or an immunogenic fragment thereof, and one or more polypeptides having an amino acid sequence comprising any one of: SEQ ID NO:1-23 and SP1574, SP1655, SP2106, SP1473, SP0605, SP1177, SP0335, SP0906, SP1828, SP2157, SP1229, SP1128, SP1836, SP1865, SP0904, SP0765, SP1634, SP0418, SP1923, SP1313, SP0775, SP0314, SP0912, SP0159, SP1412 0, SP2148, SP0372, SP1304, SP2002, SP 061061061061061, SP1988, SP0484, SP0847, SP1527, SP0542, SP0441, SP0350, SP0014, SP1965, 0117, SP0981, SP2229, SP2136, SP1179, SP1174, SP2216, SP1393, SP0641.1, SP1384 and SP 2212.

In some embodiments, the vaccine formulation comprises at least two polypeptides, each polypeptide belonging to a different group of (i) - (vii): (i) SEQ ID NO:1 or an immunogenic fragment thereof, (ii) SEQ ID NO:2-5 and 14-17 or an immunogenic fragment thereof, (iii) SEQ ID NO:6-7 and 18-19 or an immunogenic fragment thereof, (iv) one of SEQ ID NOs: 8 or an immunogenic fragment thereof, (v) SEQ ID NO: one of 9-10 and 20-21 or an immunogenic fragment thereof, (vi) SEQ ID NO:11-13, 267 and 269-270 or an immunogenic fragment thereof, and (vii) one of SEQ ID NOs: 265-one of 266 and 268 or an immunogenic fragment thereof. Examples of such combinations are listed below. Additional combinations can be obtained by replacing one of the following sequences with the corresponding consensus sequence (e.g., one of SEQ ID NOs: 14-21 or 268-270). In some embodiments, one of these polypeptides is SEQ ID NO: 265-one of 266 and 268 or an immunogenic fragment thereof. In some embodiments, the vaccine formulation further comprises pneumocolyloid. In some embodiments, the vaccine formulation further comprises CbpA 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:1 and SEQ ID NO:2

SEQ ID NO:1 and SEQ ID NO: 3

SEQ ID NO:1 and SEQ ID NO: 4

SEQ ID NO:1 and SEQ ID NO: 5

SEQ ID NO:1 and SEQ ID NO:6

SEQ ID NO:1 and SEQ ID NO:7

SEQ ID NO:1 and SEQ ID NO:8

SEQ ID NO:1 and SEQ ID NO:9

SEQ ID NO:1 and SEQ ID NO:10

SEQ ID NO:1 and SEQ ID NO:11

SEQ ID NO:1 and SEQ ID NO: 12

SEQ ID NO:1 and SEQ ID NO: 13

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:6

SEQ ID NO:2 and SEQ ID NO:7

SEQ ID NO:2 and SEQ ID NO:8

SEQ ID NO:2 and SEQ ID NO:9

SEQ ID NO:2 and SEQ ID NO:10

SEQ ID NO:2 and SEQ ID NO:11

SEQ ID NO:2 and SEQ ID NO: 12

SEQ ID NO:2 and SEQ ID NO: 13

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:6

SEQ ID NO: 3 and SEQ ID NO:7

SEQ ID NO: 3 and SEQ ID NO:8

SEQ ID NO: 3 and SEQ ID NO:9

SEQ ID NO: 3 and SEQ ID NO:10

SEQ ID NO: 3 and SEQ ID NO:11

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:6

SEQ ID NO: 4 and SEQ ID NO:7

SEQ ID NO: 4 and SEQ ID NO:8

SEQ ID NO: 4 and SEQ ID NO:9

SEQ ID NO: 4 and SEQ ID NO:10

SEQ ID NO: 4 and SEQ ID NO:11

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

SEQ ID NO: 5 and SEQ ID NO:6

SEQ ID NO: 5 and SEQ ID NO:7

SEQ ID NO: 5 and SEQ ID NO:8

SEQ ID NO: 5 and SEQ ID NO:9

SEQ ID NO: 5 and SEQ ID NO:10

SEQ ID NO: 5 and SEQ ID NO:11

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:8

SEQ ID NO:6 and SEQ ID NO:9

SEQ ID NO:6 and SEQ ID NO:10

SEQ ID NO:6 and SEQ ID NO:11

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:8

SEQ ID NO:7 and SEQ ID NO:9

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

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:9

SEQ ID NO:8 and SEQ ID NO:10

SEQ ID NO:8 and SEQ ID NO:11

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:11

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

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 embodiments, the vaccine formulation comprises at least three different polypeptides having a polypeptide sequence comprising seq id NO: 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) seq id NO:2-5 or an immunogenic fragment thereof, (iii) SEQ ID NO:6-7 or an immunogenic fragment thereof, (iv) SEQ ID NO:8 or an immunogenic fragment thereof, (v) SEQ ID NO:9-10 or an immunogenic fragment thereof, (vi) SEQ ID NO:11-13 and 267 or an immunogenic fragment thereof, and (vii) seq id NO: 265-266 or an immunogenic fragment thereof. Examples of such combinations are listed below. Additional combinations can be obtained by replacing one of the following sequences with the corresponding consensus sequence (e.g., one of SEQ ID NOs: 14-21 or 268-270). In some embodiments, one of these polypeptides is SEQ ID NO: 265-one of 266 and 268 or an immunogenic fragment thereof. In some embodiments, the vaccine formulation further comprises pneumocolyloid. In some embodiments, the vaccine formulation further comprises CbpA 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: 1. SEQ ID NO:2 and SEQ ID NO:6

SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:7

SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:8

SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:9

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

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:6

SEQ ID NO: 1. SEQ ID NO: 3 and SEQ ID NO:7

SEQ ID NO: 1. SEQ ID NO: 3 and SEQ ID NO:8

SEQ ID NO: 1. SEQ ID NO: 3 and SEQ ID NO:9

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:6

SEQ ID NO: 1. SEQ ID NO: 4 and SEQ ID NO:7

SEQ ID NO: 1. SEQ ID NO: 4 and SEQ ID NO:8

SEQ ID NO: 1. SEQ ID NO: 4 and SEQ ID NO:9

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:6

SEQ ID NO: 1. SEQ ID NO: 5 and SEQ ID NO:7

SEQ ID NO: 1. SEQ ID NO: 5 and SEQ ID NO:8

SEQ ID NO: 1. SEQ ID NO: 5 and SEQ ID NO:9

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:8

SEQ ID NO: 1. SEQ ID NO:6 and SEQ ID NO:9

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:8

SEQ ID NO: 1. SEQ ID NO:7 and SEQ ID NO:9

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:9

SEQ ID NO: 1. SEQ ID NO:8 and SEQ ID NO:10

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

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

SEQ ID NO: 2. SEQ ID NO:9 and SEQ ID NO:11

SEQ ID NO: 2. SEQ ID NO:9 and SEQ ID NO: 12

SEQ ID NO: 2. SEQ ID NO:9 and SEQ ID NO: 13

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:10

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

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

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

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 N0: 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:8

SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:9

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:8

SEQ ID NO: 5. SEQ ID NO:7 and SEQ ID NO:9

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

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

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

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

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

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

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 polypeptides having sequences comprising 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) SEQ ID NO: 14-17 or an immunogenic fragment thereof, (ii) SEQ ID NO: 18-19 or an immunogenic fragment thereof, (iii) SEQ ID NO: 20-21 or an immunogenic fragment thereof, (iv) SEQ ID NO: 268, or an immunogenic fragment thereof, and (v) one of SEQ ID NOs: 269-279 or an immunogenic fragment thereof. Examples of such combinations are listed below. The following combinations indicate consensus sequences. However, additional combinations can be obtained by replacing one of the consensus sequences with the corresponding non-consensus sequence (e.g., one of SEQ ID NO: 1-13 or 266-267). In some embodiments, one of these polypeptides is SEQ id no: 268 or an immunogenic fragment thereof. In some embodiments, the vaccine formulation further comprises pneumocolyloid. In some embodiments, the vaccine formulation further comprises CbpA 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 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

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

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 SEQ ID NO: 14-21 and 268-270, wherein 1-20 amino acid residues are removed from the N-terminus, C-terminus, or both.

In some embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 14-17, or a pharmaceutically acceptable salt thereof. In some embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ id no: 18-19. In some embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 20-21. In some embodiments, the vaccine formulation comprises a polypeptide having a sequence comprising SEQ ID NO: 268-270 in a pharmaceutically acceptable carrier or excipient.

In some aspects, the polypeptide comprising SEQ ID NO: 14-21, 268, 269 and 270 further comprises a vaccine formulation having a sequence comprising one or more of SEQ ID NOs: 1-13, 265, 266 and 267.

In certain embodiments, the vaccine formulation comprises at least three polypeptides having sequences comprising 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) - (v): (i) SEQ ID NO: 14-17 or an immunogenic fragment thereof, (ii) SEQ ID NO: 18-19 or an immunogenic fragment thereof, and (iii) SEQ ID NO: 20-21 or an immunogenic fragment thereof, (iv) 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 following combinations indicate consensus sequences. However, additional combinations can be obtained by replacing one of the consensus sequences with the corresponding non-consensus sequence (e.g., one of SEQ ID NO: 1-13 or 266-267). In some embodiments, one of these polypeptides is SEQ ID NO: 268 or an immunogenic fragment thereof. In some embodiments, the vaccine formulation further comprises pneumocolyloid. In some embodiments, the vaccine formulation further comprises CbpA 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

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

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

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

The polypeptide may comprise one or more immunogenic portions and one or more non-immunogenic portions. Immunogenic portions can be identified by different methods including protein microarrays, ELISPOT/ELISA techniques, and/or specific analysis of different deletion mutants (e.g., fragments) of the polypeptides under discussion. Immunogenic portions can also be identified by computer algorithms. Some such algorithms, such as EpiMatrix (produced by EpiVax), use a computational matrix approach. Other computational tools for identifying antigenic epitopes include PEPVAC (Promiscuous EPitope-based vaccae), a VACcine hosted on world wide web immunax.dfci.harvard/PEPVAC by Dana Farber Cancer Institute (Dana Farber Cancer Institute), MHCPred (which uses partial least squares and is hosted on world wide web www.jenner.ac.uk/MHCPred by jane Institute), and immune EPitope database algorithms hosted on world wide web animals. An immunogenic fragment of a polypeptide described herein comprises at least one immunogenic portion, as measured experimentally or identified by an algorithm. Peptides identified by the tools described above include the following:

thus, in some aspects, the present application provides immunogenic fragments of the antigens described herein. In some cases, these fragments are of a size that is close to the full-length polypeptide or the polypeptide of table 1 or 2. For example, they may lack up to one, two, three, four, five, ten, twenty, or thirty amino acids at one or both termini. In certain embodiments, the length of the polypeptide is 100-500 amino acids, or 150-450, or 200-400, or 250-250 amino acids. In some embodiments, the length of the polypeptide is 100-200, 150-250, 200-300, 250-350, 300-400, 350-450, or 400-500 amino acids. In certain embodiments, these fragments are produced by processing or partial processing of a signal sequence by an expression host, such as E.coli, an insect cell line (e.g., a baculovirus expression system), or a mammalian (e.g., human or Chinese hamster ovary) cell line. The above fragments or sub-fragments thereof (e.g., fragments of 8-50, 8-30, or 8-20 amino acid residues) preferably have one of the following biological activities, such as increasing the amount of IL-17 released by at least 1.5-fold or 2-fold or more (e.g., as an absolute measure or relative to an immune-inactive protein). The fragments may be used as polypeptides in the vaccines described herein, or may be fused to another protein, protein fragment, or polypeptide.

In some embodiments, the fragment is SEQ ID NO: 1-21 or 265-270 having 1-5, 1-10 or 1-20 amino acid residues removed from the N-terminus, C-terminus or both. In some such embodiments, the same number of residues are removed from the N-terminus and the C-terminus; in yet other embodiments, a different number of residues are removed from the N-terminus as compared to the C-terminus.

In certain aspects, the present application provides immunogenic polypeptides that are at least 90%, 95%, 97%, 98%, 99%, or 99.5% identical to a polypeptide of table 1 or 2. In certain embodiments, the vaccine formulation comprises at least two different polypeptides having a sequence comprising a sequence identical to SEQ ID NO: 1-21 or 265-270, or an immunogenic fragment thereof, with at least 90%, 95%, 98%, or 99% identity to the sequence.

In some embodiments, one or more, e.g., two, three, four, or more polypeptides, or immunogenic fragments or variants thereof, of table 1 or table 2 are provided in a mixture. In some embodiments, the mixture contains the full-length polypeptide and fragments resulting from processing or partial processing of the signal sequence by an expression host, such as e.g., e.coli, an insect cell line (e.g., baculovirus expression systems), or a mammalian (e.g., human or chinese hamster ovary) cell line.

In some embodiments, rather than being in the form of a simple physical mixture, two, three, four or more polypeptides in table 1 or 2, or immunogenic fragments or variants thereof, are covalently bound to each other, e.g., as fusion proteins. In some embodiments, the vaccine formulation is substantially free of other than having a sequence comprising SEQ id no:1-23 or 265-270, or other streptococcus pneumoniae polypeptide. In some embodiments, the vaccine formulation is substantially free of other streptococcus pneumoniae polypeptides other than the polypeptides of table 1. In some embodiments, the vaccine formulation is substantially free of other streptococcus pneumoniae polypeptides other than the polypeptides of tables 1 and/or 2.

In certain embodiments, the vaccine formulation or immunogenic composition is substantially free of polypeptides other than polypeptides having a sequence comprising SEQ ID NO:1-23 or 265-270, or other streptococcus pneumoniae polypeptide. In certain such embodiments, the vaccine formulation or immunogenic composition is substantially free of other than having a sequence consisting of SEQ ID NO:1-23 or 265-270, or other streptococcus pneumoniae polypeptides. In some embodiments, the vaccine formulation or immunogenic composition is substantially free of other streptococcus pneumoniae polypeptides other than polypeptides having an amino acid sequence comprising (or consisting of) any one of the amino acid sequences of the polypeptides of tables 1 and/or 2. Essentially, in this context, it means that 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 other streptococcus pneumoniae polypeptides.

In certain embodiments, the vaccine composition is contacted with T_H17T induced after cell_HThe 17 cell response was at least 1.5 fold higher compared to the response induced by a control group of unrelated antigens (e.g. HSV-2 protein ICP47, with the gene name US 12). In some embodiments, the vaccine formulation inhibits infection by streptococcus pneumoniae in an uninfected subject. In certain embodiments, the vaccine formulation reduces the incidence or duration of nasopharyngeal colonization by streptococcus pneumoniae in an individual infected with streptococcus pneumoniae. In some embodiments, the vaccine formulation inhibits the development of sepsis in an individual infected with streptococcus pneumoniae. In some embodiments, the vaccine formulation inhibits the development of pneumonia, meningitis, otitis media, sinusitis, or other sites or organs of streptococcus pneumoniae infection.

In certain embodiments, the present application provides nucleic acids, such as DNA, RNA, or analogs thereof, that encode one or more of the above polypeptides. The basic DNA sequence of the above-mentioned polypeptide may be modified in such a manner that it does not affect the sequence of the protein product, and such a sequence is included in the present invention. For example, the DNA sequence may be codon optimized for increased expression in a host, such as e.coli, an insect cell line (e.g., using a baculovirus expression system), or a mammalian (e.g., human or chinese hamster ovary) cell line.

In certain embodiments, the present application provides a nucleic acid (e.g., DNA, RNA, or analogs thereof) that is at least 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a gene in table 1 or 2, or a variant or portion of said gene. In some embodiments, the nucleic acid length is 600-. In some embodiments, the nucleic acid is 600-1600, 800-1800, or 1000-2000 nucleotides in length. These nucleic acids can be used, for example, for the recombinant production of the polypeptides in tables 1 and 2 or immunogenic fragments thereof.

In some embodiments, the vaccine or immunogenic composition may comprise a fusion protein and/or a fusion DNA construct. The polypeptides described herein may be used without modification. In certain embodiments, where smaller related polypeptides (e.g., fragments, etc.) are used and have a molecular weight of less than about 5000 daltons (e.g., 1500 to 5000 daltons), modifications thereof can be used to elicit a desired immune response. For example, these smaller polypeptides may be conjugated to a suitable immunogenic carrier, such as tetanus toxoid, pneumolysin, keyhole limpet hemocyanin, and the like.

In certain embodiments, the vaccine formulation comprises at least one lipidated polypeptide. Binding to the lipid moiety may be direct or indirect (e.g., via a linker). The lipid moiety may be synthetic or naturally occurring. In certain embodiments, the polypeptides in table 1 or 2 may be chemically bound to a lipid moiety. In certain embodiments, the construct may comprise a gene or polypeptide in table 1 or 2, or an immunogenic fragment or variant thereof, and a lipidated sequence, including a lipid box motif. The canonical lipid box motif is shown as SEQ ID NO: 274. the lipidation sequence may be at the N-terminus or C-terminus of the protein, and may be embedded in a signal or other sequence, or in a fusion protein. Exemplary lipidation sequences include the signal sequence of SP2108(SEQ ID NO: 275) and the signal sequence of the E.coli gene RlpB (SEQ ID NO: 276). The signal sequence may be, for example, an E.coli or S.pneumoniae signal sequence. Exemplary E.coli signal sequences include the mlpA signal sequence (Lin J. et al, "E.coli mutants having An amino acid variation in the signal sequence of the outer membrane preprolipid"; proceedings of the national academy of sciences USA, 10.1978; 75 (10): 4891-5(Lin, J. et al, "" An Escherichia coli polypeptide with amino acid alteration in the signal sequence of the outer membrane protein "Proc Natl Acad Sci U S A.1978 Oct; 75 (10): 4891-5)), the lamB signal sequence (Amel S. D. et al," Mutations that alter the cellular localization of the Escherichia coli outer membrane protein phage lambda receptor "; proceedings of the national academy of sciences 1978"; 75 "5812-6 (Emameter S. D. et al," mutation of the outer membrane protein phage lambda. 3. Proc Natl Acad Sci U S.A. 1978Dec; 75(12): 5802-6)), an MBP signal sequence (basford p.j., "use of gene fusion to study secretion of maltose binding protein into the periplasm of escherichia coli", "journal of bacteriology, month 7 of 1979; 139(1): 19-31(Bassford, P.J., "Use of gene fusion to stuck session of mass-binding protein in Escherichia coli plasmid" J bacteriol.1979 Jul; 139 (1): 19-31)). Lpp refers to exemplary E.coli signal sequences that direct lipidation (Callun P.A. et al, "Construction and evaluation of plasmid vectors for recombinant lipoprotein expression in E.coli", "plasmids," 1 month 2003; 49 (1): 18-29(Cullen, P.A. 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)). Coli signal sequences which direct lipidation are also described in Riger M. (Legain, M.) et al ("Production of lipidated meningococcal transferrin-binding protein 2in E.coli"; protein expression and purification, 10.1995; 6 (5): 570-8 ("Production of lipidated meningococcal transferrin binding protein 2in Escherichia coli". 1995 Oct.; 6 (5): 570-8), e.g.the signal sequence of the gene RlpB (SEQ ID NO: 276)). Numerous streptococcus pneumoniae signal sequences are known in the art. One such signal sequence is SEQ ID NO: 275.

in other embodiments, the construct may comprise a gene or protein in table 1 or 2, or an immunogenic fragment or variant thereof, and a tag. The tag may be located at the N-terminus or the C-terminus. For example, a tag may be added to a nucleic acid or polypeptide to aid in purification, detection, solubilization, or to impart other desirable properties to the protein or nucleic acid. For example, the purification tag can be a polypeptide, an oligopeptide, or a polypeptide that can be used for affinity purification. Examples include His, GST, TAP, FLAG, myc, HA, MBP, VSV-G, thioredoxin, V5, avidin, streptavidin, BCCP, calmodulin, Nus, S-tag, lipoprotein D, and beta galactosidase. Specific exemplary His tags include HHHHHHHHHH (SEQ ID NO: 32) and MSYYHHHHHH (SEQ ID NO: 33). In other embodiments, the polypeptide is free of a tag (e.g., a protein purification tag) and is purified by a method that is independent of affinity for the purification tag. In some embodiments, the fused portion is short. In some cases, this means that the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids at one or both termini of the polypeptide of table 1 or 2.

B. Immunogenic compositions

The disclosure also provides pharmaceutical compositions comprising immunogenic polypeptides or polynucleotides encoding the immunogenic polypeptides, and a pharmaceutical carrier. Antigens from streptococcus pneumoniae were identified by screening immune cells from mice or healthy donors infected with streptococcus pneumoniae. Since streptococcus pneumoniae is a very common disease and colonization pathogen, these donors are presumed to have been exposed to streptococcus pneumoniae at some time in their lives. Briefly, a library of streptococcus pneumoniae antigens is expressed in bacteria and mixed with Antigen Presenting Cells (APCs). These APCs in turn present polypeptides derived from streptococcus pneumoniae to lymphocytes that have been isolated from mice or from donors. Lymphocyte responses were analyzed for reactivity with streptococcus pneumoniae. Donors and mice immunized with streptococcus pneumoniae develop lymphocytes specific for the streptococcus pneumoniae antigen. Accordingly, the present disclosure encompasses compositions of streptococcus pneumoniae antigens that elicit a strong immune response against infection by streptococcus pneumoniae in immunized or infected mice or humans.

Tables 1 and 2 list the protein sequences and corresponding nucleotide sequences of Streptococcus pneumoniae antigens identified according to the screening methods described herein. These antigens were identified in the screening of mouse and human T cells. In the screening of mouse T cells, the identified antigens were subjected to at least two rounds of screening: whole genome rounds to identify pools of 4 antigens that elicit an immune response; this is followed by a deconvolution run, with individual antigens eliciting an immune response being tested and identified individually from pools identified in the whole genome run. In contrast, in the screening of human T cells, two distinct antigen pools were established, such that the polypeptides were mixed with the polypeptides that differed between the first and second pools. Thus, it is possible to determine which polypeptides are antigens by identifying which polypeptides are in the positive pool in both the first and second sets. Table 1 lists antigens (and variants thereof) identified by one of the above screening methods, which were then further tested in the mouse model described in examples 5-12. Thus, compositions according to the present disclosure may comprise one or two or more of the genes listed in table 1 or 2, or the corresponding gene products.

The immunogenic composition may also comprise a portion of the streptococcal polypeptide, such as a deletion mutant, a truncation mutant, an oligonucleotide, and a peptide fragment. In some embodiments, these portions of the polypeptide are immunogenic. The immunogenicity of a certain portion of a protein is readily determined using the same assay as used to determine the immunogenicity of a full-length protein. In some embodiments, the portion of the polypeptide has substantially the same immunogenicity as the full-length protein. In some embodiments, the immunogenicity is no more than 10%, 20%, 30%, 40%, or 50% less than the immunogenicity of a full-length protein (e.g., the polypeptides of tables 1 and 2). These polypeptide fragments may be, for example, linear, cyclic or side chain-containing.

Some embodiments of the vaccine formulations and immunogenic compositions described herein comprise immunogenic polypeptides (e.g., the polypeptides of tables 1 and 2) that contain a Membrane Translocation Sequence (MTS) to facilitate introduction of the polypeptides into mammalian cells, followed by stimulation of a cell-mediated immune response. Exemplary membrane translocation sequences include the hydrophobic region in the Kaposi fibroblast growth factor signal sequence, the MTS of alpha-, beta-or gamma-synuclein, the third helix of the antennary homeodomain, SN50, the h-region of integrin beta 3, HIVTat, pAntp, PR-39, the honeybee antibacterial peptide abaecin, the honeybee antibacterial peptide apidaecin, Bac5, Bac7, the plasmodium burgeri (p.berghei) CS protein, and those MTS described in U.S. patents 6,248,558, 6,432,680 and 6,248,558.

In certain embodiments, an antigen (e.g., a polypeptide of table 1 or 2) is covalently bound to another molecule. This may, for example, improve the half-life, solubility, bioavailability or immunogenicity of the antigen. Molecules that can be covalently bound to an antigen include carbohydrates, biotin, polyethylene glycol (PEG), polysialic acid, N-propionyl polysialic acid, nucleic acids, polysaccharides, and PLGA. There are many different types of PEG, which range in molecular weight from less than 300g/mol to more than 10,000,000 g/mol. The PEG chains may be linear, pendant or have a comb or star geometry. In some embodiments, the naturally occurring protein form is covalently bound to a moiety that stimulates the immune system. An example of such a moiety is a lipid moiety. In some cases, the lipid moiety is recognized by a Toll-like receptor (TLR), such as TLR-2 or TLR-4, and activates the innate immune system.

C. Antibodies specific for the proteins of tables 1 and 2

Another aspect disclosed herein is an antibody preparation raised against an antigenic composition (e.g., one of the proteins listed in table 1 or 2, or an immunogenic fragment thereof). For example, the disclosure provides a combination of two, three, four, or five antibodies, each antibody recognizing a different protein of table 1 or 2. Comprising any of a variety of antibodies. These antibodies include, for example, polyclonal, monoclonal, recombinant, humanized or partially humanized single chains, fabs, and fragments thereof, and the like. These antibodies may be of any isotype, e.g., IgG, different IgG isotypes such as IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, and the like; and they may be from any animal species that produces these antibodies, including goats, rabbits, mice, chickens, etc. In some embodiments, the Fab molecules are expressed and assembled in a genetically transformed host, such as e. Thus, a lambda vector system can be used to express a population of fabs with a potential diversity equal to or exceeding that of subjects who produced prior antibodies. See Haas et al (1989), Science246, 1275-81(Huse et al (1989), Science246, 1275-81).

Components of vaccines or immunogenic compositions comprising streptococcus pneumoniae antigens or antibodies recognizing these antigens

In certain embodiments, the vaccine or immunogenic composition comprises an antigen and one or more of: adjuvants, stabilizers, buffers, surfactants, controlled release components, salts, preservatives and/or antibodies specific for the antigen.

1. Adjuvant

The vaccine formulations and immunogenic compositions described herein may comprise an adjuvant. Adjuvants can be broadly divided into two categories based on their main mechanism of action: vaccine delivery systems and immunostimulating adjuvants (see, e.g., Singer et al, Current AIDS research 1: 309-20, 2003(Singh et al, Curr. HIV Res.1: 309-20, 2003)). In many vaccine formulations, the adjuvant provides a signal to the immune system such that it reacts to the antigen, and the antigen is required for specificity to drive the response to the pathogen. Vaccine delivery systems are typically particle formulations, e.g., emulsions, microparticles, Immune Stimulating Complexes (ISCOMs), nanoparticles, which may be, e.g., particles and/or matrices, and liposomes. In contrast, immunostimulatory adjuvants are sometimes pathogen-derived and exhibit pathogen-associated molecular patterns (PAMPs), such as Lipopolysaccharide (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate the innate immune system of the cell.

Alternatively, adjuvants can be classified as organic and inorganic. Inorganic adjuvants include aluminum salts such as aluminum phosphate, amorphous aluminum hydroxy phosphate sulfate and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules, including macromolecules. An example of an organic adjuvant is cholera toxin.

Adjuvants can also be classified according to the response they induce. In some embodiments, the adjuvant induces T_H1 cell or T_H2 cells are activated. In other embodiments, the adjuvant induces B cell activation. In yet other embodiments, the adjuvant induces antigen presenting cell activation. These classifications are not mutually exclusive; in some cases, the adjuvant activates more than one cell type.

In certain embodiments, the adjuvant induces T_H17 cells are activated. It can promote CD4⁺Or CD8⁺T cells secrete IL-17. In some embodiments, T is induced_H17 cell activation of adjuvant is in the following analysis to generate at least 2 times, and in some cases 10 times the experimental sample to control ratio of adjuvant. In the assay, the experimenter compared the levels of IL-17 secreted by two cell populations: (1) from the use of said adjuvants and known to induce T_H17, and (2) from animal cells treated with the adjuvant and unrelated (control) polypeptide. Induction of T_H17 the adjuvant for cell activation may cause the cell population (1) to produce more than 2-fold or more than 10-fold more IL-17 than the cell population (2). IL-17 can be measured, for example, by ELISA or ELISPOT. Certain toxins, such as cholera toxin and labile toxin (produced by enterotoxigenic E.coli or ETEC) activate T_HAnd 17, reacting. Thus, in some embodiments, the adjuvant is a toxin. Cholera toxin has been successfully used in mouse models to induce protective immunity with certain polypeptides in table 1 (see examples 5-8). Intel produces one form of labile toxin. Mutant derivatives of labile toxins which are as potent as adjuvants but toxic may also be usedIs significantly lower. Exemplary detoxified mutant derivatives of labile toxins include mutants lacking ADP ribosyltransferase activity. Specific detoxified mutant derivatives of Labile toxins include LTK7 (Dusi et al, "Mutants of E.coli Heat-Labile toxins lacking ADP ribosyltransferase activity as nontoxic mucosal adjuvants"; Proc. Natl. Acad. Sci. USA 92, p.1644 1648, p. 2.1995 (Douce et al, "Mutants of Escherichia coli Heat-Toxin-inhibiting ADP-ribosyltransferase activity act as a nonoxic, microbial add mutations" PNAS Vo1.92, p.1644-1648, February1995) and LTK63 (William et al, "natural imprints caused by Modified Heat-Labile toxins of E.coli (LTK63) provide general Protection against Infectious diseases of Lung," (Journal of immunological diseases, 2004, 173: 7435 (William. 7443, inner mutation of Escherichia coli Heat-inhibiting mutations of Escherichia coli) J. experiment 35, 2004, 173: 7435-7443)), LT-G192 (Du Si et al, "genetically detoxified mutants of heat labile toxins of E.coli", can be used as an oral adjuvant ", infection and immunity, 9 months 1999; 67(9): 4400-6(Douce et al, "genetic modified mutants of heat-stable toxin from Escherichia coli area can be changed to act as organisms" infection Immun.1999 Sep; 67 (9): 4400-6)) and LTR72 ("mucosal adjuvanticity and immunogenicity of novel mutant LTR72 of E.coli heat-labile enterotoxin that partially knocks down ADP ribosyltransferase activity", "J.Imidai.J.Med.1998, 4/6; 187(7): 1123-32 ("Mucosal adjuvancy and immunogenicity of LTR72, a novel mutation of Escherichia coli heat-lipid enterotoxin with partial knockout of ADP-ribosyltransferase activity," J Exp Med.1998Apr 6; 187 (7): 1123-32)).

In some embodiments, the adjuvant comprises a VLP (virus-like particle). One such adjuvant platform alphavirus replicon uses alphavirus to induce T_H17 cells are activated and are activated byProduced by alpha vicx corporation (Alphavax). In certain embodiments of the alphavirus replicon system, an alphavirus can be engineered to express an antigen of interest, a cytokine of interest (e.g., IL-17 or a cytokine that stimulates IL-17 production), or both, and can be produced in a helper cell line. More detailed information can be found in U.S. patent nos. 5,643,576 and 6,783,939. In some embodiments, the vaccine formulation is administered to the patient in combination with a nucleic acid encoding a cytokine.

Certain classes of adjuvants activate toll-like receptors (TLRs) to activate T_HAnd 17, reacting. TLRs are well known proteins found on leukocyte membranes and recognize foreign antigens (including microbial antigens). Administration of known TLR ligands (e.g., in the form of fusion proteins) with an antigen of interest can facilitate development of an immune response specific to the antigen of interest. One exemplary adjuvant that activates TLRs includes monophosphoryl lipid a (mpl). Traditionally, MPL has been produced as a detoxified Lipopolysaccharide (LPS) endotoxin obtained from gram-negative bacteria such as streptococcus minnesota(s). In particular, the sequential acid and base hydrolysis of LPS produces an immunologically active lipid a isolated fraction, which is MPL, and lacks carbohydrate groups and all phosphates other than one present in LPS. Many synthetic TLR agonists (especially TLR-4 agonists) in evans JT et al, "enhancement of antigen-specific immunity by TLR-4ligand MPL adjuvant and ribi.529", review by vaccine experts, month 4 2003; 2(2): 219-29(Evans JT et al, "Enhancement of anti-specific immunological vision of the TLR-4ligands MPL adjuvant and Ribi.529." Expert Rev Vaccines2003 Apr; 2 (2): 219-29). Like MPL adjuvants, these synthetic compounds activate the innate immune system through TLRs. Another type of TLR agonist is a synthetic phospholipid dimer, such as E6020 (ShibanST et al, "E6020: synthetic Toll-like receptor4 agonists as vaccine adjuvants", reviews by vaccine experts, month 10 2007; 6 (5): 773-84(Ishizaka ST et al, "E6020: a synthetic Toll-like receptor4agonist as a vaccine adjuvant," Expert Rev. vaccines.2007 Oct; 6 (5): 773-84)). Is differentTLR agonists (including TLR-4 agonists) of (a) have been produced and/or sold by, for example, the institute for infectious diseases (IRDI), corexa, Esai, Avanti Polar Lipids, Inc, and Sigma Aldrich. Another exemplary adjuvant that activates TLRs comprises a mixture of MPL, Trehalose Dimycolate (TDM), and dioctadecyldimethylammonium bromide (DDA). Another adjuvant that activates TLRs is R848 (resiquimod).

In some embodiments, the adjuvant is or comprises a saponin. Typically, the saponin is a triterpene saponin, such as those isolated from the bark of Quillaja saponaria (Quillaja saponaria). Saponins extracted from biological sources can be further isolated (e.g., by chromatography) to isolate fractions of the extract having optimal adjuvant activity and acceptable toxicity. Typical fractions known to be useful as adjuvants in quillaja extracts are fractions a and C.

A particular form of saponin that may be used in the vaccine formulations described herein is the Immune Stimulating Complex (ISCOM). ISCOMs are a recognized class of adjuvants in the art, which typically comprise a quillaja saponin fraction and a lipid (e.g., cholesterol and a phospholipid, such as phosphatidylcholine). In certain embodiments, the ISCOMs are assembled with a polypeptide or nucleic acid of interest. However, different saponin fractions may be used in different ratios. In addition, the different saponin fractions may be present together in the same particle, or each particle may have essentially only one fraction (such that a given ratio of fractions a and C is produced by mixing particles having different fractions together). In this context, "substantially" means less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or even 1%. These adjuvants may comprise a separate part a and a separate part C, mixed in the following ratio: 70-95A: 30-5C, such as 70A: 30C to 75A: 5C, 75A: 5C to 80A: 20C, 80A: 20C to 85A: 15C, 85A: 15C to 90A: 10C, 90A: 10C to 95A: 5C or 95A: 5C to 99A: 1C.

In certain embodiments, an adjuvant combination is used. Three exemplary adjuvant combinations are MPL and alum, E6020 and alum, and MPL and ISCOM.

The adjuvant may be covalently bound to the antigen. In some embodiments, the adjuvant may comprise a protein that induces an inflammatory response by activating Antigen Presenting Cells (APCs). In some embodiments, one or more of these proteins may be recombinantly fused to a selected antigen such that the resulting fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately enhances antigen presentation to T cells and initiates T cell responses (see Wu et al, Cancer research, 2005; 65(11), 4947-4954 (Wu et al, Cancer Res 2005; 65(11), pp 4947-4954)). In certain embodiments, the polypeptides described herein comprise a fusion protein of Streptococcus pneumoniae surface adhesin A (PsaA) and pULyosoid PdT and cell wall polysaccharides (PsaA: PdT-CPs) in the presence of a trivalent binding system, described in Lu et al ("trivalent binding of fusion protein to cell wall polysaccharide provides Protection against Pneumococcal colonization and deadly pneumonia", infection and immunization, 2009, 5 (77) (5) (2076-83) (Lu et al ("Protection against infection and pathogen colonization by a ternary conjugation of a fusion protein with the cell wall polysaccharide," infection with infection, May, 77 (5): 2076-83)). PdT carries three amino acid substitutions (W433F, D385N and C428G) which render the molecule non-toxic but do not interfere with its TLR-4-mediated inflammatory properties. The combination of polysaccharides and polypeptides with fusions to the TLR-4-agonist PdT results in a greatly enhanced immune response to the polypeptide. In some embodiments, one or more polypeptides described herein are used in place of PsaA in a trivalent conjugate. The trivalent binding system typically includes alum, and is usually administered parenterally. Other exemplary adjuvants that can be covalently bound to the antigen include polysaccharides, pneumolysin, synthetic peptides, lipopeptides, and nucleic acids.

Typically, the same adjuvant or mixture of adjuvants is present in each dose of vaccine. Optionally, however, the adjuvant may be administered with the first vaccine administration, but not administered at the subsequent administration (i.e., booster injection). Alternatively, a strong adjuvant may be administered with the first vaccine administration, and a weaker adjuvant or a lower dose of strong adjuvant may be administered with the subsequent administration. The adjuvant may be administered prior to, concurrently with, or after administration of the antigen to the subject (sometimes within 1, 2,6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are suitable for use in human patients, non-human animals, or both.

2. Other Components of the vaccine or immunogenic composition

In addition to the antigens and adjuvants described above, the vaccine formulation or immunogenic composition may comprise one or more additional components.

In certain embodiments, the vaccine formulation or immunogenic composition may comprise one or more stabilizers, such as sugars (e.g., sucrose, glucose, or fructose), phosphates (e.g., disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or monosodium phosphate), glutamates (e.g., monosodium L-glutamate), gels (e.g., treated gels, hydrolyzed gels, or porcine gels), amino acids (e.g., arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and alkyl esters thereof), inosine, or sodium borate.

In certain embodiments, the vaccine formulation or immunogenic composition comprises one or more buffers, such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the vaccine formulation can be administered in physiological saline (e.g., phosphate buffered physiological saline (PBS)) or distilled water.

In certain embodiments, the vaccine formulation or immunogenic composition comprises one or more surfactants, such as polysorbate 80(Tween80), Triton X-100 (polyethylene glycol tert-octylphenyl ether tert-octylphenoxypolyethoxyethanol 4- (1, 1, 3, 3-tetramethylbutyl) phenyl-polyethylene glycol) (Triton X-100); polyoxyethylene sorbitan monolaurate polyethylene glycol sorbitan monolaurate (TWEEN 20); and 4- (1, 1, 3, 3-tetramethylbutyl) phenol with formaldehyde and ethylene oxide (TYLOXAPOL). The surfactant may be ionic or non-ionic.

In certain embodiments, the vaccine formulation or immunogenic composition comprises one or more salts, such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.

In certain embodiments, a preservative is included in the vaccine formulation or immunogenic composition. In other embodiments, no preservative is used. Preservatives are most commonly used in multi-dose vaccine vials and are less commonly required in single dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.

In certain embodiments, the vaccine formulation or immunogenic composition is a controlled release formulation.

DNA vaccine

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 (e.g., the desired antigen) is produced in the patient. In some embodiments, a nucleic acid vaccine vector comprising an optimized recombinant polynucleotide can be delivered to a mammal (including a human) 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., adenovirus, liposome, papilloma virus, retrovirus, etc.) can be administered directly to a mammal for cell transduction in vivo. The nucleic acid vaccine may be formulated as a pharmaceutical composition for administration in any suitable manner, including parenteral administration. Plasmid vectors typically more efficiently transfer genes to muscle tissue. The possibility of delivering DNA vectors to mucosal surfaces by oral administration has also been reported (PLGA enveloped rotavirus and hepatitis B) and DNA plasmids have been used to introduce genes directly into other tissues. DNA vaccines have been introduced into animals primarily by intramuscular injection, by gene gun delivery, or by electroporation. After being introduced, these plasmids are usually maintained episomally without replication. Expression of the encoded protein has been shown to persist for extended periods, providing stimulation of B and T cells.

In determining the effective dose of the carrier to be administered for the treatment or prevention of infection or other condition, the physician assesses the toxicity of the carrier, the progression of the disease, and the production of anti-carrier antibodies, if any. Generally, for a typical 70 kg patient, the dose equivalent of neat nucleic acid from the vector is about 1 μ g to 1mg, and the dose of vector used to deliver the nucleic acid is calculated to yield an equivalent amount of therapeutic nucleic acid. Administration can be accomplished by single or divided administration. Toxicity and therapeutic efficacy of nucleic acid vaccine vectors can be determined in cell cultures or experimental animals using standard pharmaceutical procedures.

The nucleic acid vaccine may comprise DNA, RNA, modified nucleic acids, or combinations thereof. In some embodiments, the vaccine comprises one or more cloning or expression vectors; for example, a vaccine may comprise a plurality of expression vectors, each expression vector capable of autonomous expression of a nucleotide coding region in mammalian cells to produce at least one immunogenic polypeptide. Expression vectors typically comprise a eukaryotic promoter sequence, such as a nucleotide sequence of a strong eukaryotic promoter, operably linked to one or more coding regions. The compositions and methods herein can involve the use of any particular eukaryotic promoter, and a wide variety are known; such as CMV or RSV promoters. The promoter may be heterologous with respect to the host cell. The promoter used may be a constitutive promoter.

Vectors suitable for use in the compositions and methods of the present application may be circular or linear, single-stranded or double-stranded, and may be plasmids, cosmids, or episomes. In suitable embodiments, each nucleotide coding region is located on a separate vector; it will be appreciated, however, that one or more coding regions may be present on a single vector, and that these coding regions may be under the control of a single or multiple promoters.

Numerous plasmids can be used to produce nucleic acid vaccines. Suitable examples of nucleic acid vaccines employ constructs using plasmids VR1012 (vicat inc., San Diego Calif., San Diego), pcmvi.ubf3/2 (s. johnston, University of Texas), or pcdna3.1 (InVitrogen Corporation, Carlsbad, Calif.) as vectors. In addition, the vector construct may contain an immunostimulatory sequence (ISS), such as an unmethylated dCpG motif, which stimulates the immune system of the animal. The nucleic acid vaccine may also encode a fusion product comprising an immunogenic polypeptide. Plasmid DNA can also be delivered using attenuated bacteria as a delivery system, a method suitable for DNA vaccines for oral administration. Bacteria are transformed with an independently replicating plasmid which is released into the host cell cytoplasm after the attenuated bacteria die in the host cell.

DNA vaccines, including DNA encoding the desired antigen, can be introduced into host cells in any suitable form, including individual fragments, linearized plasmids, circular plasmids, replicable plasmids, episomes, RNA, and the like. Preferably, the gene is contained in a plasmid. In certain embodiments, the plasmid is an expression vector. Individual expression vectors capable of expressing genetic material can be produced using standard recombinant techniques. For general cloning see, e.g., manian pedicel et al, 1985molecular cloning: a Laboratory Manual or DNA Cloning, volumes I and II (D.N. Glovi eds., 1985) (Maniatis et al, 1985Molecular Cloning: A Laboratory Manual or DNA Cloning, Vol.I and II (D.N. Glover, ed., 1985)).

Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral as well as topical, transdermal, by inhalation or suppository, or to mucosal tissues, such as by lavage of vaginal, rectal, urethral, buccal, and sublingual tissues. Typical routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. The gene construct may be administered by means including, but not limited to, conventional syringes, needleless injection devices, "particle bombardment gene guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound. DNA vaccines can be delivered by any method that can be used to deliver DNA, so long as the DNA is expressed and the desired antigen is formed within the cell.

In some embodiments, the DNA vaccine is delivered by known transfection reagents such as cationic liposomes, fluorocarbon emulsions, helical tubules, gold particles, biodegradable microspheres, or cationic polymers. The helical delivery vehicle is a stable phosphoiipid calcium precipitant consisting of phosphatidylserine, cholesterol, and calcium; such non-toxic and non-inflammatory transfection reagents may be present in the digestive system. Biodegradable microspheres include polymers such as poly (lactide-co-glycolide), polyesters, which can be used to create microcapsules of DNA for transfection. Lipid-based microtubules usually consist of two spirally wound lipid layers, which are packaged by their edges connected to each other. When a thin tube is used, the nucleic acid can be arranged in its central hollow portion for delivery and controlled release into the animal.

In some embodiments, the DNA vaccine is delivered to the mucosal surface via microspheres. Bioadhesive microspheres can be prepared using different techniques and can be adapted to adhere to any mucosal tissue, including those found in the eye, nasal cavity, urinary tract, colon and gastrointestinal tract, providing the possibility of localized and systemic controlled release of vaccines. The administration of bioadhesive microspheres to specific mucosal tissues can also be used to localize the vaccine effect. In some embodiments, another method for mucosal vaccine delivery is direct administration to the mucosal surface of a plasmid DNA expression vector encoding a specific protein antigen gene.

The DNA plasmid vaccine according to the present invention is formulated according to the mode of administration to be used. In some embodiments where the DNA plasmid vaccines are injectable compositions, they are sterile and/or pyrogen-free and/or particle-free. In some embodiments, isotonic formulations are preferably used. In general, isotonic additives may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some embodiments, isotonic solutions, such as phosphate buffered saline, are preferred. In some embodiments, the stabilizing agent comprises a gel and a albumin. In some embodiments, a vasoconstrictor is added to the formulation. In some embodiments, a stabilizer, such as LGS or other polycation or polyanion is added to the formulation that makes the formulation stable at room or ambient temperature for an extended period of time.

In some embodiments, the DNA vaccine may further comprise a pharmacologically acceptable carrier or diluent. Suitable carriers for vaccines are well known to those of ordinary skill in the art and include, but are not limited to, proteins, sugars, and the like. These carriers may be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous carriers are 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 physiological saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on ringer's dextrose), and the like. Preservatives and antimicrobials include antioxidants, chelating agents, inert gases and the like. Preferred preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B.

Another method of delivering nucleic acids into animals involves the use of viral or bacterial vectors. Examples of suitable viral vectors include adenovirus, poliovirus, poxviruses (such as vaccinia, canarypox, and avipox), herpesviruses (including catfish herpesvirus), adenovirus-related vectors, and retrovirus. Exemplary bacterial vectors include attenuated forms of Salmonella (Salmonella), shigella (shigella), Edwardsiella ictaluri (edwards iella ictaluri), Yersinia ruckeri (Yersinia ruckerii), and Listeria monocytogenes (Listeria monocytogenes). In some embodiments, the nucleic acid is a vector, such as a plasmid, capable of autonomous expression of a nucleotide sequence encoding an immunogenic polypeptide.

E. Use of vaccines

The streptococcus pneumoniae vaccines described herein can be used for prophylactic and/or therapeutic treatment of streptococcus pneumoniae. Accordingly, the present application provides a method for treating a subject suffering from or susceptible to infection by streptococcus pneumoniae, the method comprising administering an effective amount of any one of the vaccine formulations described herein. In some aspects, the method inhibits colonization of streptococcus pneumoniae in an individual. In some aspects, the methods inhibit a symptom or sequelae of streptococcus pneumoniae, such as sepsis. The subject receiving the vaccination may be male or female, and may be a child or adult. In some embodiments, the subject 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 subject to induce an immune response that can help provide protection against the establishment of streptococcus pneumoniae, for example, by protecting against colonization, which is the first and necessary step in the disease. Thus, in some aspects, the methods inhibit infection by streptococcus pneumoniae in an uninplantated or uninfected subject. In another aspect, the method can reduce the duration of colonization in the colonized individual.

In some embodiments, the vaccine compositions of the present invention confer protective immunity, allowing vaccinated individuals to exhibit symptoms or sequelae that are delayedLate onset, or a reduction in the severity of symptoms or sequelae, as a result of his or her exposure to the vaccine. In certain embodiments, the severity of the symptoms or sequelae is reduced by at least 25%, 40%, 50%, 60%, 70%, 80%, or even 90%. In particular embodiments, the vaccinated individual may show no symptoms or sequelae, be colonized by streptococcus pneumoniae, or both, after exposure to streptococcus pneumoniae. Protective immunity is typically achieved by one or more of the following mechanisms: mucosal, humoral, or cellular immunity. Mucosal immunity is primarily a result of secretory iga (siga) antibodies on mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts. Iga antibodies are produced following a series of events mediated by antigen-treated cells, B and T lymphocytes, which result in the production of iga by B lymphocytes of mucosal lining tissues in vivo. Humoral immunity is typically the result of IgG and IgM antibodies in the serum. Cellular immunity can be achieved by cytotoxic T lymphocytes, or by delayed-type hypersensitivity reactions involving macrophages and T lymphocytes, as well as other mechanisms involving T cells without the need for antibodies. In particular, cellular immunity may be mediated by T_H1 or T_H17 cell mediation.

Essentially any individual has some risk of contracting streptococcus pneumoniae. However, some subpopulations have an increased risk of infection. In some embodiments, a vaccine formulation described herein (e.g., a composition comprising one or more of the polypeptides in table 1 or 2, or nucleic acids encoding such polypeptides, or antibodies reactive with such polypeptides) is administered to an immunocompromised patient.

Immune dysfunction pathology caused by medical treatment is likely to expose the subject under discussion to a higher risk of infection with streptococcus pneumoniae. It is possible to prophylactically treat an infection in an individual with an immunocompromised state before or during a treatment known to impair immune function. Before or during a treatment known to impair immune function, it is possible to prevent subsequent infection by streptococcus pneumoniae or to reduce the risk of an individual suffering from infection due to an immunocompromised state by prophylactic treatment with an antigen composition (e.g., two or more antigens in table 1 or 2, or nucleic acids encoding such antigens), or with antibodies reactive with two or more antigens in table 1 or 2. If an individual is suffering from a streptococcus pneumoniae infection, for example, after a treatment leading to an immunocompromised state, it is also possible to treat the infection by administering an antigenic composition to the individual.

The risk of pneumococcal disease or its complications is increased in the following groups and therefore subjects within the scope of one or more of these groups are eligible to receive the vaccine formulations described herein: children, especially those from 1 month to 5 years or from 2 months to 2 years of age; a child aged at least 2 years with splenomegaly, spleen dysfunction, or sickle cell disease; children at least 2 years of age with nephrotic syndrome, chronic cerebrospinal fluid leakage, HIV infection, or other conditions associated with immunosuppression.

In another embodiment, the at least one dose of the pneumococcal antigen composition is administered to an adult at increased risk of pneumococcal disease or complications thereof in each of the following groups: all persons 65 years of age; an adult with splenomegaly, spleen dysfunction, or sickle cell disease; an adult human having the following conditions: chronic cardiopulmonary disease, cirrhosis, alcoholism, chronic kidney disease, nephrotic syndrome, diabetes, chronic cerebrospinal fluid leakage, HIV infection, AIDS, and other conditions associated with immunosuppression (Hodgkin's disease), lymphoma, multiple myeloma, immunosuppression for organ transplantation); an individual having a cochlear implant; individuals with long-term health problems (such as heart and lung disease); and individuals who are taking any medication or treatment that reduces the body's resistance to infection (e.g., long-term steroids, certain cancer drugs, radiation therapy); the Alaska indigenous population and some of the American indigenous population.

2. Therapeutic use

In some therapeutic applications, a vaccine may be administered to a patient suffering from a streptococcus pneumoniae infection in an amount sufficient to treat the patient. In this context, treating a patient refers to reducing the symptoms and/or bacterial load and/or sequelae of streptococcus pneumoniae in an infected individual. In some embodiments, treating the patient refers to reducing the duration of the symptoms or sequelae, or reducing the intensity of the symptoms or sequelae. In some embodiments, the vaccine reduces the transmissibility of streptococcus pneumoniae in vaccinated patients. In certain embodiments, the above reduction is at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%.

In therapeutic embodiments, the vaccine is administered to an individual after infection. The vaccine may be administered shortly after infection, e.g., before symptoms or sequelae manifest, or may be administered during or after symptoms or sequelae manifest.

A therapeutic streptococcus pneumoniae vaccine may reduce the intensity and/or duration of different symptoms or sequelae of streptococcus pneumoniae infection. The symptoms or sequelae of streptococcus pneumoniae infection can take a variety of forms. In some cases, infected patients develop pneumonia, acute sinusitis, otitis media (ear infections), meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brain abscess.

Sepsis is a rare but life-threatening complication of streptococcus pneumoniae infection in which bacteria invade the bloodstream and produce systemic inflammation. Typically, fever is observed and white blood cell counts increase. Further description of sepsis is found in goddestan b. et al, "international conference on pediatric sepsis consensus: definition of pediatric sepsis and organ dysfunction "," pediatric critical care medicine ", 1 month 2005; 6(1): 2-8(Goldstein, B.et al, "International peer therapy session consensus: definitions for sessions and organ dysfunction in peers," Peer Crit Care Med. Jan 2005; 6 (1): 2-8).

3. Analysis of vaccination efficacy

In addition to the clinical results described above, the efficacy of vaccination with the vaccines disclosed hereinCan be determined in a number of ways. First, we can analyze IL-17 levels (in particular IL-17A) by stimulating subject-derived T cells after vaccination. These IL-17 levels can be compared to the IL-17 levels in the same subject prior to vaccination. Increased levels of IL-17 (e.g., IL-17A), such as 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more, will indicate an increased response to the vaccine. Alternatively (or in combination), we can analyze the amount of pneumococcal kill of neutrophils in the presence of T cells or antibodies from the patient. An increase in pneumococcal killing, such as a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, would indicate an increased response to the vaccine. In addition, we can measure T_H17 cell activation, wherein T_H17 an increased cell activation, such as a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, is associated with an increased response to the vaccine. We can also measure the level of antibodies specific for a vaccine, wherein an increase in the level of specific antibodies, such as a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, is associated with an increase in vaccine efficacy. In certain embodiments, two or more of these assays are used. For example, we can measure the level of IL-17 and the level of vaccine-specific antibodies. Alternatively, we can follow epidemiological markers such as the incidence, severity or duration of pneumococcal infection in vaccinated individuals compared to unvaccinated individuals.

Vaccine efficacy can also be analyzed in different model systems, such as mouse models. For example, mice of the BALB/C or C57BL/6 strain may be used. After administering the test vaccine (in single or multiple dose form) to the subject, the experimenter administers an challenge dose of streptococcus pneumoniae. In some cases, the challenge dose administered intranasally is sufficient to cause colonization (particularly nasal colonization) by streptococcus pneumoniae in unvaccinated animals; and in some cases, the challenge dose given by inhalation is sufficient to cause sepsis and high mortality in unvaccinated animals. We can then measure the reduction in colonization or reduction in mortality in vaccinated animals. Examples 5-8 and 10 show the efficacy of the polypeptides of table 1 in inhibiting nasal colonization by streptococcus pneumoniae in a mouse model following intranasal challenge. Examples 11 and 12 show the efficacy of the polypeptides of table 1 in providing protection from sepsis and death following infection of streptococcus pneumoniae by inhalation in a mouse model.

G. Use of immunogenic compositions

1. Defense against streptococcus pneumoniae infection

The immunogenic compositions of the present disclosure are designed to elicit an immune response against streptococcus pneumoniae. The compositions described herein (e.g., compositions comprising one or more polypeptides of tables 1 or 2 or nucleic acids encoding such polypeptides) can stimulate an antibody response or a cell-mediated immune response, or both, in a mammal to which they are administered. In some embodiments, the composition stimulates T_H1 offset CD4⁺T cell response, T_H17 offset CD4⁺T cell response and/or CD8⁺T cell response. In some embodiments, the composition stimulates an antibody response. In some embodiments, the composition stimulates T_H1 offset CD4⁺T cell response, T_H17 offset CD4⁺T cell response and/or CD8⁺T cell responses and antibody responses.

In certain embodiments, the compositions (e.g., compositions comprising one or more of the polypeptides of tables 1 or 2, or nucleic acids encoding such polypeptides, or antibodies reactive with such polypeptides) include a cytokine or nucleotide coding region encoding a cytokine (e.g., IL-17), providing additional stimulation of the mammalian immune system. In certain embodiments, the composition comprises a cytokine, such as IL-17.

While not wishing to be bound by theory, in some embodiments, T_H17 cellular responses are desirable in setting up immune responses to compositions disclosed herein, e.g., comprising one or more of tables 1 or 2A composition of a plurality of polypeptides. In certain embodiments, effective T_HThe 17 response is beneficial for clearing pneumococcal infection. For example, mice lacking the IL-17A receptor showed a reduced whole cell-based vaccine protection against pneumococcal challenge (Lu et al, "Interleukin-17A mediated acquired immunity against pneumococcal colonization", "public science library. pathogens," 2008. 9.19.; 4(9)) (Lu et al, "Interleukin-17A mediators acquired immunity to pneumococcal metabolism," PLoS Pathology.2008Sep19; 4 (9)).

Accordingly, provided herein are methods of increasing IL-17 production by administering to a subject a composition described herein (e.g., a composition comprising one or more polypeptides of table 1 or 2). In addition, the present application provides for activating T by administering the composition to a subject_H17 cells. In certain embodiments, an increase in IL-17A levels increases the amount of pneumococci killed by neutrophils or neutrophil-like cells, e.g., by inducing recruitment and activation of neutrophils or neutrophil-like cells. In certain embodiments, such pneumococcal killing is independent of antibody and complement. However, specific antibody production and complement activation may be useful additional mechanisms that help clear pneumococcal infection.

Immunogenic compositions comprising immunogenic polypeptides or polynucleotides encoding these immunogenic polypeptides and a pharmaceutical carrier are also provided.

In some cases, the immunogenic composition comprises a polypeptide encoding SEQ ID NO: 1-13, 265, 266, and 267, as determined from one or more of SEQ ID nos: 24-31, 271, 272, and 273. In some embodiments, the nucleic acids are expressed in an immunized individual to produce an encoded streptococcus pneumoniae antigen, and the streptococcus pneumoniae antigen so produced can produce an immunostimulatory effect in the immunized individual.

Such immunostimulatory nucleic acid-containing compositions can include, for example, an origin of replication and a promoter that drives a promoter encoding the amino acid sequence of SEQ ID NO: 1-13, 265, 266, and 267. Such compositions may also comprise a bacterial plasmid vector into which a promoter (sometimes a strong viral promoter) is inserted, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1-13, 265, 266, and 267, and a polyadenylation/transcription termination sequence. In some cases, the nucleic acid is DNA.

H. Diagnostic use

The present application provides, inter alia, a rapid, economical, sensitive and specific method for detecting streptococcus pneumoniae in a patient. In this respect, it would be beneficial for all hospitals and physicians to examine and treat patients with or at risk of having a streptococcus pneumoniae infection. The test kit can be simple enough to be constructed in any local hospital laboratory, and the antibody and antigen-binding portions thereof can be readily tailored to all hospitals for the treatment of patients having or at risk of having a streptococcus pneumoniae infection. As used herein, "patient" refers to an individual (e.g., a human) having a streptococcus pneumoniae infection or having the potential to suffer a streptococcus pneumoniae infection. The patient may be an individual (e.g., a human) having a streptococcus pneumoniae infection, having a likelihood of suffering from a streptococcus pneumoniae infection, having recovered from a streptococcus pneumoniae infection, and/or having an unknown infection status.

In some embodiments, we can perform a diagnostic assay using two or more antibodies, each antibody binding to one of the antigens of table 1 or 2 to detect streptococcus pneumoniae in an individual. In some embodiments, one of these antigens is SEQ ID NO: 265. 266 or 268. The disclosure also provides methods of analyzing a phenotype of a biological sample from a patient suspected of having a streptococcus pneumoniae infection: (a) obtaining a biological sample from a patient; (b) contacting the sample with two or more streptococcus pneumoniae specific antibodies or antigen binding portions thereof under conditions that allow the antibodies or antigen binding portions to bind to an epitope of streptococcus pneumoniae; wherein binding indicates the presence of Streptococcus pneumoniae in the sample. In some embodiments, the binding to the biological sample and the binding of the same antibody to a negative control tissue are compared, wherein the patient is identified as likely to have a streptococcus pneumoniae infection if the biological sample shows the presence of streptococcus pneumoniae compared to the negative control tissue. In some cases, binding of one antibody indicates the presence of streptococcus pneumoniae; in other cases, binding of two or more antibodies indicates the presence of streptococcus pneumoniae. The above test may be suitably adapted to detect other bacterial infections, for example by using antibodies immunologically active against homologues of one of the proteins described in table 1 (from another bacterial species). In some embodiments, antibodies raised against the streptococcus pneumoniae proteins in table 1 or 2 will also bind to homologues in another streptococcus species, particularly when these homologues have a high percentage of sequence identity.

Alternatively, we can use the antigens of Table 1 or 2 (e.g., SEQ ID NOS: 265, 266, or 268) to detect anti-Streptococcus pneumoniae antibodies in an individual. The disclosure also provides methods of analyzing a phenotype of a biological sample from a patient suspected of having a streptococcus pneumoniae infection: (a) obtaining a biological sample from a patient; (b) contacting the sample with two or more streptococcus pneumoniae specific antigens or portions thereof selected from table 1 or 2 under conditions that allow binding of the antigens (or portions thereof) to any host antibodies present in the sample; wherein binding indicates the presence of anti-streptococcus pneumoniae antibodies in the sample. In some embodiments, the binding to a biological sample and the binding of the same antigen to a negative control tissue are compared, wherein if the biological sample shows the presence of anti-streptococcus pneumoniae antibodies as compared to the negative control tissue, the patient is identified as likely to have (1) a streptococcus pneumoniae infection, or (2) a streptococcus pneumoniae infection in the past. In some cases, detection of one antibody indicates that streptococcus pneumoniae is currently or previously infected; in other cases, detection of two or more antibodies indicates that streptococcus pneumoniae has been infected now or in the past. The above tests may be suitably adapted to detect other bacterial infections, for example by using homologues of the proteins described in table 1 (from another bacterial species (e.g. streptococcal species)).

In some embodiments, the immune cell response of mammalian cells can be quantified in vitro. Methods for such quantification include administering a composition disclosed herein to mammalian T cells in vitro, and quantifying changes in the amount of cytokines produced by the mammalian T cells in response to the composition. In these methods, the cytokine can be, for example, IL-17.

The binding of an anti-Streptococcus pneumoniae antibody to an antigen (e.g., a polypeptide of Table 1 or 2, such as SEQ ID NO:265, 266 or 268) can be measured using any suitable method. These methods include ELISA (enzyme linked immunosorbent assay), western blotting, competition assays, and dot hybridization. The detection step may be, for example, chemiluminescence, fluorescence, or colorimetric analysis. One suitable method for measuring antibody-protein binding is the Luminex xMAP system, in which a polypeptide is bound to microspheres containing a dye. Some systems, including the xMAP system, can measure several different labels in a multiplexed manner and can be used to measure antibody levels at once. In some embodiments, other systems are used to analyze multiple markers in a multiplexed manner. For example, feature analysis (profiling) may be performed using any of the following systems: antigen microarrays, bead microarrays, nano-barcode particle technology, arrayed proteins from cDNA expression libraries, protein in situ arrays, protein arrays of live transformants, universal protein arrays, lab-on-a-chip microfluidics, and peptides on pins (peptides on pins). Another type of clinical assay is the chemiluminescence assay, which detects antibody binding. In some such assays, including the vitreos Eci anti-HCV assay, antibodies are bound to a solid support composed of microparticles in liquid suspension, and a surface fluorometer is used to quantify the fluorescent product produced by the enzyme.

In some embodiments, if the biological sample shows the presence of streptococcus pneumoniae (e.g., by detecting one or more polypeptides of table 1 or 2, such as SEQ ID NOs 265, 266, or 268, or an antibody that binds to one of the polypeptides), we can administer to the patient a therapeutically effective amount of the compositions and therapies described herein. The biological sample may include, for example, blood, semen, urine, vaginal fluid, mucus, saliva, stool, urine, cerebrospinal fluid, or a tissue sample. In some embodiments, the biological sample is an organ intended for transplantation. In certain embodiments, prior to the detecting step, the biological sample is subjected to culture conditions that promote the growth of streptococcus pneumoniae.

The diagnostic tests herein (e.g., those that detect the polypeptides of Table 1 or 2, such as SEQ ID NOS: 265, 266, or 268, or an antibody that binds one of the polypeptides) can be used to detect Streptococcus pneumoniae in a variety of samples, including samples taken from patients and samples obtained from other sources. For example, diagnostic tests may be used to detect streptococcus pneumoniae: food, beverage or a component of food and beverage; articles such as medical instruments, medical devices (e.g., cochlear implants and pacemakers), shoes, clothing, furniture (including hospital furniture), and drapes (including hospital drapes); or a sample taken from the environment (e.g., a plant sample). In some embodiments, the tests herein can be performed on samples taken from animals, such as agricultural animals (cattle, pigs, chickens, goats, horses, etc.), companion animals (dogs, cats, birds, etc.) or wild animals. In certain embodiments, the tests herein may be performed on samples taken from cell cultures, such as human cell cultures producing therapeutic proteins, bacterial cultures intended to produce useful biomolecules, or cell cultures grown for research purposes.

The present disclosure also provides a method of determining the location of a streptococcus pneumoniae infection in a patient, the method comprising: (a) administering to a patient a pharmaceutical composition comprising a tagged streptococcus pneumoniae antibody, or antigen binding portion thereof, and (b) detecting the tag, wherein binding indicates that streptococcus pneumoniae infection is in a particular location in the patient. Such diagnosis may also include comparing the level of binding of the patient to a control group. In certain embodiments, the method further comprises, if the patient has a streptococcus pneumoniae infection, treating the infection by administering to the patient a therapeutically effective amount of a streptococcus pneumoniae binding antibody, or antigen binding portion thereof. In certain embodiments, the method further comprises, if the patient has a streptococcus pneumoniae infection, treating the infection by administering to the patient a therapeutically effective amount of a streptococcus pneumoniae protein, or immunogenic portion thereof, of table 1 or 2. The method may further comprise determining the location and/or volume of streptococcus pneumoniae in the patient. The method may be used to assess the spread of streptococcus pneumoniae in a patient and determine whether local therapy is appropriate.

In some embodiments, the anti-streptococcus pneumoniae antibodies or T cells described herein can be used to make a prognosis of the course of infection. In some embodiments, the anti-streptococcus pneumoniae antibodies or T cells herein can be detected in a sample taken from a patient. If antibodies or T cells are present at normal levels, this will indicate that the patient has developed an immune response against Streptococcus pneumoniae. If antibodies or T cells are not present, or are present at a reduced level, this will indicate that the patient is unable to produce an adequate response against Streptococcus pneumoniae and a more aggressive treatment needs to be recommended. In some embodiments, the presence of reduced levels of antibodies or T cells refers to the presence of antibodies at levels that are 50%, 20%, 10%, 5%, 2%, or 1% less than the levels of antibodies or T cells typical in patients with a normal immune system. Antibodies can be detected by affinity for any of the antigens described herein (e.g., those in tables 1 and/or 2), e.g., using ELISA. T cells can be detected by in vitro reaction to any of the antigens described herein (e.g., those in tables 1 and/or 2), for example, using ELISA or ELISPOT assays.

In some embodiments, detection of specific Streptococcus pneumoniae antigens (e.g., those in tables 1 and/or 2, such as SEQ ID NOs: 265, 266, or 268) can be used to predict the progression and symptoms of Streptococcus pneumoniae infection in a patient. Those skilled in the art will appreciate that the methods herein are not limited to the detection of streptococcus pneumoniae. Other embodiments include detecting related bacteria, including bacteria having proteins homologous to the proteins described in table 1 or 2. These related bacteria include other strains such as streptococcus.

I. Dosage and route of administration

1. Dosage form, dosage and timing

The amount of antigen in each dose of vaccine or immunogenic composition is selected to be an effective amount that induces a prophylactic or therapeutic response as described above, either in a single dose or over multiple doses. Preferably, the dose is free of significant adverse side effects in a typical vaccinee. This amount will vary depending on which particular antigen is employed. In general, it is expected that a dose will contain 1-1000. mu.g of each protein, in some cases 2-100. mu.g, for example 4-40. mu.g. In some aspects, the vaccine formulation comprises 1-1000 μ g of the polypeptide and 1-250 μ g of the adjuvant. In some embodiments, the appropriate amount of antigen to be delivered will depend on the age, weight, and health (e.g., immune compromised state) of the subject. When present, the adjuvant will typically be present in an amount of from 1. mu.g to 250. mu.g, for example from 50 to 150. mu.g, from 75 to 125. mu.g or 100. mu.g per dose.

In some embodiments, only one dose of vaccine is administered to achieve the above results. In other embodiments, after the primary vaccination, the subject receives one or more booster vaccinations for a total of two, three, four, or five vaccinations. Advantageously, the number is three or less. Booster vaccinations may be administered, for example, about 1 month, 2 months, 4 months, 6 months or 12 months after the primary vaccination, such that one vaccination regimen involves administration at 0, 0.5-2 and 4-8 months. It may be advantageous to administer separate doses of the vaccine, which doses may be administered by the same or different routes.

The vaccines and immunogenic compositions described herein may be in a variety of dosage forms. In certain embodiments, the composition is provided in solid or powder (e.g., lyophilized) form; it may also be provided in solution form. In certain embodiments, the dosage form is provided as a dose of the lyophilized composition and at least one separate diluent in a sterile container.

In some embodiments, the compositions will be administered in an ascending dose manner, such that compositions administered sequentially contain a higher concentration of the composition than previously administered. In some embodiments, the compositions will be administered in such a way that successively administered compositions contain lower concentrations than previously administered.

In therapeutic applications, a composition is administered to a patient suffering from a disease in an amount sufficient to treat the patient. Therapeutic applications of the compositions described herein include reducing transmission, slowing disease progression, reducing bacterial viability or replication, or inhibiting expression of proteins required for toxicity, such as reducing levels of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% that would occur in an individual not treated with the composition.

In prophylactic embodiments, the compositions are administered to a human or other mammal to induce an immune response that can inhibit the establishment of an infectious disease or other condition. In some embodiments, the composition may partially block the bacteria from establishing an infection.

In some embodiments, these compositions are administered in combination with an antibiotic. Such co-administration is particularly appropriate when administering the pharmaceutical composition to a patient who has recently been exposed (or suspected of having recently been exposed) to streptococcus pneumoniae. Various antibiotics are used to treat pneumococcal infections, including penicillin, amoxicillin/clavulanic acid, cefuroxime, cefotaxime, ceftriaxone, and vancomycin. Appropriate antibiotics can be selected based on the type and severity of infection and any known antibiotic resistance of the infection (Yatenus MR, "Drug-resistant Streptococcus pneumoniae: rational antibiotic selection", J.Med.3.5 1999; 106 (5A): 19S-25S (Jacobs MR "Drug-resistant Streptococcus pneumoniae: rational antibiotic choice" Am J. Med.1999May 3; 106 (5A): 19S-25S)).

2. Route of administration

The vaccine formulations and pharmaceutical compositions herein can be delivered by administration to an individual, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, subdermal, transdermal, intracranial, intranasal, transmucosal, transanal, vaginal, oral, buccal routes or they can be inhaled) or they can be administered by topical administration. In some embodiments, the route of administration is intramuscular. In other embodiments, the route of administration is subcutaneous. In yet other embodiments, the route of administration is transmucosal. In certain embodiments, the route of administration is through the dermis or intradermally.

Certain routes of administration are particularly suitable for vaccine formulations and immunogenic compositions comprising the indicated adjuvants. In particular, dermal administration is one suitable route of administration for streptococcus pneumoniae vaccines that include a toxin (e.g., cholera toxin or labile toxin); in other embodiments, the administration is intranasal. Vaccines formulated with alphavirus replicons may be administered, for example, by intramuscular or subcutaneous routes. Vaccines comprising monophosphoryl lipid a (mpl), Trehalose Dimycolate (TDM) and dioctadecyl dimethyl ammonium bromide (DDA) are suitable for (inter alia) intramuscular and subcutaneous administration. The vaccine comprising resiquimod may be administered, for example, topically or subcutaneously.

3. Preparation

The vaccine formulation or immunogenic composition may be suitable for administration to a human patient, and the preparation of the vaccine or immunogenic composition may follow USFDA guidelines. In some embodiments, the vaccine formulation or immunogenic composition is suitable for administration to a non-human animal. In some embodiments, the vaccine or immunogenic composition is substantially free of endotoxin or exotoxin. Endotoxins may include pyrogens, such as Lipopolysaccharide (LPS) molecules. The vaccine or immunogenic composition may also be substantially free of inactive protein fragments that may cause fever or other side effects. In some embodiments, the composition contains less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% endotoxin, exotoxin, and/or inactivated protein fragment. In some embodiments, the vaccine or immunogenic composition has a lower level of pyrogens than industrial, tap 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 pyrogen may be inactivated or destroyed prior to administration to the patient. Vaccine materials such as water, buffers, salts and other chemicals can also be screened and pyrogen removed. All materials in the vaccine can be sterile, and each batch of vaccine can be tested for sterility. Thus, in certain embodiments, the endotoxin level in the vaccine is below the level set by the USFDA, e.g., the product of an intrathecal injection composition is 0.2 Endotoxin (EU)/kg; the product of the non-intrathecal injection composition is 5EU/kg and sterile water is 0.25-0.5 EU/mL.

In certain embodiments, the formulation comprises less than 50%, 20%, 10%, or 5% (by dry weight) contaminating protein. In certain embodiments, the desired molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins that may substantially mask, diminish, confound, or alter the properties of the component proteins as purified preparations or in their function in the subject reconstituted mixture). In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of the same type of biological macromolecules are present (although water, buffers, and other small molecules, particularly molecules having a molecular weight of less than 5000, may be present). In some embodiments, vaccines or immunogenic compositions comprising purified subunit proteins contain less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of the proteins from host cells expressing these subunit proteins relative to the amount of purified subunit. In some embodiments, the desired polypeptide is substantially free of nucleic acids and/or carbohydrates. For example, in some embodiments, the vaccine or immunogenic composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNA and/or RNA. In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dry weight) of the same type of biological macromolecule is present in the formulation (although water, buffers, and other small molecules, particularly molecules having a molecular weight of less than 5000, may be present).

It is preferred that the vaccine or immunogenic composition has low or no toxicity within a reasonable risk-benefit ratio. In certain embodiments, the vaccine or immunogenic composition comprises a concentration that is lower than the LD of the vaccinated animal₅₀The composition of the measured values. LD₅₀Measurements can be obtained in mouse or other experimental model systems and extrapolated to humans and other animals. For assessing LD of compounds in humans and other animals₅₀Methods of (a) are well known in the art. The vaccine formulation or immunogenic composition and any component thereof may have a LD greater than 100g/kg, greater than 50g/kg, greater than 20g/kg, greater than 10g/kg, greater than 5g/kg, greater than 2g/kg, greater than 1g/kg, greater than 500mg/kg, greater than 200mg/kg, greater than 100mg/kg, greater than 50mg/kg, greater than 20mg/kg or greater than 10mg/kg in rats₅₀The value is obtained. Vaccine formulations or immunogenic compositions comprising a toxin (e.g., botulinum toxin), which may be used as an adjuvant, should contain significantly less LD₅₀The botulinum toxin of (1).

Formulations suitable for introduction into a vaccine formulation or pharmaceutical composition vary depending on the route of administration. Formulations suitable for parenteral administration (e.g., by intra-articular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes) include aqueous and non-aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which can contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. These formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules or vials.

Injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type described above. In the case of adoptive transfer of therapeutic T cells, these cells may be administered intravenously or parenterally.

Formulations suitable for oral administration may consist of: (a) a liquid solution, such as an effective amount of the polypeptide or encapsulated nucleic acid suspended in a diluent, such as water, saline, or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of active ingredient in liquid, solid, granular or gel form; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. The tablet form may comprise one or more of the following ingredients: lactose, sucrose, mannitol, sorbitol, calcium phosphate, 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, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Buccal tablet forms can contain the active ingredient in a flavoring agent, typically sucrose and acacia or tragacanth, as well as tablets, emulsions, gels, and the like containing the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia, and in addition to the active ingredient, carriers known in the art. The pharmaceutical composition may be encapsulated, for example in liposomes, or in a formulation that provides slow release of the active ingredient.

Antigens may be formulated as aerosols (e.g., they may be "nebulized") for administration by inhalation, alone or in combination with other suitable components. The aerosol formulation may be placed in a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol formulations may be delivered orally or nasally.

Suitable formulations for vaginal or rectal administration include, for example, suppositories, which consist of the polypeptide or encapsulated nucleic acid and a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. Furthermore, it is also possible to use gel rectal capsules consisting of the polypeptide or encapsulated nucleic acid and a matrix comprising, for example, liquid triglycerides, polyethylene glycols and paraffin hydrocarbons.

J. Preparation and storage of vaccine formulations and immunogenic compositions

The streptococcus pneumoniae vaccines and immunogenic compositions described herein can be produced using a variety of techniques. For example, the polypeptide may be produced in a suitable host cell using recombinant DNA techniques. Suitable host cells may be bacterial, yeast, mammalian or other types of cells. The host cell may be modified to express an exogenous copy of one of the genes for the polypeptide of interest. Typically, the gene is operably linked to appropriate regulatory sequences, such as a strong promoter and polyadenylation sequence. In some embodiments, the promoter is inducible or repressible. Depending on how one wishes to purify the polypeptide, other regulatory sequences may provide for secretion or excretion of the polypeptide of interest or retention of the polypeptide of interest in the cytoplasm or cell membrane. The gene may be present in an extrachromosomal plasmid or integrated into the host genome. It will be appreciated by those skilled in the art that it is not necessary to use a nucleic acid that is 100% identical to a naturally occurring sequence. In fact, some variation from these sequences is tolerated and may be desirable. For example, the nucleic acid can 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 enhance expression in a particular host. The nucleic acid may be produced, for example, by PCR or by chemical synthesis.

Once the recombinant cell line is produced, the polypeptide can be isolated therefrom. Separation can be achieved, for example, by affinity purification techniques or physical separation techniques (e.g., size columns).

In another aspect of the disclosure, methods of manufacture are provided, the methods comprising admixing one or more polypeptides, or immunogenic fragments or variants thereof, with a carrier and/or adjuvant.

In some embodiments, the antigens included in the immunological formulation and immunogenic composition may be produced in cell culture. One method includes providing one or more expression vectors and cloning nucleotides encoding one or more polypeptides selected from polypeptides having the amino acid sequences of tables 1 or 2 (e.g., SEQ ID NOS: 265, 266, or 268), then expressing and isolating the polypeptides.

The immunogenic polypeptides and nucleic acid compositions expressing these polypeptides described herein can be packaged in packages, dispensing devices, and kits for administering the nucleic acid compositions to a mammal. For example, a packaging or dispensing device containing one or more unit dosage forms is provided. Typically, instructions for administration of the compound will be provided with the package, along with appropriate instructions on the label that the compound is suitable for use in treating the indicated conditions (such as those disclosed herein).

Examples V. examples

Example 1 antigen identification and Combined murine Screen

Each open reading frame predicted in the genome of streptococcus pneumoniae TIGR4 is cloned into an expression vector comprising a tag capable of being presented by the Major Histocompatibility Complex (MHC). Each construct was then expressed in e.coli and full-length expression was verified by a surrogate assay that identified the tag in an MHC background. The screening is described in more detail in international application WO 2010/002993. To facilitate screening of large libraries, the libraries were pooled such that there were four induced library clones in each well. To screen T cells from mice immunized against streptococcus pneumoniae, aliquots of the pooled library were added to peritoneal-derived macrophages. Macrophages are allowed to bind to the tagged streptococcus pneumoniae antigen via MHC. Macrophages were washed with PBS after 2 hours at 37 ℃. Macrophages were then fixed with 1% paraformaldehyde for 15min and washed extensively with PBS. Will 10⁵Individual T cells were added to 200 μ L RP-10 medium per well. These T cells were previously killed from lungs that had been usedMice immunized 2 times with Streptococcus pneumoniae and cholera toxin adjuvant were isolated. The assay plates were incubated at 37 ℃ for 72 hours. The amount of IL-17 in the supernatant of each well was determined by using an IL-17ELISA assay. The threshold for positive results was set at two standard deviations above the mean of all samples.

Example 2 deconvolution of Positive murine pools

A second screen was used to determine which antigen(s) of the four clones in each well induced the positive response observed in the pooled screen described in example 1. All clones in each positive pool were pulsed individually onto peritoneal macrophages in duplicate wells. Pulsed macrophages were screened using T cells isolated from immunized mice of the same genetic background as the primary screen using the IL-17 assay described in example 1. Individual antigens that induced an average response in duplicate wells that exceeded the average of negative control samples by more than two standard deviations were considered positive responses. 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, SP0906, SP1828, SP2157, SP1229, SP1128, SP1836, SP1865, SP0904, SP0882, SP0765, SP1634, SP0418, SP1923, SP1313, SP0775, SP0314, SP0912, SP0159, SP0910, SP2148, SP1412, SP0372, SP1304, SP2002, SP0612, SP1988, SP0484, SP0847, SP1527, SP 42, SP0441, SP 050350, SP0014, SP1965, SP 0111527 and SP2108 were confirmed using this method.

Example 3 antigen identification and pooled human screening

Isolation of CD4+ T cells and CD14 from peripheral blood obtained from human donors⁺A monocyte. These monocytes were differentiated into dendritic cells by culturing them in a medium containing GM-CSF and IL-4, essentially as described for Tyde TF and Jansen PJ (1997 "Isolation and passaging of human dendritic cells", A guide to immunology, suppl 23: 7.32.1-7.32.16) (Tedder TF and Jansen PJ (1997 "Isolation and generation of human dendritic cells", Current Protocolsin Immunology Supp 23: 7.32.1-7.32.16)). After five days of culture, dendritic cells were seeded into 384-well plates. Make CD4⁺T cells expand non-specifically in culture to ensure sufficient numbers.

Each open reading frame predicted in the genome of streptococcus pneumoniae TIGR4 is cloned into an expression vector comprising a tag capable of being presented by the Major Histocompatibility Complex (MHC). Each construct was then expressed in e.coli and full-length expression was verified by a surrogate assay that identified the tag in an MHC background. To facilitate screening of large libraries, the libraries were pooled such that there were four induced library clones in each well. To screen for human T cells, aliquots of the pooled library were added to dendritic cells seeded in 384-well plates. After 2 hours at 37 ℃, dendritic cells were fixed with 1% paraformaldehyde for 15min and washed extensively with phosphate buffer and lysine buffer. 40,000 CD4 in 70. mu.L of RP-10 medium⁺T cells were added to each well of the 384-well plate. These assay plates were incubated at 37 ℃ for 3 days. The amount of IL-17 in the supernatant of each well was determined by using an IL-17ELISA assay. The threshold for positive results was set at two standard deviations above the mean of all samples, two standard deviations above the mean of the negative control, or 1.78 times the absolute deviation of the median of the data set, at different iterations of the screen. The positive pools were then deconvoluted as described in example 4.

Example 4 deconvolution of Positive human pools

For all antigens, deconvolution was performed by comparing the results of the two sets of pool screens. In this method, two different sets of pools are prepared, such that the polypeptide is between the first pool and the second pool and the three different polypeptides are together. Thus, it is possible to determine which polypeptides are antigens by identifying which polypeptides are in the positive pool in both the first and second sets. In this deconvolution method, a pool is identified as positive if it is 1.78 times the absolute deviation of the number of bits in the dataset.

If the antigen is positive in at least two repeated second screens, it is identified as a positive hit. The antigens SP2108, SP0641, SP1393, SP0024, SP0641.1, SP1072, SP1384 and SP2032 were identified using the methods described above.

Example 5

SP2108, SP0148 and SP1634 polypeptides

The SP2108 polypeptide (SEQ ID NO: 9), the SP0148 polypeptide (SEQ ID NO: 7) and the S P1634 polypeptide (see Table 2) were formulated as vaccine compositions using 4 μ g of the polypeptide in combination with 1 μ g of cholera toxin adjuvant (CT). For the combination, 4. mu.g of each polypeptide was used. The composition was administered intranasally three times at one week intervals to C57BL/6 mice. The subjects were then allowed to rest for 3 weeks and at that time a blood draw was taken for examination of immunogenicity. For this assay, heparinized whole blood was collected from the retro-orbital sinus (retrograde orbital sinus). Whole PBMCs were stimulated with killed non-capsulated whole cell streptococcus pneumoniae (WCC) or a combination of the three polypeptides in round bottom tubes for three days. Supernatants were then collected and IL-17 levels were assessed by ELISA. Cholera Toxin (CT) alone or an unrelated antigen from HSV (003) was used as a negative control. The results of the IL-17 immunogenicity analysis are shown in figures 1 and 2, where the left panel shows the data in a dispersed format and the right panel shows the data as mean with standard deviation. The subjects were allowed to rest for an additional 2 weeks, during which time they were challenged by intranasal administration of live, pod-bearing streptococcus pneumoniae. The subjects were sacrificed one week later and the number of Colony Forming Units (CFU) was counted from the nasal wash. The results of the colonization analysis are shown in fig. 3.

Example 6

SP0882 and SP0314 polypeptides

This example used the same protocol 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 the SP0314 polypeptide (see Table 2) were tested in parallel with two of the three polypeptides tested in example 5. The results of the IL-17 immunogenicity analysis are shown in FIGS. 4 and 5. The results of the colonization analysis are shown in fig. 6.

Example 7

SP1072, SP0641N, and SP0024 polypeptides

This example used a similar protocol to example 5, except that two doses of the vaccine composition were administered, each one week apart. The vaccine composition comprises 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, mice were challenged intranasally with live streptococcus pneumoniae type 6B. One week later, the bacterial load in each mouse was assessed by plating nasal lavage fluid onto selective medium and counting the resulting CFUs. The number of CFUs isolated from each mouse was plotted for each immune group. The results of this colonization analysis are shown in fig. 7. Statistically significant results (═ p values < 0.05) are indicated in the figures.

Example 8

SP0148, SP0314, SP0882, and SP2108 polypeptides tested in BALB/c mice

To determine whether similar immune responses were seen for different mouse genotypes, the immunization compositions were administered to BALB/c mice. The vaccine composition comprises 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 similar protocol to example 5, these mice were immunized, challenged intranasally with streptococcus pneumoniae, and CFU numbers were recorded. The results of this colonization experiment are shown in fig. 8.

Example 9

SP1912, SP2108 and SP0148 polypeptides: IL-17A immunogenicity assay

The polypeptides SP1912(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). Mice were given these vaccine compositions twice, each at one week intervals. Positive controls were killed non-capsulated whole cells + CT (wcb) and negative controls were CT alone or recombinant protein without CT (except SP 1912). Three weeks after the last immunization, peripheral blood was collected from the retro-orbital sinus and evaluated in a whole blood analysis. Briefly, heparinized whole blood was diluted in culture medium and then incubated for six days in duplicate with a) immunoprotein or B) whole cell vaccine. Supernatants were collected and IL-17A levels were measured by ELISA. The results of the IL-17A immunogenicity assay are shown in FIG. 9. Each symbol in the figure represents a response from a separate mouse, and the line represents the median response of the group.

Example 10

SP1912, SP2108 and SP0148 polypeptides: analysis of colonization

Animals were immunized with vaccine preparations 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, then 10 for four weeks after the last immunization (and one week after retro-orbital blood sampling)⁷Individual live streptococcus pneumoniae type 6B were challenged intranasally. Seven days after challenge, the animals were euthanized and lavaged of the nasopharyngeal cavity and cultured on permissive media to assess streptococcus pneumoniae titers. The results are shown in fig. 10 as Colony Forming Units (CFU) of bacteria per lavage. Each symbol represents the titer of an individual mouse response, and the horizontal line represents the median of the group. (value of p < 0.05).

Example 11

SP1912 polypeptide: inspiratory attack (septicemia analysis)

The ability of the polypeptide SP1912 to protect mice from sepsis was evaluated. A group of ten mice were immunized subcutaneously three times, two weeks apart, with a vaccine composition comprising an SP1912 polypeptide adsorbed to alum (SEQ ID NO: 265) or a pneumocolyloid (PdT). The positive control isKilled non-capsular whole cell streptococcus pneumoniae + alum (WCB), and the negative control was alum alone. Three weeks after the last immunization, blood was collected for assessment of IL-17A response and antibody levels, and then 10 weeks later⁷Individual live strains 0603 (type 6B) streptococcus pneumoniae were subjected to inspiratory challenge in these mice. The survival of these animals was monitored for eight days. The results of the inspiratory challenge are shown in fig. 11 as survival curves for each immune group.

Example 12

pneumocolyloid PdT, SP0148 and SP0641N polypeptides: inspiratory attack (septicemia analysis)

The ability of polypeptide SP0148 to protect mice from sepsis when immunized alone or in combination with SP0641N and/or pneumocolyloid (pdt) was evaluated. Groups of ten mice were immunized subcutaneously three times, two weeks apart, with a vaccine composition comprising polypeptide SP0148(SEQ ID NO: 7) alone or polypeptide SP0148(SEQ ID NO: 7) in combination with polypeptides SP0641N (SEQ ID NO: 13) and/or PdT adsorbed to alum. The positive control was killed non-capsulated whole cell streptococcus pneumoniae + alum (WCB) and the negative control was alum alone. Three weeks after the last immunization, blood was collected for assessment of IL-17 and antibodies, and then 10 weeks later⁷Individual live bacterial strains 0603 (type 6B) streptococcus pneumoniae were subjected to inspiratory challenge in mice. The survival of these animals was monitored for eight days. Data are shown in figure 12 as survival curves for each immunization group.

Example 13

SP1912, SP2108 and SP0148 polypeptides: analysis of colonization

Additional studies were performed essentially as described in example 10, for a total of four separate studies. Briefly, animals were immunized with vaccine preparations as described in example 9, which contained the polypeptides SP1912(SEQ ID NO: 265), SP2108(SEQ ID NO: 9), SP0148(SEQ ID NO: 7) or alternatively SP2108 plus SP0148, and cholera toxin adjuvant (CT). With killed non-capsulated whole cell streptococcus pneumoniaeBacteria plus CT (WCB) or CT alone immunized control animals. Four weeks after the last immunization, 10 is used⁷Live Streptococcus pneumoniae type 6B challenged these immunized animals intranasally. Seven days after challenge, the animals were euthanized and nasopharyngeal lavage performed and cultured on permissive media to assess streptococcus pneumoniae titers. The combined results of the four studies are shown in fig. 13 as Colony Forming Units (CFU) of bacteria per lavage. Each symbol represents the titer from an individual mouse response, and the horizontal line represents the median of the group. (value of p < 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 immunized twice subcutaneously with vaccine compositions comprising alum-adsorbed SP1912 polypeptide (SEQ ID NO: 265), SP0148 polypeptide (SEQ ID NO: 7), or both polypeptides, each at two week intervals. Control animals were immunized with alum only. Three weeks after the last immunization, heparinized blood was collected by cardiac puncture and IL-17A levels were assessed in a whole blood assay. Briefly, heparinized whole blood is diluted in culture medium and then incubated with one or more immune proteins for six days. Supernatants were collected and IL-17A levels were measured by ELISA. The results of the IL-17A immunogenicity assay are shown in FIG. 14. Each symbol in the figure represents a response from a separate mouse, and the line represents the median response of the group.

Example 15

SP1912 and SP0148 polypeptides: analysis of colonization

Animals were immunized subcutaneously three times, two weeks apart, with vaccine formulations containing different doses of the polypeptide SP0148(SEQ ID NO: 7) plus minus S P1912(SEQ ID NO: 265) adsorbed to alum. Immunization of control animals with killed non-capsulated whole cell Streptococcus pneumoniae plus Alum (WCV) or Alum alone. Four weeks after the last immunization, 10 is used⁷Live Streptococcus pneumoniae type 6B challenged these immunized animals intranasally. Seven days after challenge, the animals were euthanized and nasopharyngeal lavage performed and cultured on permissive media to assess streptococcus pneumoniae titers. The results are shown in fig. 15 as Colony Forming Units (CFU) of bacteria per lavage. Each symbol represents the titer from an individual mouse response, and the horizontal line represents the median of the group. The number of animals in the group that were protected from colonization is indicated at the top of the figure.

Example 16

SP1912, SP0148, and SP2108 polypeptides: analysis of colonization

In two separate studies, animals were immunized subcutaneously three times, two weeks apart, with vaccine preparations containing polypeptides SP0148(SEQ ID NO: 7) and SP0148 plus SP1912(SEQ ID NO: 265) adsorbed to alum, or in addition SP2108(SEQ ID NO: 9), SP2108 plus SP0148, and SP2108 plus SP 1912. Control animals were immunized with killed non-capsulated whole cell streptococcus pneumoniae plus alum (WCV) or alum alone. Four weeks after the last immunization, 10 is used⁷Live Streptococcus pneumoniae type 6B challenged these immunized animals intranasally. Seven days after challenge, the animals were euthanized and nasopharyngeal lavage performed and cultured on permissive media to assess streptococcus pneumoniae titers. The combined results of the two studies are shown in figure 16 as Colony Forming Units (CFU) of bacteria per lavage. Each symbol represents the titer from an individual mouse response, and the horizontal line represents the median of the group. The number of animals protected from colonization and the corresponding percentage of animals protected from colonization of the group of animals are indicated at the top of the figure. (. p < 0.05,. p < 0.01,. p < 0.001, Dunn Multiple Comparison Test compared to alum control).

Example 17

pneumocolyloid L460D, PspA derivative PR + NPB, SP1912, SP0148, and SP2108 polypeptide: analysis of colonization

Animals were immunized subcutaneously three times, two weeks apart, with vaccine preparations comprising the polypeptides SP0148(SEQ ID NO: 7), SP2108(SEQ ID NO: 9), SP0148 plus SP2108, and a combination of SP0148 plus SP2108 with SP1912(SEQ ID NO: 265) or the known Streptococcus pneumoniae antigens L460D plus PR + NPD adsorbed to alum. Two independent studies were performed. Control animals were immunized with alum only. Four weeks after the last immunization, 10 is used⁷Live Streptococcus pneumoniae type 6B challenged these immunized animals intranasally. Seven days after challenge, the animals were euthanized and nasopharyngeal lavage performed and cultured on permissive media to assess streptococcus pneumoniae titers. The results of the second study are shown in figure 17 as Colony Forming Units (CFU) of bacteria per lavage. Each symbol represents the titer from an individual mouse response, and the horizontal line represents the median of the group. The number of animals in the group that were protected from colonization is indicated at the top of the figure.

The table below shows the absolute number of animals protected from colonization and the corresponding percentage in the four studies described in examples 16 and 17.

Example 18

PspA, SP0148 and SP2108 passive antibody transfer and inspiratory challenge (sepsis assay)

Monoclonal antibody specific to PspA, to SP0148, SP2108, or both, were used on a group of ten miceThe combination of the two is injected by specific heat-inactivated rabbit serum. Antibody and antiserum concentrations and total injection volume were adjusted with Normal Rabbit Serum (NRS) and PBS. Control animals were injected with NRS or serum against killed non-capsulated whole cell streptococcus pneumoniae (WCB). One day after injection, 10⁶Mice were challenged with inspiration by live Streptococcus pneumoniae type WU-2 (ST-3). The survival of these animals was monitored for eight days. Data are shown in figure 18 as survival curves for each immunization group.

Figure 19 shows the percentage of animals protected from sepsis in the studies described in examples 12 and 18, as well as in two additional studies.

Sequence listing

SEQ ID NO：1

SP0024

The putative conserved protein Streptococcus pneumoniae TIGR4 of > gi |14971488| gb | AAK74215.1|

MSYFEQFMQANQAYVALHGQLNLPLKPKTRVAIVTCMDSRLHVAQALGLALGDAHILRNAGGRVTEDMIRSLVISQQ

QMGTREIVVLHHTDCGAQTFENEPFQEYLKEELGVDVSDQDFLPFQDIEESVREDMQLLIESPLIPDDVIISGAIYN

VDTGSMTVVEL

SEQ ID NO：2

SP0882

(> gi 14972356| gb | AAK75009.1| conserved hypothetical protein (Streptococcus pneumoniae TIGR4)

MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDI

SRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNI

TQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYIDSSLCYY

HDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW

SEQ ID NO：3

SP0882N

MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDI

SRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVNEVVKPFI

SEQ ID NO：4

SP0882 with exogenous signal sequence

MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQN

VFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPF

IDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEE

ADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW

SEQ ID NO：5

SP0882N with exogenous signal sequence

MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQN

VFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPF

I

SEQ ID NO：6

SP0148 lacking Signal sequence

MCSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYN

MAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNY

TKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKD

GTLEKLSKQFFGDTYLPAEADIKE

SEQ ID NO：7

SP0148 (277 amino acids with N-terminal E) comprising a signal sequence

MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSDKY

DVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDDIGGKSTEVVQAT

TSAKQLEAYNAEHTDNPTILNYTKADFQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPTVYPL

LAQGQDELKSFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIKE

SEQ ID NO：8

SP1072

' gi |14972547| gb | AAK75185.1| DNA primer enzyme Streptococcus pneumoniae TIGR4

MVDKQVIEEIKNNANIVEVIGDVISLQKAGRNYLGLCPFHGEKTPSFNVVEDKQFYHCFGCGRSGDVFKFIEEYQGV

PFIEAVQILGQRVGIEVEKPLYSEQKSASPHQALYDMHEDAAKFYHAILMTTTMGEEARNYLYQRGLTDEVLKHFWI

GLAPPERNYLYQRLSDQYREEDLLDSGLFYLSDANQFVDTFHNRIMFPLTNDQGKVIAFSGRIWQKTDSQTSKYKNS

RSTAIFNKSYELYHMDRAKRSSGKASEIYLMEGFMDVIAAYRAGIENAVASMGTALSREHVEHLKRLTKKLVLVYDG

DKAGQAATLKALDEIGDMPVQIVSMPDNLDPDEYLQKNGPEDLAYLLTKTRISPIEFYIHQYKPENSENLQAQIEFL

EKIAPLIVQEKSIAAQNSYIHILADSLASFDYTQIEQIVNESRQVQRQNRMEGISRPTPITMPVTKQLSAIMRAEAH

LLYRMMESPLVLNDYRLREDFAFATPEFQVLYDLLGQYGNLPPEVLAEQTEEVERAWYQVLAQDLPAEISPQELSEV

EMTRNKALLNQDNMRIKKKVQEASHVGDTDTALEELERLISQKRRME

SEQ ID NO：9

SP2108 comprising a signal sequence

(> gi |14973620| gb | AAK76167.1| maltose/maltodextrin ABC transporter, maltose/maltodextrin binding protein (Streptococcus pneumoniae TIGR4)

MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGD

ALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYY

NKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIV

GINYAKSWYEKWPKGMQDTEGAGNLIQTQFQEGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKA

WVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVW

DPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE

SEQ ID NO：10

SP2108 lacking signal sequence

MCGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMA

PYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSK

YAFAGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWYEKWPKGMQDTEGAG

NLIQTQFQEGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVIPQAVKNLEASQKFVDFLVAT

EQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAAND

AVTLIKETIKQKFGE

SEQ ID NO：11

SP0641M

MSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQG

AGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKL

DETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEAL

NSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGV

IQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGLTPSPLVLRSAEEGLISIVNT

NKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGREENAPESKDNQDPATKIRGQF

EPIAEGQYFYKFKYRLTKDYPWQVSYIPVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQYKNETLFARDQKEH

PEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTIYEIKGAGDLRGKIIEVIALDGSSNFT

KIHRIKFANQADEKGMISYYLVDPDQDSSKYQ

SEQ ID NO：12

SP0641

(> gi |14972117| gb | AAK74791.1| serine protease, subtilisin family [ Streptococcus pneumoniae TIGR4]

MKKSTVLSLTTAAVILAAYAPNEVVLADTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEKEV

VSKNPVIDNNTSNEEAKIKEENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLY

TYDRIFNGSAIETTPDNLDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNI

DTGTDYRHKAMRIDDDAKASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILA

GNDTEQDIKNFNGIDGIAPNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAI

RALRKAGIPMVVATGNYATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYR

NIGAFFDKSKITTNEDGTKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNT

VNYYNRDNWTELPAMGYEADEGTKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNK

PNVGDEKEIDFKFAPDTDKELYKEDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYGYMSGTSMAT

PIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQGAGLINVAN

ALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKLDETYKDEK

SPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEALNSNGKKIN

FQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGVIQNRKDKN

TTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGLTPSPLVLRSAEEGLISIVNTNKEGENQR

DLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGREENAPESKDNQDPATKIRGQFEPIAEGQY

FYKFKYRLTKDYPWQVSYIPVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQYKNETLFARDQKEHPEKFDEIA

NEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTIYEIKGAGDLRGKIIEVIALDGSSNFTKIHRIKFA

NQADEKGMISYYLVDPDQDSSKYQKLGEIAESKFKNLGNGKEGSLKKDTTGVEHHHQENEESIKEKSSFTIDRNIST

IRDFENKDLKKLIKKKFREVDDFTSETGKRMEEYDYKYDDKGNIIAYDDGTDLEYETEKLDEIKSKIYGVLSPSKDG

HFEILGKISNVSKNAKVYYGNNYKSIEIKATKYDFHSKTMTFDLYANINDIVDGLAFAGDMRLFVKDNDQKKAEIKI

RMPEKIKETKSEYPYVSSYGNVIELGEGDLSKNKPDNLTKMESGKIYSDSEKQQYLLKDNIILRKGYALKVTTYNPG

KTDMLEGNGVYSKEDIAKIQKANPNLRALSETTIYADSRNVEDGRSTQSVLMSALDGFNIIRYQVFTFKMNDKGEAI

DKDGNLVTDSSKLVLFGKDDKEYTGEDKFNVEAIKEDGSMLFIDTKPVNLSMDKNYFNPSKSNKIYVRNPEFYLRGK

ISDKGGFNWELRVNESVVDNYLIYGDLHIDNTRDFNIKLNVKDGDIMDWGMKDYKANGFPDKVTDMDGNVYLQTGYS

DLNAKAVGVHYQFLYDNVKPEVNIDPKGNTSIEYADGKSVVFNINDKRNNGFDGEIQEQHIYINGKEYTSFNDIKQI

IDKTLNIKIVVKDFARNTTVKEFILNKDTGEVSELKPHRVTVTIQNGKEMSSTIVSEEDFILPVYKGELEKGYQFDG

WEISGFEGKKDAGYVINLSKDTFIKPVFKKIEEKKEEENKPTFDVSKKKDNPQVNHSQLNESHRKEDLQREEHSQKS

DSTKDVTATVLDKNNISSKSTTNNPNKLPKTGTASGAQTLLAAGIMFIVGIFLGLKKKNQD

SEQ ID NO：13

SP0641N

MVVLADTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAKIKEEN

SNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDNLDKIK

QIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDAKASMR

FKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGIAPNAQ

IFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNYATSAS

SSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDGTKAPS

KLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGYEADEG

TKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDTDKELY

KEDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYG

SEQ ID NO：14

SP0882 consensus sequence

MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESF

I Y

IGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAE

Y H E E

FVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQ

EK

EAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVH

I H R

LDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW

SEQ ID NO：15

SP0882N consensus sequence

MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESF

I Y

IGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAE

Y H E E

FVMEVVKPFI

SEQ ID NO：16

SP0882 consensus sequence with exogenous signal sequence

MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYE

T T V I

KDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNE

Y Y H

YAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNIT

E E

QFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLM

EK I H

DGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW

R

SEQ ID NO：17

SP0882N consensus sequence with exogenous signal sequence

MSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYE

T T V I

KDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNE

Y Y H

YAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFI

E E

SEQ ID NO：18

SP0148 consensus sequence lacking Signal sequence

MCSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELTGYEIEVVRAIFKDSDKYDVKFE

Q S R N N X

KTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAPIAQNPNVLVVKKDDSSIKSLDD

I E

IGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKADLQQIMVRLSDGQFDYKIFDKIG

F

VETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELKSFVDKRIKELYKDGTLEKLSKQ

Y S

FFGDTYLPAEADIK(E)

SEQ ID NO：19

SP0148 consensus sequence comprising a Signal sequence

MKKIVKYSSLAALALVAAGVLAACSGGAKKEGEAASKKEIIVATNGSPKPFIYEENGELT

G L Q S R N

GYE IEVVRAIFKDSDKYDVKFEKTEWSGVFAGLDADRYNMAVNNLSYTKERAEKYLYAAP

N X I

IAQNPNVLVVKKDDSSIKSLDDIGGKSTEVVQATTSAKQLEAYNAEHTDNPTILNYTKAD

E

LQQIMVRLSDGQFDYKIFDKIGVETVIKNQGLDNLKVIELPSDQQPYVYPLLAQGQDELK

F Y S

SFVDKRIKELYKDGTLEKLSKQFFGDTYLPAEADIK(E)

SEQ ID NO：20

SP2108 consensus sequence lacking a Signal sequence

MCGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVAKAYEKEAGVKVTLKTGDALGGLD

A I

KLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLSDGAKTDDTTKSLVTAANGKVYGA

I X T

PAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAFAGEDGKTTAFLADWTNFYYTYGL

A

LAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWYEKWPKGMQDTEGAGNLIQTQFQE

G P A X H

GKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEYAAFGGGKAWVIPQAVKNLEASQK

A

FVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDELTTAVIKQFKNTQPLPNISQMSA

S A S

VWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQKFGE

SEQ ID NO：21

SP2108 consensus sequence comprising a signal sequence

MSSKFMKSAAVLGTATLASLLLVACGSKTADKPADSGSSEVKELTVYVDEGYKSYIEEVA

T T V A

KAYEKEAGVKVTLKTGDALGGLDKLSLDNQSGNVPDVMMAPYDRVGSLGSDGQLSEVKLS

I I X

DGAKTDDTTKSLVTAANGKVYGAPAVIESLVMYYNKDLVKDAPKTFADLENLAKDSKYAF

T

AGEDGKTTAFLADWTNFYYTYGLLAGNGAYVFGQNGKDAKDIGLANDGSIVGINYAKSWY

A G P A X

EKWPKGMQDTEGAGNLIQTQFQEGKTAAIIDGPWKAQAFKDAKVNYGVATIPTLPNGKEY

H

AAFGGGKAWVIPQAVKNLEASQKFVDFLVATEQQKVLYDKTNEIPANTEARSYAEGKNDE

A S A

LTTAVIKQFKNTQPLPNISQMSAVWDPAKNMLFDAVSGQKDAKTAANDAVTLIKETIKQK

S

FGE

SEQ ID NO：22

SP1634

Gii 14973124 gB AAK75714.1 hypothetical protein SP _1634 Streptococcus pneumoniae TIGR4

DANIFDYLKDVAYDSYYDLPLNELDILTLIEITYLSFDNLVSTLPQRLLDLAPQVPRDPTMLTSKNRLQLLDELAQH

KRFKNCKLSHFINDIDPELQKQFAAMTYRVSLDTYLIVFRGTDDSIIGWKEDFHLTYMKEIPAQKHALRYLKNFFAH

HPKQKVILAGHSKGGNLAIYAASQIEQSLQNQITAVYTFDAPGLHQELTQTAGYQRIMDRSKIFIPQGSIIGMMLEI

PAHQIIVQSTALGGIAQHDTFSWQIEDKHFVQLDKTNSDSQQVDTTFKEWVATVPDEELQLYFDLFFGTILDAGISS

INDLASLKALEYIHHLFVQAQSLTPEERETLGRLTQLLIDTRYQAWKNR

SEQ ID NO：23

SP0314

Gt gi |14971788| gb | AAK74491.1| Hyaluronidase Streptococcus pneumoniae

TIGRAMQTKTKKLIVSLSSLVLSGFLLNHYMTIGAEETTTNTIQQSQKEVQYQQRDTKNLVENGDFGQTEDGSSPWT

GSKAQGWSAWVDQKNSADASTRVIEAKDGAITISSHEKLRAALHRMVPIEAKKKYKLRFKIKTDNKIGIAKVRIIEE

SGKDKRLWNSATTSGTKDWQTIEADYSPTLDVDKIKLELFYETGTGTVSFKDIELVEVADQLSEDSQTDKQLEEKID

LPIGKKHVFSLADYTYKVENPDVASVKNGILEPLKEGTTNVIVSKDGKEVKKIPLKILASVKDAYTDRLDDWNGIIA

GNQYYDSKNEQMAKLNQELEGKVADSLSSISSQADRTYLWEKFSNYKTSANLTATYRKLEEMAKQVTNPSSRYYQDE

TVVRTVRDSMEWMHKHVYNSEKSIVGNWWDYEIGTPRAINNTLSLMKEYFSDEEIKKYTDVIEKFVPDPEHFRKTTD

NPFKALGGNLVDMGRVKVIAGLLRKDDQEISSTIRSIEQVFKLVDQGEGFYQDGSYIDHTNVAYTGAYGNVLIDGLS

QLLPVIQKTKNPIDKDKMQTMYHWIDKSFAPLLVNGELMDMSRGRSISRANSEGHVAAVEVLRGIHRIADMSEGETK

QCLQSLVKTIVQSDSYYDVFKNLKTYKDISLMQSLLSDAGVASVPRPSYLSAFNKMDKTAMYNAEKGFGFGLSLFSS

RTLNYEHMNKENKRGWYTSDGMFYLYNGDLSHYSDGYWPTVNPYKMPGTTETDAKRADSDTGKVLPSAFVGTSKLDD

ANATATMDFTNWNQTLTAHKSWFMLKDKIAFLGSNIQNTSTDTAATTIDQRKLESGNPYKVYVNDKEASLTEQEKDY

PETQSVFLESFDSKKNIGYFFFKKSSISMSKALQKGAWKDINEGQSDKEVENEFLTISQAHKQNRDSYGYMLIPNVD

RATFNQMIKELESSLIENNETLQSVYDAKQGVWGIVKYDDSVSTI SNQFQVLKRGVYTIRKEGDEYKIAYYNPETQE

SAPDQEVFKKLEQAAQPQVQNSKEKEKSEEEKNHSDQKNLPQTGEGQSILASLGFLLLGAFYLFRRGKNN

SEQ ID NO：24

SP0882N DNA

ATGAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGTGT

ACGTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGGCAAA

ATGTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCGGATATC

AGTCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAGTTCCAAGA

ATCTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTGGTCAAGCCTT

TTATC

SEQ ID NO：25

SP0882 (nucleotide) with exogenous signal sequence

ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGCAT

GAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGTGTAC

GTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGGCAAAAT

GTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCGGATATCAG

TCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAGTTCCAAGAAT

CTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTGGTCAAGCCTTTT

ATCGATGAGACCTATCGTACAAAAGCAGACTGCCAGCATACGGCTATGATTGGTTCCTCACTAGGAGGCAATATTAC

CCAGTTTATCGGTTTGGAATACCAAGACCAAATTGGTTGCTTGGGCGTTTTTTCATCTGCAAACTGGCTCCACCAAG

AAGCCTTTAACCGCTATTTCGAGTGCCAGAAACTATCGCCTGACCAGCGCATCTTCATCTATGTAGGAACAGAAGAA

GCAGATGATACAGACAAGACCTTGATGGATGGCAATATCAAACAAGCCTATATCGACTCGTCGCTTTGCTATTACCA

TGATTTGATAGCAGGGGGAGTACATCTGGATAATCTTGTGCTAAAAGTTCAGTCTGGTGCCATCCATAGTGAAATCC

CTTGGTCAGAAAATCTACCAGATTGTCTGAGATTTTTTGCAGAAAAATGGTAA

SEQ ID NO：26

SP0882N (nucleotide) with exogenous signal sequence

ATGTCATCTAAATTTATGAAGAGCGCTGCGGTGCTTGGAACTGCTACACTTGCTAGCTTGCTTTTGGTAGCTTGCAT

GAATCAATCCTACTTTTATCTAAAAATGAAAGAACACAAACTCAAGGTTCCTTATACAGGTAAGGAGCGCCGTGTAC

GTATTCTTCTTCCTAAAGATTATGAGAAAGATACAGACCGTTCCTATCCTGTTGTATACTTTCATGACGGGCAAAAT

GTTTTTAATAGCAAAGAGTCTTTCATTGGACATTCATGGAAGATTATCCCAGCTATCAAACGAAATCCGGATATCAG

TCGCATGATTGTCGTTGCTATTGACAATGATGGTATGGGGCGGATGAATGAGTATGCGGCTTGGAAGTTCCAAGAAT

CTCCTATCCCAGGGCAGCAGTTTGGTGGTAAGGGTGTGGAGTATGCTGAGTTTGTCATGGAGGTGGTCAAGCCTTTT

ATC

SEQ ID NO：27

SP0148 (nucleotide) lacking signal sequence

ATGTGCTCAGGGGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACC

AAAGCCATTTATCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATT

CTGACAAATATGATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAAT

ATGGCTGTCAACAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCC

TAATGTCCTTGTCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCG

TTCAAGCCACTACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTAT

ACTAAGGCAGACTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATCGG

TGTTGAAACAGTGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAACCGTACG

TTTACCCACTTCTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTATAAAGAT

GGAACTCTTGAAAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAAGAGTAA

SEQ ID NO：28

SP0148 (nucleotide) comprising a Signal sequence

ATGAAAAAAATCGTTAAATACTCATCTCTTGCAGCCCTTGCTCTTGTTGCTGCAGGTGTGCTTGCGGCTTGCTCAGG

GGGTGCTAAGAAAGAAGGAGAAGCAGCTAGCAAGAAAGAAATCATCGTTGCAACCAATGGATCACCAAAGCCATTTA

TCTATGAAGAAAATGGCGAATTGACTGGTTACGAGATTGAAGTCGTTCGCGCTATCTTTAAAGATTCTGACAAATAT

GATGTCAAGTTTGAAAAGACAGAATGGTCAGGTGTCTTTGCTGGTCTTGACGCTGATCGTTACAATATGGCTGTCAA

CAATCTTAGCTACACTAAAGAACGTGCGGAGAAATACCTCTATGCCGCACCAATTGCCCAAAATCCTAATGTCCTTG

TCGTGAAGAAAGATGACTCTAGTATCAAGTCTCTCGATGATATCGGTGGAAAATCGACGGAAGTCGTTCAAGCCACT

ACATCAGCTAAGCAGTTAGAAGCATACAATGCTGAACACACGGACAACCCAACTATCCTTAACTATACTAAGGCAGA

CTTCCAACAAATCATGGTACGTTTGAGCGATGGACAATTTGACTATAAGATTTTTGATAAAATCGGTGTTGAAACAG

TGATCAAGAACCAAGGTTTGGACAACTTGAAAGTTATCGAACTTCCAAGCGACCAACAACCGTACGTTTACCCACTT

CTTGCTCAGGGTCAAGATGAGTTGAAATCGTTTGTAGACAAACGCATCAAAGAACTTTATAAAGATGGAACTCTTGA

AAAATTGTCTAAACAATTCTTCGGAGACACTTATCTACCGGCAGAAGCTGATATTAAAGAGTAA

SEQ ID NO：29

SP2108 (nucleotides) lacking a signal sequence

ATGTGCGGAAGCAAAACTGCTGATAAGCCTGCTGATTCTGGTTCATCTGAAGTCAAAGAACTCACTGTATATGTAGA

CGAGGGATATAAGAGCTATATTGAAGAGGTTGCTAAAGCTTATGAAAAAGAAGCTGGAGTAAAAGTCACTCTTAAAA

CTGGTGATGCTCTAGGAGGTCTTGATAAACTTTCTCTTGACAACCAATCTGGTAATGTCCCTGATGTTATGATGGCT

CCATACGACCGTGTAGGTAGCCTTGGTTCTGACGGACAACTTTCAGAAGTGAAATTGAGCGATGGTGCTAAAACAGA

CGACACAACTAAATCTCTTGTAACAGCTGCTAATGGTAAAGTTTACGGTGCTCCTGCCGTTATCGAGTCACTTGTTA

TGTACTACAACAAAGACTTGGTGAAAGATGCTCCAAAAACATTTGCTGACTTGGAAAACCTTGCTAAAGATAGCAAA

TACGCATTCGCTGGTGAAGATGGTAAAACTACTGCCTTCCTAGCTGACTGGACAAACTTCTACTATACATATGGACT

TCTTGCCGGTAACGGTGCTTACGTCTTTGGCCAAAACGGTAAAGACGCTAAAGACATCGGTCTTGCAAACGACGGTT

CTATCGTAGGTATCAACTACGCTAAATCTTGGTACGAAAAATGGCCTAAAGGTATGCAAGATACAGAAGGTGCTGGA

AACTTAATCCAAACTCAATTCCAAGAAGGTAAAACAGCTGCTATCATCGACGGACCTTGGAAAGCTCAAGCCTTTAA

AGATGCTAAAGTAAACTACGGAGTTGCAACTATCCCAACTCTTCCAAATGGAAAAGAATATGCTGCATTCGGTGGTG

GTAAAGCTTGGGTCATTCCTCAAGCCGTTAAGAACCTTGAAGCTTCTCAAAAATTTGTAGACTTCCTTGTTGCAACT

GAACAACAAAAAGTATTATATGATAAGACTAACGAAATCCCAGCTAATACTGAGGCTCGTTCATACGCTGAAGGTAA

AAACGATGAGTTGACAACAGCTGTTATCAAACAGTTCAAGAACACTCAACCACTGCCAAACATCTCTCAAATGTCTG

CAGTTTGGGATCCAGCGAAAAATATGCTCTTTGATGCTGTAAGTGGTCAAAAAGATGCTAAAACAGCTGCTAACGAT

GCTGTAACATTGATCAAAGAAACAATCAAACAAAAATTTGGTGAATAA

SEQ ID NO：30

SP0641M (nucleotides)

ATGTCAGGAACTAGTATGGCGACTCCAATCGTGGCAGCTTCTACTGTTTTGATTAGACCGAAATTAAAGGAAATGCT

TGAAAGACCTGTATTGAAAAATCTTAAGGGAGATGACAAAATAGATCTTACAAGTCTTACAAAAATTGCCCTACAAA

ATACTGCGCGACCTATGATGGATGCAACTTCTTGGAAAGAAAAAAGTCAATACTTTGCATCACCTAGACAACAGGGA

GCAGGCCTAATTAATGTGGCCAATGCTTTGAGAAATGAAGTTGTAGCAACTTTCAAAAACACTGATTCTAAAGGTTT

GGTAAACTCATATGGTTCCATTTCTCTTAAAGAAATAAAAGGTGATAAAAAATACTTTACAATCAAGCTTCACAATA

CATCAAACAGACCTTTGACTTTTAAAGTTTCAGCATCAGCGATAACTACAGATTCTCTAACTGACAGATTAAAACTT

GATGAAACATATAAAGATGAAAAATCTCCAGATGGTAAGCAAATTGTTCCAGAAATTCACCCAGAAAAAGTCAAAGG

AGCAAATATCACATTTGAGCATGATACTTTCACTATAGGCGCAAATTCTAGCTTTGATTTGAATGCGGTTATAAATG

TTGGAGAGGCCAAAAACAAAAATAAATTTGTAGAATCATTTATTCATTTTGAGTCAGTGGAAGAAATGGAAGCTCTA

AACTCCAACGGGAAGAAAATAAACTTCCAACCTTCTTTGTCGATGCCTCTAATGGGATTTGCTGGGAATTGGAACCA

CGAACCAATCCTTGATAAATGGGCTTGGGAAGAAGGGTCAAGATCAAAAACACTGGGAGGTTATGATGATGATGGTA

AACCGAAAATTCCAGGAACCTTAAATAAGGGAATTGGTGGAGAACATGGTATAGATAAATTTAATCCAGCAGGAGTT

ATACAAAATAGAAAAGATAAAAATACAACATCCCTGGATCAAAATCCAGAATTATTTGCTTTCAATAACGAAGGGAT

CAACGCTCCATCATCAAGTGGTTCTAAGATTGCTAACATTTATCCTTTAGATTCAAATGGAAATCCTCAAGATGCTC

AACTTGAAAGAGGATTAACACCTTCTCCACTTGTATTAAGAAGTGCAGAAGAAGGATTGATTTCAATAGTAAATACA

AATAAAGAGGGAGAAAATCAAAGAGACTTAAAAGTCATTTCGAGAGAACACTTTATTAGAGGAATTTTAAATTCTAA

AAGCAATGATGCAAAGGGAATCAAATCATCTAAACTAAAAGTTTGGGGTGACTTGAAGTGGGATGGACTCATCTATA

ATCCTAGAGGTAGAGAAGAAAATGCACCAGAAAGTAAGGATAATCAAGATCCTGCTACTAAGATAAGAGGTCAATTT

GAACCGATTGCGGAAGGTCAATATTTCTATAAATTTAAATATAGATTAACTAAAGATTACCCATGGCAGGTTTCCTA

TATTCCTGTAAAAATTGATAACACCGCCCCTAAGATTGTTTCGGTTGATTTTTCAAATCCTGAAAAAATTAAGTTGA

TTACAAAGGATACTTATCATAAGGTAAAAGATCAGTATAAGAATGAAACGCTATTTGCGAGAGATCAAAAAGAACAT

CCTGAAAAATTTGACGAGATTGCGAACGAAGTTTGGTATGCTGGCGCCGCTCTTGTTAATGAAGATGGAGAGGTTGA

AAAAAATCTTGAAGTAACTTACGCAGGTGAGGGTCAAGGAAGAAATAGAAAACTTGATAAAGACGGAAATACCATTT

ATGAAATTAAAGGTGCGGGAGATTTAAGGGGAAAAATCATTGAAGTCATTGCATTAGATGGTTCTAGCAATTTCACA

AAGATTCATAGAATTAAATTTGCTAATCAGGCTGATGAAAAGGGGATGATTTCCTATTATCTAGTAGATCCTGATCA

AGATTCATCTAAATATCAA

SEQ ID NO：31

SP0641N (nucleotides)

ATGGTAGTCTTAGCAGACACATCTAGCTCTGAAGATGCTTTAAACATCTCTGATAAAGAAAAAGTAGCAGAAAATAA

AGAGAAACATGAAAATATCCATAGTGCTATGGAAACTTCACAGGATTTTAAAGAGAAGAAAACAGCAGTCATTAAGG

AAAAAGAAGTTGTTAGTAAAAATCCTGTGATAGACAATAACACTAGCAATGAAGAAGCAAAAATCAAAGAAGAAAAT

TCCAATAAATCCCAAGGAGATTATACGGACTCATTTGTGAATAAAAACACAGAAAATCCCAAAAAAGAAGATAAAGT

TGTCTATATTGCTGAATTTAAAGATAAAGAATCTGGAGAAAAAGCAATCAAGGAACTATCCAGTCTTAAGAATACAA

AAGTTTTATATACTTATGATAGAATTTTTAACGGTAGTGCCATAGAAACAACTCCAGATAACTTGGACAAAATTAAA

CAAATAGAAGGTATTTCATCGGTTGAAAGGGCACAAAAAGTCCAACCCATGATGAATCATGCCAGAAAGGAAATTGG

AGTTGAGGAAGCTATTGATTACCTAAAGTCTATCAATGCTCCGTTTGGGAAAAATTTTGATGGTAGAGGTATGGTCA

TTTCAAATATCGATACTGGAACAGATTATAGACATAAGGCTATGAGAATCGATGATGATGCCAAAGCCTCAATGAGA

TTTAAAAAAGAAGACTTAAAAGGCACTGATAAAAATTATTGGTTGAGTGATAAAATCCCTCATGCGTTCAATTATTA

TAATGGTGGCAAAATCACTGTAGAAAAATATGATGATGGAAGGGATTATTTTGACCCACATGGGATGCATATTGCAG

GGATTCTTGCTGGAAATGATACTGAACAAGACATCAAAAACTTTAACGGCATAGATGGAATTGCACCTAATGCACAA

ATTTTCTCTTACAAAATGTATTCTGACGCAGGATCTGGGTTTGCGGGTGATGAAACAATGTTTCATGCTATTGAAGA

TTCTATCAAACACAACGTTGATGTTGTTTCGGTATCATCTGGTTTTACAGGAACAGGTCTTGTAGGTGAGAAATATT

GGCAAGCTATTCGGGCATTAAGAAAAGCAGGCATTCCAATGGTTGTCGCTACGGGTAACTATGCGACTTCTGCTTCA

AGTTCTTCATGGGATTTAGTAGCAAATAATCATCTGAAAATGACCGACACTGGAAATGTAACACGAACTGCAGCACA

TGAAGATGCGATAGCGGTCGCTTCTGCTAAAAATCAAACAGTTGAGTTTGATAAAGTTAACATAGGTGGAGAAAGTT

TTAAATACAGAAATATAGGGGCCTTTTTCGATAAGAGTAAAATCACAACAAATGAAGATGGAACAAAAGCTCCTAGT

AAATTAAAATTTGTATATATAGGCAAGGGGCAAGACCAAGATTTGATAGGTTTGGATCTTAGGGGCAAAATTGCAGT

AATGGATAGAATTTATACAAAGGATTTAAAAAATGCTTTTAAAAAAGCTATGGATAAGGGTGCACGCGCCATTATGG

TTGTAAATACTGTAAATTACTACAATAGAGATAATTGGACAGAGCTTCCAGCTATGGGATATGAAGCGGATGAAGGT

ACTAAAAGTCAAGTGTTTTCAATTTCAGGAGATGATGGTGTAAAGCTATGGAACATGATTAATCCTGATAAAAAAAC

TGAAGTCAAAAGAAATAATAAAGAAGATTTTAAAGATAAATTGGAGCAATACTATCCAATTGATATGGAAAGTTTTA

ATTCCAACAAACCGAATGTAGGTGACGAAAAAGAGATTGACTTTAAGTTTGCACCTGACACAGACAAAGAACTCTAT

AAAGAAGATATCATCGTTCCAGCAGGATCTACATCTTGGGGGCCAAGAATAGATTTACTTTTAAAACCCGATGTTTC

AGCACCTGGTAAAAATATTAAATCCACGCTTAATGTTATTAATGGCAAATCAACTTATGGC

SEQ ID NO：32

HHHHHH

SEQ ID NO：33

MSYYHHHHHH

SEQ ID NO：265

SP1912

MNGMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIF

EDGRHYTFVYENEDLVYEEEVL

SEQ ID NO：266

SP1912L

MRYLATLLLSLAVLITAGCKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIFEDGRHYT

FVYENEDLVYEEEVL

SEQ ID NO：267

SP0641.1

DTSSSEDALNISDKEKVAENKEKHENIHSAMETSQDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAKIKEENSNKSQ

GDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDNLDKIKQIEGI

SSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDAKASMRFKKED

LKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGIAPNAQIFSYK

MYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNYATSASSSSWD

LVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDGTKAPSKLKFV

YIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGYEADEGTKSQV

FSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDTDKELYKEDII

VPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYGYMSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLK

GDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQGAGLINVANALRNEVVATFKNTDSKGLVNSYGSISL

KEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKLDETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDT

FTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEALNSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAW

EEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGVIQNRKDKNTTSL

SEQ ID NO：268

SP1912 consensus sequence

MNGMKAKKMWMAGLALLGIGSLALATKKVADDRKLMKTQEELTEIVRDHFSDMGEIATLYVQVYESSLESLVGGVIF

H A L I L S

EDGRHYTFVYENEDLVYEEEVL

I

SEQ ID NO：269

SP641N consensus sequence

MVVLADTSSSEDALNISDKEKVA-----ENKEKHENIHSAMETSQDFKEKKTAVIKEKEVVSKNPVIDNNTSNEEAK

- N S VVDKET KD N I K TE TI EG A T TK R

L

IKEENSNKSQGDYTDSFVNKNTENPKKEDKVVYIAEFKDKESGEKAIKELSSLKNTKVLYTYDRIFNGSAIETTPDN

D- Q H Q S Q N G Q

NAH SA G RL G

LDKIKQIEGISSVERAQKVQPMMNHARKEIGVEEAIDYLKSINAPFGKNFDGRGMVISNIDTGTDYRHKAMRIDDDA

T I

KASMRFKKEDLKGTDKNYWLSDKIPHAFNYYNGGKITVEKYDDGRDYFDPHGMHIAGILAGNDTEQDIKNFNGIDGI

APNAQIFSYKMYSDAGSGFAGDETMFHAIEDSIKHNVDVVSVSSGFTGTGLVGEKYWQAIRALRKAGIPMVVATGNY

ATSASSSSWDLVANNHLKMTDTGNVTRTAAHEDAIAVASAKNQTVEFDKVNIGGESFKYRNIGAFFDKSKITTNEDG

Q N

TKAPSKLKFVYIGKGQDQDLIGLDLRGKIAVMDRIYTKDLKNAFKKAMDKGARAIMVVNTVNYYNRDNWTELPAMGY

EADEGTKSQVFSISGDDGVKLWNMINPDKKTEVKRNNKEDFKDKLEQYYPIDMESFNSNKPNVGDEKEIDFKFAPDT

N

DKELYKEDIIVPAGSTSWGPRIDLLLKPDVSAPGKNIKSTLNVINGKSTYG

SEQ ID NO：270

SP641M consensus sequence

MSGTSMATPIVAASTVLIRPKLKEMLERPVLKNLKGDDKIDLTSLTKIALQNTARPMMDATSWKEKSQYFASPRQQG

K T

AGLINVANALRNEVVATFKNTDSKGLVNSYGSISLKEIKGDKKYFTIKLHNTSNRPLTFKVSASAITTDSLTDRLKL

V

DETYKDEKSPDGKQIVPEIHPEKVKGANITFEHDTFTIGANSSFDLNAVINVGEAKNKNKFVESFIHFESVEEMEAL

Y R A

NSNGKKINFQPSLSMPLMGFAGNWNHEPILDKWAWEEGSRSKTLGGYDDDGKPKIPGTLNKGIGGEHGIDKFNPAGV

S TD K ME

IQNRKDKNTTSLDQNPELFAFNNEGINAPSSSGSKIANIYPLDSNGNPQDAQLERGLTPSPLVLRSAEEGLISIVNT

R D D Q VH E T

NKEGENQRDLKVISREHFIRGILNSKSNDAKGIKSSKLKVWGDLKWDGLIYNPRGREENAPESKDNQDPATKIRGQF

K V G

EPIAEGQYFYKFKYRLTKDYPWQVSYIPVKIDNTAPKIVSVDFSNPEKIKLITKDTYHKVKDQYKNETLFARDQKEH

PEKFDEIANEVWYAGAALVNEDGEVEKNLEVTYAGEGQGRNRKLDKDGNTIYEIKGAGDLRGKIIEVIALDGSSNFT

S A

KIHRIKFANQADEKGMISYYLVDPDQDSSKYQ

DH K A E

SEQ ID NO：271

SP1912 (nucleotide)

ATGAATGGTATGAAAGCTAAAAAAATGTGGATGGCAGGCTTGGCTCTGCTAGGTATCGGAAGCCTTGCTCTTGCTAC

GAAAAAAGTTGCAGATGACCGTAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCG

ACATGGGGGAAATTGCGACCCTTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTT

GAGGATGGCCGTCATTATACCTTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTATGA

SEQ ID NO：272

SP1912L (nucleotide)

ATGAGATACCTGGCAACATTGTTGTTATCTCTGGCGGTGTTAATCACCGCCGGGTGCAAAAAAGTTGCAGATGACCG

TAAGCTCATGAAGACTCAGGAAGAGTTGACAGAGATTGTGCGAGACCATTTTTCCGACATGGGGGAAATTGCGACCC

TTTATGTTCAAGTTTACGAAAGCAGTCTGGAGAGCTTGGTTGGTGGCGTCATTTTTGAGGATGGCCGTCATTATACC

TTTGTCTATGAAAATGAAGACCTAGTCTATGAGGAGGAAGTCTTATGA

SEQ ID NO：273

SP0641.1 (nucleotide)

GACACATCTAGCTCTGAAGATGCTTTAAACATCTCTGATAAAGAAAAAGTAGCAGAAAATAAAGAGAAACATGAAAA

TATCCATAGTGCTATGGAAACTTCACAGGATTTTAAAGAGAAGAAAACAGCAGTCATTAAGGAAAAAGAAGTTGTTA

GTAAAAATCCTGTGATAGACAATAACACTAGCAATGAAGAAGCAAAAATCAAAGAAGAAAATTCCAATAAATCCCAA

GGAGATTATACGGACTCATTTGTGAATAAAAACACAGAAAATCCCAAAAAAGAAGATAAAGTTGTCTATATTGCTGA

ATTTAAAGATAAAGAATCTGGAGAAAAAGCAATCAAGGAACTATCCAGTCTTAAGAATACAAAAGTTTTATATACTT

ATGATAGAATTTTTAACGGTAGTGCCATAGAAACAACTCCAGATAACTTGGACAAAATTAAACAAATAGAAGGTATT

TCATCGGTTGAAAGGGCACAAAAAGTCCAACCCATGATGAATCATGCCAGAAAGGAAATTGGAGTTGAGGAAGCTAT

TGATTACCTAAAGTCTATCAATGCTCCGTTTGGGAAAAATTTTGATGGTAGAGGTATGGTCATTTCAAATATCGATA

CTGGAACAGATTATAGACATAAGGCTATGAGAATCGATGATGATGCCAAAGCCTCAATGAGATTTAAAAAAGAAGAC

TTAAAAGGCACTGATAAAAATTATTGGTTGAGTGATAAAATCCCTCATGCGTTCAATTATTATAATGGTGGCAAAAT

CACTGTAGAAAAATATGATGATGGAAGGGATTATTTTGACCCACATGGGATGCATATTGCAGGGATTCTTGCTGGAA

ATGATACTGAACAAGACATCAAAAACTTTAACGGCATAGATGGAATTGCACCTAATGCACAAATTTTCTCTTACAAA

ATGTATTCTGACGCAGGATCTGGGTTTGCGGGTGATGAAACAATGTTTCATGCTATTGAAGATTCTATCAAACACAA

CGTTGATGTTGTTTCGGTATCATCTGGTTTTACAGGAACAGGTCTTGTAGGTGAGAAATATTGGCAAGCTATTCGGG

CATTAAGAAAAGCAGGCATTCCAATGGTTGTCGCTACGGGTAACTATGCGACTTCTGCTTCAAGTTCTTCATGGGAT

TTAGTAGCAAATAATCATCTGAAAATGACCGACACTGGAAATGTAACACGAACTGCAGCACATGAAGATGCGATAGC

GGTCGCTTCTGCTAAAAATCAAACAGTTGAGTTTGATAAAGTTAACATAGGTGGAGAAAGTTTTAAATACAGAAATA

TAGGGGCCTTTTTCGATAAGAGTAAAATCACAACAAATGAAGATGGAACAAAAGCTCCTAGTAAATTAAAATTTGTA

TATATAGGCAAGGGGCAAGACCAAGATTTGATAGGTTTGGATCTTAGGGGCAAAATTGCAGTAATGGATAGAATTTA

TACAAAGGATTTAAAAAATGCTTTTAAAAAAGCTATGGATAAGGGTGCACGCGCCATTATGGTTGTAAATACTGTAA

ATTACTACAATAGAGATAATTGGACAGAGCTTCCAGCTATGGGATATGAAGCGGATGAAGGTACTAAAAGTCAAGTG

TTTTCAATTTCAGGAGATGATGGTGTAAAGCTATGGAACATGATTAATCCTGATAAAAAAACTGAAGTCAAAAGAAA

TAATAAAGAAGATTTTAAAGATAAATTGGAGCAATACTATCCAATTGATATGGAAAGTTTTAATTCCAACAAACCGA

ATGTAGGTGACGAAAAAGAGATTGACTTTAAGTTTGCACCTGACACAGACAAAGAACTCTATAAAGAAGATATCATC

GTTCCAGCAGGATCTACATCTTGGGGGCCAAGAATAGATTTACTTTTAAAACCCGATGTTTCAGCACCTGGTAAAAA

TATTAAATCCACGCTTAATGTTATTAATGGCAAATCAACTTATGGCTATATGTCAGGAACTAGTATGGCGACTCCAA

TCGTGGCAGCTTCTACTGTTTTGATTAGACCGAAATTAAAGGAAATGCTTGAAAGACCTGTATTGAAAAATCTTAAG

GGAGATGACAAAATAGATCTTACAAGTCTTACAAAAATTGCCCTACAAAATACTGCGCGACCTATGATGGATGCAAC

TTCTTGGAAAGAAAAAAGTCAATACTTTGCATCACCTAGACAACAGGGAGCAGGCCTAATTAATGTGGCCAATGCTT

TGAGAAATGAAGTTGTAGCAACTTTCAAAAACACTGATTCTAAAGGTTTGGTAAACTCATATGGTTCCATTTCTCTT

AAAGAAATAAAAGGTGATAAAAAATACTTTACAATCAAGCTTCACAATACATCAAACAGACCTTTGACTTTTAAAGT

TTCAGCATCAGCGATAACTACAGATTCTCTAACTGACAGATTAAAACTTGATGAAACATATAAAGATGAAAAATCTC

CAGATGGTAAGCAAATTGTTCCAGAAATTCACCCAGAAAAAGTCAAAGGAGCAAATATCACATTTGAGCATGATACT

TTCACTATAGGCGCAAATTCTAGCTTTGATTTGAATGCGGTTATAAATGTTGGAGAGGCCAAAAACAAAAATAAATT

TGTAGAATCATTTATTCATTTTGAGTCAGTGGAAGAAATGGAAGCTCTAAACTCCAACGGGAAGAAAATAAACTTCC

AACCTTCTTTGTCGATGCCTCTAATGGGATTTGCTGGGAATTGGAACCACGAACCAATCCTTGATAAATGGGCTTGG

GAAGAAGGGTCAAGATCAAAAACACTGGGAGGTTATGATGATGATGGTAAACCGAAAATTCCAGGAACCTTAAATAA

GGGAATTGGTGGAGAACATGGTATAGATAAATTTAATCCAGCAGGAGTTATACAAAATAGAAAAGATAAAAATACAA

CATCCCTG

SEQ ID NO：274

Canonical lipid box motifs

[LIVMFESTAGPC]-[LVIAMFTG]-[IVMSTAGCP]-[AGS]-C

SEQ ID NO：275

SP2108 Signal sequence

MSSKFMKSAAVLGTATLASLLLVAC

SEQ ID NO：276

Coli RlpB signal sequence

MRYLATLLLSLAVLITAG[C]

SEQ ID NO：301

Immunogenic PspA/PspC polypeptides comprising a coiled coil structure (PR + NPB)

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVA

KLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSG

HMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNE

PEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQ

QPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMWCRQACGRTRAPPPPPLRSGC

SEQ ID NO：302

Immunogenic PspA/PspC polypeptides (PR only) comprising a coiled coil structure

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVA

KLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSG

HMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADLKKAVNE

PETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKP

APAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMWCRQACGRTRAPPPPPLRSG

SEQ ID NO：303

Immunogenic PspA/PspC polypeptides (PR + NPB) lacking coiled-coil structure

DLKKAVNEPEKPAEEPENPAPAPKPAPAPQPEKPAPAPAPKPEKSADQQAEEDYARR

SEEEYNRLTQQQPPKAEKPAPAPVPKPEQPAPAPKTGWGQENGMW

SEQ ID NO：304

Immunogenic PspA/PspC polypeptides (PR only) lacking coiled-coil structure

DLKKAVNEPETPAPAPAPAPAPAPTPEAPAPAPAPAPKPAPAPKPAPAPKPAPAPKPA

PAPKPAPAPKPAPAPAPAPKPEKPAEKPAPAPKPETPKTGWKQENGMW

SEQ ID NO：305

Immunogenic PspA/PspC polypeptides (PR + NPB) lacking coiled-coil structure

MAKKAELEKTPEKPAEEPENPAPAPQPEKSADQQAEEDYARRSEEEYNRLTQQQPPKA

SEQ ID NO：306

Non-proline block (NPB)

EKSADQQAEEDYARRSEEEYNRLTQQQ

SEQ ID NO：307

Non-proline block (NPB)

DQQAEEDYARRSEEEYNRLTQQQ

SEQ ID NO：308

Non-proline block (NPB)

MEKSADQQAEEDYARRSEEEYNRLTQQQ

SEQ ID NO：309

Amino terminal boundary of PR region

DLKKAVNE

SEQ ID NO：310

Carboxy-terminal boundaries of PR regions

(K/G)TGW(K/G)QENGMW

Claims (35)

1. A vaccine formulation comprising a pharmaceutically acceptable carrier and a polypeptide having an amino acid sequence comprising SEQ id No. 265 or 268 or an immunogenic fragment thereof.

2. The vaccine formulation of claim 1, wherein the polypeptide comprises an exogenous signal sequence.

3. The vaccine formulation of claim 2, wherein the polypeptide has an amino acid sequence comprising SEQ ID NO 266 or an immunogenic fragment thereof.

4. The vaccine formulation of claim 1, wherein the polypeptide has an amino acid sequence consisting of SEQ ID NO 265 or 268.

5. The vaccine agent of claim 1, wherein said polypeptide has an amino acid sequence consisting of SEQ ID NO 266.

6. The vaccine formulation of claim 1, further comprising a first polypeptide having an amino acid sequence comprising one of SEQ ID NOs 1-23, 267 and 269-270 or an immunogenic fragment thereof.

7. The vaccine formulation of claim 6, further comprising a second polypeptide having an amino acid sequence comprising any one of SEQ ID NOs 1-23, 267 and 269-270 or an immunogenic fragment thereof.

8. The vaccine formulation of claim 7, wherein said first and said second polypeptides belong to different groups of (i) - (viii):

(i) 1 or an immunogenic fragment thereof,

(ii) 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) 8 or an immunogenic fragment thereof,

(v) 9-10 and 20-21 or an immunogenic fragment thereof,

(vi) 11-13, 267 and 269-270 or immunogenic fragments thereof,

(vii) 22 or an immunogenic fragment thereof, and

(viii) 23 or an immunogenic fragment thereof.

9. The vaccine formulation of claim 6, comprising a polypeptide having an amino acid sequence comprising SEQ ID NO 6.

10. The vaccine formulation of claim 6, comprising a polypeptide having an amino acid sequence comprising SEQ ID NO 7.

11. The vaccine formulation of claim 6, comprising a polypeptide having an amino acid sequence comprising SEQ ID NO 9.

12. The vaccine formulation of claim 6, comprising a polypeptide having an amino acid sequence comprising SEQ ID NO 10.

13. The vaccine formulation of claim 7, wherein the vaccine formulation comprises a polypeptide consisting of SEQ ID NO 6 or 7 and a polypeptide consisting of SEQ ID NO 9 or 10.

14. The vaccine formulation of any one of claims 1 to 13, wherein the polypeptide of SEQ ID NO 265, 266 or 268 is a truncated fragment having 1-20 amino acid residues removed from the N-terminus, C-terminus, or both termini.

15. The vaccine formulation of any one of claims 1 to 14, substantially free of other streptococcus pneumoniae polypeptides than polypeptides having amino acid sequences comprising any one of SEQ ID NOs 1-23 and 265-270.

16. The vaccine formulation of claim 1, further comprising one or more polypeptides having an amino acid sequence comprising SEQ ID NO 22 or 23 or an immunogenic fragment thereof.

17. The vaccine formulation of any one of claims 1 to 16, wherein one or more polypeptides are conjugated to an immunogenic carrier.

18. The vaccine formulation of any one of claims 1 to 17, comprising at least one lipidated polypeptide.

19. The vaccine formulation of any one of claims 1 to 18, further comprising an adjuvant.

20. The vaccine formulation of claim 19, wherein the adjuvant is a toll-like receptor (TLR) agonist.

21. The vaccine formulation of claim 19, wherein the adjuvant is alum.

22. The vaccine formulation of claim 19, wherein the vaccine formulation comprises 1-1000 μ g of each polypeptide and 1-250 μ g of the adjuvant.

23. The vaccine formulation of any one of claims 1 to 22, which is on contact T_H17 cells later induced T than induced by control unrelated antigen_H17 cell response is at least 1.5 fold greaterT_H17 cell response.

24. The vaccine formulation of any one of claims 1-23, wherein the vaccine formulation inhibits infection by Streptococcus pneumoniae in an uninfected subject.

25. The vaccine formulation of any one of claims 1-24, wherein the vaccine formulation inhibits colonization of streptococcus pneumoniae in an individual.

26. The vaccine formulation of any one of claims 1-25, wherein the vaccine formulation inhibits streptococcus pneumoniae symptoms.

27. The vaccine formulation of claim 26, wherein the vaccine formulation inhibits streptococcus pneumoniae-induced sepsis.

28. A method for treating a subject suffering from or susceptible to infection by streptococcus pneumoniae infection comprising administering an effective amount of a vaccine formulation according to any one of claims 1 to 27.

29. The method of claim 28, wherein the method inhibits infection by streptococcus pneumoniae in an uninfected subject.

30. The method of claim 28, wherein the method inhibits colonization of streptococcus pneumoniae in the subject.

31. The method of claim 28, wherein the method inhibits streptococcus pneumoniae symptoms.

32. The method of claim 28, wherein the method inhibits streptococcus pneumoniae-induced sepsis.

33. The method of claim 28, wherein the method treats the subject with one dose.

34. The method of claim 28, wherein the method treats the subject within three doses.

35. The method of claim 28, wherein the subject is a human.