CROSS-REFERENCE TO RELATED APPLICATIONS The present applications claims priority to U.S. Serial No. 61/289,236, filed December

22, 2009; and U.S. Serial No. 61/325,660, filed April 19, 2010, which are incorporated by reference herein in their entireties.

FIELD OF INVENTION

The present invention relates to the field of immunology and, in particular, to

Streptococcus pneumoniae antigens and their use in immunization.

BACKGROUND

Streptococcus pneumoniae is a rather ubiquitous human pathogen, frequently found in the upper respirator}- tract of health}- children and adults. These bacteria can infect several organs including the lungs, the central nervous system (CNS), the middle ear, and the nasal tract and cause a range of diseases (i.e., symptomatic infections) such as for example, sinus infection, otitis media, bronchitis, pneumonia, meningitis, and bacteremia (septicemia). Pneumococcal meningitis, the most severe form of these pneumococcal diseases, is associated with significant mortality and morbidity despite antibiotic treatment (Quagliarello et. al. ( 1992) N. Engl. J. Med. 327: 864-872). Children under the age of two and the elderly are particular!}' susceptible to symptomatic pneumococcal infections.

Currently, there are two available types of pneumococcal vaccines. The first includes capsular polysaccharides from 23 types of S. pneumoniae, which together represent the capsular types of about 90% of strains causing pneumococcal infection. This vaccine, however, is not very immunogenic in young children, an age group with heightened susceptibility to

pneumococcal infection as the}' do not generate a good immune response to polysaccharide antigens prior to 2 years of age. In adults the vaccine has been shown to be about 60% efficacious against bacteremic pneumonia, but it is less efficacious in adults at higher risk of pneumococcal infection because of age or underlying medical conditions (Fedson, and Musher 2004, ^" Pneumococcal Polysaccharide Vaccine ^" , pp. 529-588; In Vaccines. S.A. Plotikin and W.A. Orenstein (eds.), W.B. Saunders and Co., Philadelphia, PA; Shapiro et. al., N. Engl. J. Med. 325 : 1453-1460 ( 1991)).

The second available type are conjugate vaccines. These vaccines which include serotype specific capsular poh saccharide antigens conjugated to a protein carrier, elicit serotype- specific protection (9). Currently available are 7-valent and 13-valent conjugate vaccines: the 7- valent includes 7 poh saccharide antigens (derived from the capsules of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and the 13-valent includes 13 poh saccharide antigens (derived from the capsules of serotypes 1, 3, 5, 6A, 7F and 19 A, in addition to those covered by the 7-valent). A 9-valent and 1 1-valent conjugate vaccine have also been developed and each includes polysaccharides specific for serotypes not covered by the 7-valent (i.e., serotypes 1 and 5 in the 9- valent and types 3 and 7F in the 1 1-valent).

The manufacture of conjugate vaccines is complex and costly due in part to the need to produce 7 (or 9 or 11) different polysaccharides each conjugated to the protein carrier. Such vaccines also do not do a good job of covering infections in the developing world where serotypes of Streptococcus pneumoniae not covered by the conjugate vaccines are very common (Di Fabio et al, Pediatr. Infect. Dis. J. 20:959-967 (2001); Mulholland, Trop. Med. Int. Health 10:497-500 (2005)). The use of the 7-valent conjugate vaccine has also been shown to have led to an increase in colonization and disease w ith strains of capsule types not represented by the 7 polysaccharides included in the vaccine (Bogaert et al.. Lancet Infect. Dis. 4: 144-154 (2004); Eskola et aL N. Engl. J. Med. 344-403-409 (2001); Mbelle et al., J. Infect. Dis. 180 : 1 171-1 176 ( 1999)).

As an alternative to the poh saccharide based vaccines currently available, a number of S. pneumoniae antigens have been suggested as possible candidates for a protein-based vaccine against S. pneumoniae. To date, however, no such vaccine is currently available on the market. Therefore, a need remains for effective treatments for S. pneumoniae.

SUMMARY

Immunogenic compositions and methods for eliciting an immune response against Streptococcus infections (such as e.g., S. pneumoniae) are described. More particularly, the present disclosure relates to immunogenic compositions comprising immunogenic PcpA poh peptides and/or immunogenic poh peptides of the poh histidine triad famih' (PhtX: PhtA, B, D, E), methods for their production and their use. Immunogenic PcpA and PhtX polypeptides (e.g. PhtD), including fragments of PcpA and PhtD and variants of each, and nucleic acids that encode the poh peptides are also provided. Immunogenic compositions comprising immunogenic PcpA poh peptides and/or immunogenic poh peptides of the polyhistidine triad family (PhtX: PhtA, B, D, E), and/or detoxified pneumolysis Further provided, are methods of preparing antibodies against Streptococcus poh peptides and methods for treating and/or preventing Streptococcus infection (e.g., S. pneumoniae infection) using such antibodies.

Also provided are compositions, such as pharmaceutical compositions (e.g., vaccine compositions), including one or more immunogenic PcpA poh peptides, PhtX poh peptides and/or detoxified pneumolysin proteins. Optionally, the compositions can include an adjuvant. The compositions ma}' also include one or more pharmaceutically acceptable excipients, which increase the thermal stability of the polypeptides/proteins relative to a composition lacking the one or more pharmaceutically acceptable excipients. In one example, the one or more pharmaceutically acceptable excipients increase the thermal stability of PcpA, PhtX and/or detoxified pneumolysin protein by 0.5°C or more, relative to a composition lacking the one oi ^¬ more pharmaceutically acceptable excipients. The compositions can be in liquid form, dry powder form, freeze dried, spray dried and or foam dried. The one or more pharmaceutically acceptable excipients can be for example, selected from the group consisting of buffers, tonicity agents, simple carbohydrates, sugars, carbohydrate polymers, amino acids, oligopeptides, polyamino acids, polyhydric alcohols and ethers thereof, detergents, lipids, surfactants, antioxidants, salts, human serum albumin, gelatins, formaldehyde, or combinations thereof.

Also provided are methods of inducing an immune response to S. pneumoniae in a subject, which involve administering to the subject a composition as described herein. Use of the compositions of the invention in inducing an immune response to S. pneumoniae in a subject, or in preparation of medicaments for use in this purpose is also provided.

The invention provides several advantages. For example, administration of the compositions of the present invention to a subject elicits an immune response against infections by a number of strains of S. pneumoniae. In addition, the multivalent compositions of the present invention include specific combinations of immunogenic poh peptides of S. pneumoniae which when administered do not experience antigenic interference and ma}' provide additive effects. Use of the excipients described herein can result in increased thermal stability of the

polypeptides/proteins within the compositions.

Other features and advantages of the invention will be apparent from the following Detailed Description, the Drawings and the Claims. BRIEF DESCRIPTION OF FIGURES

The present invention will be further understood from the follow ing description with reference to the drawings, in which:

Figure 1 Depicts the serum anti-protein IgG antibody titres of mice immunized with

5 varying doses of PcpA and PhtD (Example 2). In this study, recombinant PhtD and PcpA w ere combined with AIOOH adjuvant as monovalent or bivalent formulations. Balb/c mice were immunized subcutaneoush' 3 times at 3 weeks interval, and blood was collected prior to the first immunization and following the first, second and third immunizations. IgG titers were assessed by end-point ELISAs. All mice that had received PcpA and PhtD proteins generated antigenic ⁾ specific antibody responses after immunization.

Figure 2 a to d Depicts the serum anti-protein IgG antibody titres of rats immunized with 50 μg antigen/dose of PcpA and/or PhtD. In this stud}', rats were immunized on days 0, 21 and 42 with either a control of Tris Buffered Saline ( 10 mM Tris pH 7.4, 150 mM NaCl), aluminum hydroxide adjuvanted bivalent PhtD and PcpA, unadjuvanted bivalent PhtD and PcpA oi ^¬ ls aluminum hydroxide adjuvanted PcpA using 50 μg antigen/dose. Sera from pretest, da}' 44 and da}' 57 bleeds were tested for antibody titers to PhtD and PcpA specific IgG antibody titers by ELISA.

Figure 3 Depicts the survival percentage for each group of mice immunized (Example 5).

In this study, a bivalent formulation of recombinant PhtD and PcpA w as evaluated using an 20 intranasal challenge model. Immunized animals were challenged with a lethal dose of an S. pneumoniae strain (MD, 14453 or 941192).

Figure 4a, 4b. Figure 4a depicts the total antigen-specific IgG titres measured by endpoint dilution ELISA and geometric mean titres (+/- SD) for each group. Figure 4b depicts total antigen-specific titres measured by quantitative ELISA. In this stud}' (Example 7), bivalent 25 compositions of PhtD and PcpA were prepared (using two different lots of each of PhtD and PcpA) and formulated with phosphate treated AIOOH (2mM). Groups of 6 female CBA/j mice were immunized intramuscularly or subcutaneoush' three times at 3 week intervals with the applicable formulation. Mice were challenged a lethal dose of S. pneumoniae strain MD following the third (final) bleed.

30 Figure 5 Depicts the survival percentage for each group. In this stud}' (Example 6), bivalent compositions of PhtD and PcpA were prepared (using two different lots of each of PhtD and PcpA) and formulated with phosphate treated AIOOH (2mM). Groups of 6 female CBA/j mice were immunized intramuscularly or subcutaneous!}' three times at 3 week intervals with the applicable formulation. Mice were challenged a lethal dose of S. pneumoniae strain MD following the third bleed.

Figure 6 Depicts Recognition of PcpA and PhtD on bacterial surface by Corresponding Rabbit Antisera on Various Pneumococcal Strains Grown in Mn2+ Depleted Media (Example 9).

Figure 7 Depicts Binding of Purified Human Anti-PcpA and Anti-PhtD Antibodies to proteins (PcpA, PhtD) on bacterial cell surface of Strain WU2 (Example 9).

Figure 8 Depicts % survival observed per log dilution of sera administered (Example 10).

Figure 9 Depicts summary of the total IgG titers measured by ELISA (Example 11) Figure 10a to f The stability of PcpA and PhtD in monovalent and bivalent formulations

(formulated with AIO(OH) or phosphate treated AIO(OH) (PTH). Formulations were prepared using AIO(OH) or PTH w ith a final concentration of 2mM phosphate and then incubated at various temperatures (i.e., 5°C, 25°C, 37°C or 45°C). Intact antigen concentration was then assessed by RP-HPLC.

Figure 11 Stability of PhtD and PcpA under stress conditions as evaluated by ELISA. Bivalent formulations at 100 μg/mL w ere incubated at 37°C for 12 w eeks and the antigenicity was evaluated by ELISA.

Figure 12A Studies of excipient effects on the stability of PcpA (stored at 50°C for three day s) in the presence of 10% sorbitol (■), 10% trehalose (·), 10% sucrose (Δ), TBS pH 9.0 (♦), and TBS pH 7.4 (o) by RP-HPLC.

Figure 12B Studies of excipient effects on the antigenicity of PcpA (stored at 50°C for three day s) in the presence of 10% sorbitol, 10% trehalose, 10% sucrose, TBS pH 9.0, and TBS pH 7.4 by quantitative ELISA sandwich. Formulations were stored at 50°C for three day s. Antigenicity w as evaluated for each formulation at time zero (white bars) and following three da}' storage (black bars).

Figure 13 Effect of pH on the physical stability of adjuvanted proteins. PcpA (A), PhtD (B) and PlyDl (C) were adjuvanted with aluminum hydroxide or aluminum phosphate at different pH values and the Tm values w ere obtained by derivative analysis of the fluorescence traces.

Figure 14 Depicts the total antigen-specific IgG titres measured by endpoint dilution ELISA and geometric mean titres (+/- SD) for each group. Figures 15 A, B, C Depicts the total antigen-specific IgG titres elicited as measured by ELISA per antigen dose administered to mice.

DETAILED DESCRIPTION OF INVENTION

Compositions and methods for eliciting an immune response against S. pneumoniae and for treating and preventing disease caused by S. pneumoniae in mammals, such as for example in humans are described. Provided are immunogenic compositions comprising immunogenic PcpA polypeptides and/or immunogenic polypeptides of the polyhistidine triad family (PhtX: PhtA, PhtB, PhtD, PhtE), methods for their production and their use. The compositions may include detoxified pneumoh sin or immunogenic fragments thereof. Methods include passive and active immunization approaches, which include administration (e.g, subcutaneous, intramuscular) of immunogenic compositions comprising one or more substantial!}' purified Streptococcal (e.g., S. pneumoniae) polypeptides, antibodies to the polypeptides themselves, or a combination thereof. The invention also includes Streptococcus sp. (e.g. , S. pneumoniae) polypeptides, immunogenic compositions (e.g. , vaccines) comprising Streptococcal polypeptides, methods of producing such compositions, and methods of producing Streptococcal (e.g. , S. pneumoniae) antibodies. These methods and compositions are described further, below.

The compositions of the invention include one, two, three or more immunogenic polypeptides. The compositions ma}' include for example, individual!}' or in combination, an immunogenic poh peptide of PcpA; an immunogenic poh peptide of a member of the poh histidine triad family of proteins (e.g. , PhtA, PhtB, PhtD, and PhtE, referenced herein as PhtX proteins); a detoxified pneumoh sin poh peptide. Immunogenic fragments and fusions of these polypeptides ma}' also be included in the compositions (e.g., a fusion of PhtB and PhtE). These immunogenic polypeptides ma}' optionally be used in combination with pneumococcal saccharides or other pneumococcal polypeptides.

In one multi-component example, the immunogenic composition includes an immunogenic PcpA poh peptide and one or more immunogenic PhtX polypeptides. A preferred embodiment of such a composition comprises an immunogenic PhtD poh peptide and an immunogenic PcpA poh peptide. In another example, the composition includes an immunogenic PcpA poh peptide, an immunogenic PhtX poh peptide (e.g., PhtD) and detoxified pneumoh sin. Certain emodiments of the immunogenic composition (in e.g., bivalent and trivalent form) are described in the Examples herein. Polypeptides

Immunogenic PcpA polypeptides comprise the full-length PcpA amino acid sequence (in the presence or absence of the signal sequence), fragments thereof and variants thereof. PcpA polypeptides suitable for use in the compositions described herein include, for example, those of GenBank Accession No. CAB04758 from S. pneumoniae strain B6, GenBank Accession No. NP_from S. pneumoniae strain TIGR4 and GenBank Accession No. NP_359536 from

S.pneumoniae strain R6, and those from S. pneumoniae strain 14453.

The amino acid sequence of full length PcpA in the S. pneumoniae 14453 genome is SEQ ID NO. 2. Preferred PcpA polypeptides for use with the invention comprise an amino acid sequence having 50% or more identity (e.g, 60, 65, 70, 75. 80, 85, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99, 99.5% or more) to SEQ ID NO:2 or SEQ ID NO:7. Preferred polypeptides for use with the invention comprise a fragment of at least 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more consecutive amino acids of SEQ ID NO:2. Preferred fragments comprise an epitope from SEQ ID NO.2. Other preferred fragments lack one or more amino acids from the N-terminus of SEQ ID NO. 2 (e.g., 1, 2, 3, 4,5,6,7,8, 9, 10, 15,20, 25 or more) and/or one or more amino acids from the C -terminus of SEQ ID NO:2 while retaining at least one epitope of SEQ ID NO:2. Further preferred fragments lack the signal sequence from the N-terminus of SEQ ID NO:2. A preferred PcpA polypeptide is SEQ ID NO: 7.

Optionally, immunogenic polypeptides of PcpA comprise one or more leucine rich regions (LRRs). These LLRs are present in naturally occurring PcpA or have about 60 to about 99% sequence identity, including, for example, 80%,85%,90% or 95% sequence identity to the naturally occurring LRRs. LRRs in the mature PcpA protein (i.e., the protein lacking the signal peptide) can be found in certain sequences disclosed in WO 2008/022302 (e.g., SEQ ID NOs: 1,2, 41 and 45 of WO 2008/022302).

An immunogenic polypeptide of PcpA optionally lacks the choline binding domain anchor sequence typically present in the naturally occurring mature PcpA protein. The naturally occurring sequence of the choline binding anchor of the mature PcpA protein is disclosed in WO 2008/022302 as SEQ ID NO:52. More particularly, an immunogenic polypeptide comprises an N-terminal region of naturally occurring PcpA with one or more amino acid substitutions and about 60 to about 99% sequence identity or an}' identity in between, e.g. 80, 85, 90 and 95% identity, to the naturally occurring PcpA. The N-terminal region ma}' comprise the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NOs: 1, 2,3,4,41 or 45 of WO2008/022302), in the presence or absence of one or more conservative amino acid substitutions and in the presence or absence of the signal sequence. The N-terminal region ma}' comprise an amino acid sequence having about 60 to about 99% sequence identity (or an}' identity in between 80 to 99% identity) to SEQ ID NOs: 1 or 7 (set out in the Sequence Listing herein) or SEQ ID NOs: 1, 2,3,4, or 41 of WO2008/022302.

Immunogenic fragments of SEQ ID NOs: 2 and 7 comprise 5, 10, 20, 30, 40, 50, 60, 70,

80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190 and 191 amino acid residues of SEQ ID NOs: 2 and 7 or an}- number of amino acid residues between 5 and 191. Examples of immunogenic fragments of PcpA are disclosed in WO 2008/022302.

Optionally, immunogenic polypeptides of PcpA lack the LRRs. Examples of immunogenic polypeptides lacking the LRR are disclosed in WO 2008/022302 as SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

Immunogenic PhtX polypeptides suitable for the compositions of the invention comprise the full-length PhtA, PhtB, PhtD or PhtE amino acid sequence (in the presence or absence of the signal sequence), immunogenic fragments thereof, variants thereof and fusion proteins thereof. PhtD polypeptides suitable for use in the compositions described herein include, for example, those of GenBank Accession Nos. AAK06760, YP816370 and NP35851, among others. The amino acid sequence of full length PhtD in the S. pneumoniae 14453 genome is SEQ ID NO: 1. A preferred polypeptide of PhtD (derived from the S. pneumonaie 14453 genome) is SEQ ID NO:5.

The immunogenic fragments of PhtX polypeptides of the present invention are capable of eliciting an immune response specific for the corresponding full length mature amino acid sequence.

Immunogenic PhtX (e.g., PhtD) polypeptides include the full length protein with the signal sequence attached, the mature full length protein with the signal peptide (e.g., 20 amino acids at N-terminus) removed, variants of PhtX (naturally occurring or otherwise, e.g, synthetically derived) and immunogenic fragments of PhtX (e.g, fragments comprising at least 15 or 20 contiguous amino acids present in the naturally occurring mature PhtX protein).

Examples of immunogenic fragments of PhtD are disclosed in PCT publication

WO2009/012588.

Preferred PhtD polypeptides for use with the invention comprise an amino acid sequence having 50% or more identity (e.g, 60, 65, 70, 75. 80, 85, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99, 99.5% or more) to SEQ ID NO: 1 or to SEQ ID NO:5. Preferred polypeptides for use with the invention comprise a fragment of at least 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more consecutive amino acids of SEQ ID NO: l . Preferred fragments comprise an epitope from SEQ ID NO.1 or to SEQ ID NO:5. Other preferred fragments lack one or more amino acids from the N-terminus of SEQ ID NO. 1 (e.g., 1, 2, 3, 4,5,6,7,8, 9, 10, 15,20, 25 or more) and/or one or amino acids from the C-terminus of SEQ ID NO: 1 while retaining at least one epitope of SEQ ID NO: 1. Further preferred fragments lack the signal sequence from the N-terminus of SEQ ID NO: 1. A preferred PhtD poh peptide is SEQ ID NO:5.

Pneumoh sin (Ply) is a cytolytic-activating toxin implicated in multiple steps of pneumococcal pathogenesis, including the inhibition of ciliary beating and the disruption of tight junctions between epithelial cells (Hirst et al. Clinical and Experimental Immunology (2004)). Several pneumolyses are known and (following detoxification) would be suitable for use in the compositions described herein including, for example GenBank Accession Nos. Q04IN8, P0C2J9, Q7ZAK5, and AB021381, among others. In one embodiment. Ply has the amino acid sequence shown in SEQ ID NO.10.

Immunogenic pneumoh sin polypeptides for use with the invention include the full length protein with the signal sequence attached, the mature full length protein with the signal peptide removed, variants of pneumoh sin (naturally occurring or otherwise, e.g., synthetically derived) and immunogenic fragments of pneumoh sin (e.g, fragments comprising at least 15 or 20 contiguous amino acids present in the naturally occurring mature pneumoh sin protein).

Immunogenic variants and fragments of the immunogenic pneumoh sin polypeptides of the present invention are capable of eliciting an immune response specific for the corresponding full length mature amino acid sequence. The immunogenic pneumoh sin polypeptides of the present invention are detoxified; that is, the}' lack or have reduced toxicity as compared to the mature wild-type pneumoh sin protein produced and released by S. pneumoniae. The immunogenic pneumoh sin polypeptides of the present invention ma}' be detoxified for example, chemically (e.g., using formaldehyde treatment) or genetically (e.g., recombinantly produced in a mutated form).

Preferred examples of the immunogenic detoxified pneumoh sin for use in the present invention are disclosed in PCT Publication No. WO 2010/071986. As disclosed in that application, the detoxified pneumoh sin ma}' be a mutant pneumoh sin protein comprising amino acid substitutions at positions 65, 293 and 428 of the wild type sequence. In a preferred detoxified pneumoh sin protein, the three amino acid substitutions comprise T ₆₅ ->C, and A preferred immunogenic and detoxified pneumolysin polypeptide is SEQ ID NO:9.

Preferred pneumoysin polypeptides for use with the invention comprise an amino acid sequence having 50% or more identity (e.g., 60, 65, 70, 75, 80, 85, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more) to SEQ ID NO:9 or to SEQ ID NO: 10. Preferred pohpeptides for use with the invention comprise a fragment of at least 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more consecutive amino acids of SEQ ID NO:9 or 10. Preferred fragments comprise an epitope from SEQ ID NO.9 or to SEQ ID NO: 10. Other preferred fragments lack one or more amino acids from the N-terminus of SEQ ID NO. 9 or 10 (e.g., 1, 2, 3, 4,5,6,7,8, 9, 10, 15,20, 25 or more) and/or one or amino acids from the C -terminus of SEQ ID NO: 9 or 10 while retaining at least one epitope of SEQ ID NO: 9 or 10. Further preferred fragments lack the signal sequence from the N-terminus of SEQ ID NO: 10.

The immunogenic poh peptides of PcpA, PhtX (e.g., PhtD), and pneumolysin described herein, and fragments thereof, include variants. Such variants of the immunogenic poh peptides described herein are selected for their immunogenic capacity using methods well known in the art and ma}' comprise one or more conservative amino acid modifications. Variants of the immunogenic poh peptides (of PcpA, PhtD, pneumolysin) include amino acid sequence having about 60 to about 99% sequence identity (or any identity in between 60 and 99% identity) to the disclosed sequences (i.e., SEQ ID NO:2 or 7 (PcpA); SEQ ID NO: 1 or 5 (PhtD); SEQ ID NO: 9 or 10 (PI)'))- Amino acid sequence modifications include substitutional, insertional or deletional changes. Substitutions, deletions, insertions or an}- combination thereof ma)' be combined in a single variant so long as the variant is an immunogenic poh peptide. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarih' will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically no more than about from 2 to 6 residues are deleted at an}- one site within the protein molecule. These variants ordinarih' are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in a recombinant cell culture. Techniques for making substitution mutations are predetermined sites in DNA having a known sequence are well known and include, but are not limited to. Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues but can occur at a number of different locations at once. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table and are referred to as consen ative substitutions. Others are well known to those of skill in the art.

As used herein, the amino acid substitution ma}' be consen ative or non-conservative. Consen ative amino acid substitutions ma}' involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position and, in particular, does not result in decreased immunogenicity. Suitable consen ative amino acid substitutions are shown in the Table 1 below.

TABLE 1

The specific amino acid substitution selected ma}' depend on the location of the site selected. In certain embodiments, nucleotides encoding polypeptides and /or fragments are substituted based on the degeneracy of the genetic code (i.e, consistent with the "Wobble ^" hypothesis). Where the nucleic acid is a recombinant DNA molecule useful for expressing a poh peptide in a cell (e.g., an expression vector), a Wobble-type substitution will result in the expression of a poh peptide with the same amino acid sequence as that originalh' encoded by the DNA molecule. As described above, however, substitutions ma}' be consen ative, or non- conservative, or any combination thereof. A skilled artisan will be able to determine suitable variants of the poh peptides and /or fragments provided herein using well-known techniques.

Analogs can differ from naturally occurring S. pneumoniae poh peptides in amino acid sequence and/or by virtue of non-sequence modifications. Non-sequence modifications include changes in acetylation, methylation, phosph} orylation, carboxylation, or glycosylation. A ^" modification ^" of a polypeptide of the present invention includes poh peptides (or analogs thereof, such as, e.g. fragments thereof) that are chemically or enzymatically derived at one or more constituent amino acid. Such modifications can include, for example, side chain modifications, backbone modifications, and N- and C- terminal modifications such as, for example, acetylation, hydroxylation, methylation, amidation, and the attachment of carbonhydrate or lipid moieties, cofactors, and the like, and combinations thereof. Modified poh peptides of the invention ma}' retain the biological activity of the unmodified poh peptides or ma}' exhibit a reduced or increased biological activity.

Structural similarity of two poh peptides can be determined by aligning the residues of the two poh peptides (for example, a candidate polypeptide and the polypeptide of, for example, SEQ ID NO: 2) to optimize the number of identical amino acids along the length of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate polypeptide is the polypeptide being compared to the reference polypeptide. A candidate polypeptide can be isolated, for example, from a microbe, or can be produced using a recombinant techniques, or chemically or enzaymatically synthesized.

A pair-wise comparison analysis of amino acids sequences can be carried out using a global algorithm, for example, Needleman-Wunsch. Alternative!}', poh peptides ma}' be compared using a local alignment algorithm such as the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol. Lett, 174 247-250 ( 1999), and available on the National Centre for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters ma}' be used, including matrix = BLOSUM62; open gap penalty = 1 1, extension gap penalty = 1, gap x dropoff = 50, expect 10, wordsize = 3, and filter on. The Smith and Waterman algorithm is another local alignment tool that can be used ( 1988).

In the comparison of two amino acid sequences, structural similarly ma}' be referred to by percent ^" identity ^" or ma}' be referred to by percent ^" similarity." ^" Identity" refers to the presence of identical amino acids. ^" Similarity" refers to the presences of not only identical amino acid but also the presence of conservative substitutions. A conservative substitution for an amino acid in a polypeptide of the inv ention ma}' be selected from other members of the class to which the amino acid belongs, shown on Table 1.

The nucleic acids encoding the immunogenic polypeptides ma}' be isolated for example, but without limitation from wild type or mutant S. pneumoniae cells or alternatively, ma}' be obtained directly from the DNA of an S. pneumoniae strain carrying the applicable DNA gene (e.g., pcpA, phtD, ply), by using the polymerase chain reaction (PCR) or by using alternative standard techniques that are recognized by one skilled in the art. Possible strains of use include for example, S. pneumoniae strains TIGR4 and 14453. In preferred embodiments the

polypeptides are recombinant!}' derived from S. pneumoniae strain 14453. Preferred examples of the isolated nucleic acid molecules of the present invention have nucleic acid sequences set out in SEQ ID NOs: 3, 4, 6 and 8. Sequence-conservative variants and function-conservative variants of these sequences are encompassed by the present invention.

The polypeptides of the present invention can be produced using standard molecular biology techniques and expression systems (see for example. Molecular Cloning: A Laboratory Manual, Third Edition by Sambrook et. al.. Cold Spring Harbor Press, 2001). For example, a fragment of a gene that encodes an immunogenic polypeptide ma}' be isolated and the polynucleotide encoding the immunogenic polypeptide ma}' be cloned into an}' commercially available expression vector (such as, e.g., pBR322, and pUC vectors (New England Biolabs, Inc., Ipswich, MA)) or expression /purification vectors (such as e.g., GST fusion vectors (Pfizer, Inc., Piscataway, N.J.)) and then expressed in a suitable prokaryotic, viral or eukaryotic host.

Purification ma}' then be achieved by conventional means, or in the case of a commerical expression/purification system, in accordance with manufacturer's instructions.

Alternatively, the immunogenic polypeptides of the present invention, including variants, ma}' be isolated for example, but without limitation, from wild -type or mutant S. pneumoniae cells, and through chemical synthesization using commercially automated procedures, such as for example, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or solution synthesis.

Polypeptides of the present invention preferably have immunogenic activity.

^" Immunogenic activity ^" refers to the ability of a polypeptide to elicit an immunological response in a subject. An immunological response to a polypeptide is the development in a subject of a cellular and / or antibody-mediated immune response to the polypeptide. Usually, an immunogical response includes but is not limited to one or more of the following effects: the product of antibodies, B cells, helper T cells, suppressor T cells and / or cytotoxic T cells, directed to an epitope or epitodes of the polypeptide. The term "Epitope ^" refers to the site on an antigen to which specific B cells and / or T cells respond so that antibody is produced. The immunogenic activity ma}' be protective. The term "Protective immunogenic activity ^" refers to the ability of a polypeptide to elicit an immunogical response in a subject that prevents or inhibits infection by S. pneumoniae (resulting in disease).

C '(impositions

The disclosed immunogenic S. pneumoniae polypeptides are used to produce immunogenic compositions such as, for example, vaccine compositions. An immunogenic composition is one that, upon administration to a subject (e.g., a mammal), induces or enhances an immune response directed against the antigen contained within the composition. This response ma}' include the generation of antibodies (e.g, through the stimulation of B cells) or a T cell-based response (e.g., a cytolytic response). These responses ma}' or ma}' not be protective oi ^¬ neutralizing. A protective or neutralizing immune response is one that is detrimental to the infectious organism corresponding to the antigen (e.g, from which the antigen was derived) and beneficial to the subject (e.g., by reducing or preventing infection). As used herein, protective or neutralizing antibodies ma}' be reactive to the corresponding wild-type S. pneumoniae polypeptide (or fragment thereof) and reduce or inhibit the lethality of the corresponding wild- type S. pneumoniae polypeptide when tested in animals. An immunogenic composition that, upon administration to a host, results in a protective or neutralizing immune response ma}' be considered a vaccine.

The compositions include immunogenic polypeptides in amounts sufficient to elicit an immune response when administered to a subject. Immunogenic compositions used as vaccines comprise an immunogenic polypeptide in an immunologically effective amount, as well as any other components, as needed. By 'immunologically effective amount', it is meant that the administration of that amount to a subject, either in a single dose or as part of a series, is effective for treatment or prevention.

In compositions that are comprised of two, three or more immunogenic polypeptides (e.g., PcpA, PhtD, and/or detoxified pneumolysin), the polypeptide components are preferably compatible and are combined in appropriate ratios to avoid antigenic interference and to optimize an}- possible synergies. For example, the amounts of each component can be in the range of about 5 μg to about 500 μg per dose, 5 μg to about 100 μg per dose; or 25 μg to about 50 μg per dose. Preferably the range can be 5 or 6 μg to 50 μg per antigenic component per dose. In one example, a composition includes 25 μg of an immunogenic poh peptide of PhtX (e.g., PhtD) and 25 μg of an immunogenic poh peptide of PcpA. The composition, in a different example, also includes 25 μg of pneumolysin (e.g. detoxified pneumolysin; PlyD l (SEQ ID NO:9).

In the Examples set out below, in animal models, various antigen ratios were compared for a two- component vaccine composition of PhtX (e.g., PhtD) and PcpA, and for a three- component vaccine composition of PcpA, PhtX (e.g., PhtD) and detoxified pneumolysin (e.g., PlyD l). Surprisingly, statistically significant antigenic interference was not observed at the antigen ratios tested. Also, surprisingly antigen-specific antibodies elicited in response to immunization with the bivalent composition (or trivalent composition) w ere found to act in an additive manner in a passive immunization stud}' in mice using rabbit sera. Thus, in a multi- component composition these components ma}' be present in equivalent amounts (e.g. 1 : 1, 1 : 1 : 1). The components ma}' be present in other ratios having regard to the estimated minimum antigen dose for each antigen (e.g., PcpA:PhtX(PhtD):Pneumolysin, about 1 : 1 : 1 to about 1 :5 :25). In one example, a trivalent composition comprises PcpA, PhtD and pneumolysin (e.g. PlyD l) in amounts (ug/dose) at a ratio of PcpA:PhtD:pneumoh sin of 1 :4: 8. In a different example, the ratio of PcpA:PhtD:pneumoh sin is 1 : 1 : 1.

Compositions of the invention can be administered by an appropriate route such as for example, percutaneous (e.g., intramuscular, intravenous, intraperitoneal or subcutaneous), transdermal, mucosal (e.g., intranasal) or topical, in amounts and in regimes determined to be appropriate by those skilled in the art. For example, 1- 250 g or 10-100 μg of the composition can be administered. For the purposes of prophylaxis or therapy, the composition can be administered 1, 2, 3, 4 or more times. In one example, the one or more administrations ma}' occur as part of a ^" prime-boost ^" protocol. When multiple doses are administered, the doses can be separated from one another by, for example, one week, one month or several months.

Compositions (e.g., vaccine compositions) of the present invention ma}' be administered in the presence or absence of an adjuvant. Adjuvants generally are substances that can enhance the immunogenicity of antigens. Adjuvants ma}' play a role in both acquired and innate immunity (e.g., toll-like receptors) and ma}' function in a variety of ways, not all of which are understood.

Man}- substances, both natural and synthetic, have been shown to function as adjuvants. For example, adjuvants ma}' include, but are not limited to, mineral salts, squalene mixtures, muram} 1 peptide, saponin derivatives, mycobacterium cell wall preparations, certain emulsions. monophosphon i lipid A, mycolic acid derivatives, nonionic block copoh mer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, immunostimulating complexes (ISCOMs), cytokine adjuvants, MF59 adjuvant, lipid adjuvants, mucosal adjuvants, certain bacterial exotoxins and other components, certain oligonucleotides, PLG, and others. These adjuvants ma}' be used in the compositions and methods described herein.

In certain embodiments, the composition is administered in the presence of an adjuvant that comprises an oil-in- ater emulsion comprising at least squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, a hydrophobic nonionic surfactant, wherein said oil-in-water emulsion is obtainable by a phase inversion temperature process and w herein 90% of the population by volume of the oil drops has a size less than 200 nm, and optionally less than 150 nm. Such an adjuvant is described in WO2007006939 (Vaccine

Composition Comprising a Thermoinversable Emulsion) which is incorporate herein in its entirety. The composition ma}' also include the product E6020 (having CAS Number 287180-63- 6), in addition to, or instead of the described squalene oil-in-water emulsion. Product E6020 is described in US2007/0082875 (which is incorporated herein by reference in its entirety).

In certain embodiments, the composition includes a TLR agonist (e.g. , TLR4 agonist) alone or together in combination with an adjuvant. For example, the adjuvant ma}' comprise a TLR4 agonist (e.g. , TLA4), squalene, an aqueous solvent, a nonionic hydrophilic surfactant belonging to the poh oxyethylene alkyl ether chemical group, a nonionic hydrophobic surfactant and which is thermoreversible. Examples of such adjuvants are described in WO2007080308 (Thermoreversible Oil-in-Water Emulsion) which is incorporated herein in its entirety. In one embodiment, the composition is adjuvanted with a combination of CpG and an aluminum salt adjuvant (e.g.. Alum).

Aluminum salt adjuvants (or compounds) are among the adjuvants of use in the practice of the invention. Examples of aluminum salt adjuvants of use include aluminum hydroxide (e.g., crystalline aluminum oxyh} droxide AIO(OH), and aluminum hydroxide Al(OH) ₃ . Aluminum hydroxide is an aluminum compound comprising Al ³⁺ ions and hydrox} 1 groups (-OH).

Mixtures of aluminum hydroxide with other aluminum compounds (e.g., hydroxyphosphate or hydroxysulfate) ma}' also be of use where the resulting mixture is an aluminum compound comprising rrydroxyl groups. In particular embodiments, the aluminum adjuvant is aluminum oxyhydroxide (e.g., Alhydrogel*). It is well known in the art that compositions with aluminum salt adjuvants should not be exposed to extreme temperatures, i.e. below freezing (0°C) or extreme heat (e.g., > 70 °C) as such exposure ma}' adversely affect the stability and the immunogenicity of both the adsorbed antigen and adjuvant.

The inventors have noted that the degradation rate of PcpA and PhtD polypeptides when adjuvanted with aluminum hydroxide adjuvant (AIO(OH)) is high (as discussed in the examples below). The inventors have found that adjuvanting PcpA and PhtD polypeptides with an aluminum compound comprising hydroxide groups (e.g., aluminum hydroxide adjuvant) that has been pretreated with phosphate, carbonate, sulfate, carboxylate, diphosphonate or a mixture of two or more of these compounds, increases the stability of these polypeptides. Thus, provided herein are formulations of compositions comprising an immunogenic PcpA polypeptide or an immunogenic PhtX polypeptide (e.g., PhtD) and an aluminum compound comprising hy droxide groups that has been treated with phosphate, carbonate, sulfate, carboxy late, diphosphonate or a mixture of two or more of these compounds, w here the treatment increases the stability of the immunogenic polypeptide relative to a composition where the polypeptide is adsorbed to an untreated aluminum compound. In preferred embodiments the aluminum compound is treated with phosphate. Multivalent compositions comprising both immunogenic polypeptides of PcpA and PhtX (e.g., PhtD) and an aluminum compound comprising hydroxide groups that has been treated with phosphate, carbonate, sulfate, carboxylate, diphosphonate or a mixture of two or more of these compounds, w here the treatment increases the stability of the immunogenic polypeptides relative to a composition where the polypeptide is adsorbed to an untreated aluminum compound are also provided.

In a particular embodiment of the invention, the aluminum compound (e.g., aluminum hydroxide adjuvant) is treated with phosphate, carbonate, sulfate, carboxylate, diphosphonate, or a mixture of two or more of these compounds. By treating the aluminum compound in this way a number of the hydroxyl groups (-OH) in the aluminum compound are replaced w ith the corresponding ion with which it is being treated (e.g., phosphate (P0 ₄ )). This replacement lowers the PZC of the aluminum compound and the pH of the compound's microenvironment. The phosphate, carbonate, sulfate, carboxylate, or diphosphonate ions are added in an amount sufficient to low er the pH of the microenvironment to a lev el at w hich the antigen is stabilized (i.e., the rate of antigen hydrolysis is decreased). The amount necessary will depend on a number of factors such as, for example, the antigen involved, the antigen's isoelectric point, the antigen's concentration, the adjuvanting method utilized, and the amount and nature of any additional antigens present in the formulation. Those skilled in the art in the field of vaccines are capable of assessing the relevant factors and determining the concentration of phosphate, carbonate. sulfate, carboxylate, diphosphonate to add to the aluminum compound to increase the stability of the antigen (and therefore, can prepare the corresponding formulation and composition). For example, titration studies (i.e. , adding increasing concentrations of phosphate, etc., to aluminum compound) ma}' be performed.

Phosphate compounds suitable for use include an}- of the chemical compounds related to phosphoric acid (such as for example, inorganic salts and organic esters of phosphoric acid). Phosphate salts are inorganic compounds containing the phosphate ion (P0 ₄ ³ ), the hydrogen phosphate ion (HP0 ² ) or the dihydrogen phosphate ion (H ₂ P0 ⁴ ) along with any cation.

Phosphate esters are organic compounds in which the hydrogens of phosphoric acid are replaced by organic groups. Examples of compounds that ma}' be used in place of phosphate salts include anionic amino acids (e.g., glutamate, aspartate) and phospholipids.

Carboxylate compounds suitable for use include an}- of the organic esters, salts and anions of carboxylic acids (e.g. , malic acid, lactic acid, fumaric acid, glutaric acid, EDTA, and EGTA). Sulfer anions suitable for use include an}- compound containing the sulfate (S0 ₄ radical) such as salts or esters of sulfuric acid (e.g., sodium sulfate, ammonium sulfate, sulfite, metabisulfite, thiosulfate). Examples of disphosphonate compounds suitable for use include clodronate, pamidronate, tiludronate, and alendronate.

In a preferred embodiment of the invention, phosphate is added to aluminum hydroxide adjuvant in the form of a salt. Preferabh', the phosphate ions are provided by a buffer solution comprising disodium monosodium phosphate.

In the preferred practice of the present invention, as exemplified herein, the aluminum compound (e.g., aluminum oxyhydroxide) is treated with phosphate (for example, by a process as described in the examples). In this process, an aqueous suspension of aluminum oxyhydroxide (approximate!}' 20 mg/mL) is mixed with a phosphate buffer solution (e.g., approximate!}' 400 mol/L). The preferable final phosphate concentration is from about 2 mM to 20mM. The mixture is then diluted with a buffer (e.g., Tris-HCl, Tris-HCl with saline, HEPES) to prepare a suspension of aluminum oxyhydroxide and phosphate (P0 ₄ ). Preferabh' the buffer is 10 mM Tris-HCl and 150 mM NaCl at a pH of about 7.4. The suspension is then mixed for

approximateh' 24 hr at room temperature. Preferabh' the concentration of elemental aluminum in the final suspension is within a range from about 0.28 mg/mL to 1.68 mg/mL. More preferabh', the concentration of elemental aluminum is about 0.56 mg/mL. Immunogenic polypeptides of Pep A, PhtD and detoxified pneumo sin (individually or in combination) ma}' then be adsorbed to the treated aluminum hydroxide. Preferably,

approximately 0.2-0.4 mg/mL of antigen is mixed with the suspension of treated aluminum hydroxide adjuvant (e.g. , at room temperature or at 2-8°C, in an orbital mixer, for approximately 30 min, or approximately 12- 15 hours, or approximately 24 hours).

The percentage of antigen adsorption ma}' be assessed using standard methods known in the art. For example, an aliquot of the antigen/adjuvant preparation ma}' be removed and centrifuged (e.g, at 10,000 rpm) to separate the unadsorbed protein (pellet) from the adjuvant suspension (supernatant). The concentration of protein in the supernatant ma}' be determined using the bicinchoninic acid protein assay (BCA) or reverse phase- high performance liquid chromatography (RP-HPLC). The percentage of adsorption is calculated as follows: %A=100- ([PrSN] x 100/[PrCtr]) where, [PrSN] is the concentration of protein in supernatant and [PfCtr] is the concentration in the corresponding unadjuvanted control. In preferred embodiments, the % adsorption ranges from about 70% to about 100%. In more preferred embodiments the % adsorption is at least about 70%.

In one embodiment of adjuvanted immunization, immunogenic polypeptides and / or fragments thereof ma}' be covalenth' coupled to bacterial poh saccharides to form poh saccharide conjugates. Such conjugates ma}' be useful as immunogens for eliciting a T cell dependent immunogenic response directed against the bacterial poh saccharide conjugated to the polypeptides and /or fragments thereof.

The disclosed formulations are stable when stored for prolonged time periods at conventional refrigeration temperatures, e.g., about 2 °C to about 8°C. The formulations exhibit little or no particle agglomeration, no significant decrease in antigen concentration and retain a significant level of immunogenicity and/or antigenicity for at least 6 months or 12 months and preferably for 18 months. The phrase ^" no significant decrease in antigen concentration ^" is intended to mean that the composition retains at least 50%, 60%, or 70% of the original antigen concentration, more preferably at least about 80%, 85%, or 90% of the original antigen concentration, more preferably at least about 91%, 92%, 98%, 99% or more of the antigen concentration present when first formulated. Antigen concentration ma}' be measured, for example, by an RP-HPLC, SDS-PAGE or ELISA-based method.

A stable formulation or an immunogenic composition comprising a stable formulation maintains a substantial degree of structural integrity (e.g., maintains a substantial amount of the original antigen concentration, etc.). Stability ma}' be assessed by measuring for example, the concentration of antigen present (e.g, by RP-HPLC) or by assessing antigen degradation for example by SDS-PAGE analysis. The antigen concentration in the formulation ma}' be compared with that of the formulation as prepared with the same aluminum compound albeit untreated (i.e., not treated with phosphate or carbonate ions). Stability prediction and/or comparison tools include for example. Stability

System™ (by ScienTek Software, Inc.), which use Arrhenius Treatment to predict rate constant at storage temperature (2 C-8 C). Standard assays for measuring the antigen concentration, and immunogenicity are known in the art and are described in the Examples. Protective efficacy may be assessed by for example evaluating the survival rates of immunized and non-immunized subjects following challenge with a disease causing pathogen or toxin corresponding to the particular antigen present in the formulation.

The immunogenic compositions of the present invention are preferably in liquid form, but the}' ma}' be h ophilized (as per standard methods) or foam dried (as described in

WO2009012601, Antigen- Adjuvant Compositions and Methods). A composition according to one embodiment of the invention is in a liquid form. An immunization dose ma}' be formulated in a volume of between 0.5 and 1.0 ml. Liquid formulations ma}' be in an}' form suitable for administration including for example, a solution, or suspension. Thus, the compositions can include a liquid medium (e.g., saline or water), which ma}' be buffered.

The pH of the formulation (and composition) is preferably between about 6.4 and about 8.4. More preferably, the pH is about 7.4. An exemplaiy pH range of the compositions is 5-10, e.g., 5-9, 5-8, 5.5-9, 6-7.5, or 6.5-7. The pH may be maintained by the use of a buffer.

The pharmaceutical formulations of the immunogenic compositions of the present invention ma}' also optionally include one or more excipients (e.g., diluents, thickeners, buffers, preservatives, surface active agents, adjuvants, detergents and/or immunostimulants) which are well known in the art. Suitable excipients will be compatible with the antigen and with the aluminum adjuvant as is known in the art. Examples of diluents include binder, disintegrants, or dispersants such as starch, cellulose derivatives, phenol, polyeth} lene gh col, propylene gh col or glycerin. Pharmaceutical formulations ma}' also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents and anesthetics. Examples of detergents include a Tween (polysorbate) such as Tween 80. Suitable excipients for inclusion in the composition of the invention are known in the art.

The invention provides compositions including PcpA, PhtX (e.g., PhtD) and/or detoxified pneumolysin proteins and one or more pharmaceutical!}' acceptable excipients that provide beneficial properties to the compositions (e.g., increase the stability of one or more of the proteins of the compositions). The compounds or excipients that can be included in the compositions of the invention include for example, buffers (e.g., glycine, histidine); tonicity agents (e.g, mannitol); carbohydrates, such as sugars or sugar alcohols (e.g., sorbitol, trehalose, or sucrose; 1- 30%) or carbohydrate polymers (e.g., dextran); amino acids, oligopeptides or polyamino acids (up to 100 mM); polyhydric alcohols (e.g., glycerol, and concentrations of up to 20%); detergents, lipids, or surfactants (e.g., T een 20, T een 80, or pluronics, with concentrations of up to 0.5%); antioxidants; salts (e.g., sodium chloride, potassium chloride, magnesium chloride, or magnesium acetate, up to 150 mM); or combinations thereof.

Examples of excipients that can be used in the compositions of the invention include those that are listed in Table 11, and the examples below. In various examples, the excipients ma}' be those that result in increased thermal stability (e.g., of at least 0.5, e.g., 0.5-5, 1-4, or 2-3) as measured by, e.g., the assay s described below (e.g., extrinsic fluorescence of SYPRO Orange).

Exemplar}- excipients and buffers include sorbitol (e.g., 4-20%, 5-10%), (see Table 11). These excipients can be used in the invention in the concentrations listed in Table 11.

Alternatively, the amounts can be varied by, e.g., 0.1-10 fold, as is understood in the art. Other carbohy drates, sugar alcohols, surfactants and amino acids that are known in the art can also be included in the composition of the invention.

The excipients and buffers can be used individually or in combination. The pH of such a composition can be, e.g., 5.5-8.0 or 6.5-7.5, and the composition can be stored at, e.g., 2-8°C, in liquid or lyophilized form. In variations of the composition, the sorbitol can be replaced with sucrose (e.g., 4-20%, or 5-10%), or trehalose (e.g., 4-20%, or 5-10%). Other variations of the compositions are included in the invention and involve use of other components listed herein. Based on the above, an exemplary' composition of the invention includes 10% sorbitol, pH 7.4.

In one embodiment, a monovalent PlyD 1 composition may include per dose, in the range of 5 to 50 μg of antigen, PTH adjuvant (with about 0.56 mg/mL elemental Aluminum containing 2 mM sodium phosphate buffer at about pH 7.5), in about: 10 mM Tris HCl, and about 150 mM NaCl, at about pH 7.4.

In another embodiment, a monovalent PhtD composition may include per dose, in the range of 5 to50 μg of antigen, PTH adjuvant (with about 0.56 mg/mL elemental Aluminum containing 2 mM sodium phosphate buffer at about pH 7.5), in about: 10 mM Tris HCl, and about 150 mM NaCl, at about pH 7.4. In a further embodiment, a monovalent PcpA composition ma}' include per dose, in the range of 5 to 50 μg of antigen, PTH adjuvant (with about 0.56 mg/mL elemental Aluminum containing 2 mM sodium phosphate buffer at about pH 7.5), in about: 10 mM Tris HCl, and about 150 mM NaCl, at about pH 7.4.

In another embodiment, a bivalent formulation composition ma}' include per dose, two proteins (selected from the following: PhtD, PlyD l or PcpA), each in the range of 5 to 50 μg/dose, PTH adjuvant (with about 0.56 mg/mL elemental Aluminum containing 2 mM sodium phosphate buffer at about pH 7.5), in about: 10 mM Tris HCl, and about 150 mM NaCl, at about pH 7.4.

In yet a further embodiment, a trivalent formulation composition can include per dose, three proteins (PhtD, PlyD l, PcpA), each in the range of 5 to 50 μg/dose, PTH adjuvant (with about 0.56 mg/mL elemental Aluminum containing 2 mM sodium phosphate buffer at about pH 7.5), in about: 10 mM Tris HCl, and about 150 mM NaCl, at about pH 7.4.

In another example, the compositions include sorbitol, or sucrose, which have been shown to provide benefits with respect to stability (see below). The amounts of these components can be, for example, 5-15%, 8-12% or 10% sorbitol or sucrose. A specific example in which these components are present at 10% is described below. In a preferred embodiment the compositions include 10% sorbitol or 10% sucrose.

The invention also includes methods of identifying excipients that can be used to generate compositions including S. pneumoniae proteins (e.g., PcpA, PhtX (e.g., PhtD), detoxified pneumolysin) having improved properties. These methods involve screening assays, such as those described further below, which facilitate the identification of conditions resulting in increased stabilits of one or more of the protein components of the compositions. These methods include stability assay s as described further below . Further, the invention includes the use of other assay s for identify ing desirable formulations, including solubility, immunogenicity and viscosity assays.

A composition according to one embodiment of the invention may be prepared by (i) treating an aluminum hydroxide adjuvant with phosphate, carbonate, sulfate, carboxy late, diphosphonate or a mixture of two or more of these compounds, and (ii) mixing the treated aluminum hydroxide adjuvant with an immunogenic PcpA polypeptide and/or an immunogenic PhtX poly peptide. In preferred embodiments, the immunogenic PhtX poly peptide is PhtD.

Immunogenic compositions (e.g. vaccines) containing one or more of the S. pneumoniae polypeptides of the present invention may be used to prevent and/or treat S. pneumoniae infections. The prophylactic and therapeutic methods of the invention involve vaccination with one or more of the disclosed immunogenic polypeptides in, for example, earn ing out the treatment itself, in preventing subsequent infection, or in the production of antibodies for subsequent use in passive immunization.

The immunogenic compositions of the invention find use in methods of preventing or treating a disease, disorder, condition or symptoms associated with or resulting from a S.

pneumonaie infection The terms disease disorder and condition are used interchangeably herein. Specifically the prophylactic and therapeutic methods comprise administration of a

therapeutically effective amount of a pharmaceutical composition to a subject. In particular embodiments, methods for preventing or treating S. pneumoniae are provided.

As used herein, preventing a disease or disorder is intended to mean administration of a therapeutically effective amount of a pharmaceutical composition of the invention to a subject in order to protect the subject from the development of the particular disease or disorder associated with S. pneumonaie.

By treating a disease or disorder is intended administration of a therapeutically effective amount of a pharmaceutical composition of the invention to a subject that is afflicted with a disease caused by S. pneumonaie or that has been exposed to S. pneumonaie where the purpose is to cure, heal, alleviate, releave, alter, remedy, ameliorate, improve, or affect the condition or the symptoms of the disease.

A therapeutically effective amount refers to an amount that provides a therapeutic effect for a given condition and administration regimen. A therapeutically effective amount can be determined by the ordinary skilled medical w orker based on patient characteristics (age, weight, gender, condition, complications other diseases etc.). The therapeutically effective amount will be further influenced by the route of administration of the composition.

Also disclosed, is a method of reducing the risk of a pneumococcal disease in a subject comprising administering to the subject an immunogenic composition comprising one or more of the disclosed immunogenic polypeptides. Pneumococcal diseases (i.e., symptomatic infections) include, for example, sinus infection, otitis media, bronchitis, pneumonia, meningitis, hemolytic uremia and bacteremia (septicemia). The risk of an}- one or more of these infections ma}' be reduced by the methods described herein. Preferred methods include a method of reducing the risk of invasive pneumococcal disease and/or pneumonia in a subject comprising administering to the subject an immunogenic composition comprising an immunogenic Pep A poh peptide and an immunogenic PhtX (e.g., PhtD) poh peptide. In other preferred methods, the composition also includes detoxified pneumolysin (e.g., PlyDl).

The present disclosure also provides methods of eliciting an immune response in a mammal by administering the immunogenic compositions, or formulations thereof, to subjects. This ma}' be achieved by the administration of a pharmaceuticalh' acceptable formulation of the compositions to the subject to effect exposure of the immunogenic poh peptide and/or adjuvant to the immune system of the subject. The administrations ma}' occur once or ma}' occur multiple times. In one example, the one or more administrations ma}' occur as part of a so-called "prime- boost" protocol. Other administration systems ma}' include time-release, delayed release or sustained release deliver}- systems.

Immunogenic compositions ma}' be presented in a kit form comprising the immunogenic composition and an adjuvant or a re constitution solution comprising one or more

pharmaceutical!}' acceptable diluents to facilitate re constitution of the composition for administration to a mammal using conventional or other devices. Such a kit would optionally include the device for administration of the liquid form of the composition (e.g. hypodermic syringe, microneedle array) and/or instructions for use.

The compositions and vaccines disclosed herein ma}' also be incorporated into various deliver}- systems. In one example, the compositions ma}' be applied to a "microneedle array" or "microneedle patch" deliver}' system for administration. These microneedle arrays or patches generalh' comprise a plurality of needle-like projections attached to a backing material and coated with a dried form of a vaccine. When applied to the skin of a mammal, the needle-like projections pierce the skin and achieve deliver}' of the vaccine, effecting immunization of the subject mammal.

Definitions

The term "antigen ^" as used herein refers to a substance that is capable of initiating and mediating the formation of a corresponding immune bod}' (antibody) when introduced into a mammal or can be bound by a major histocompatibility complex (MHC) and presented to a T- cell. An antigen ma}' possess multiple antigenic determinants such that the exposure of the mammal to an antigen ma}' produce a plurality of corresponding antibodies with differing specificities. Antigens ma}' include, but are not limited to proteins, peptides, polypeptides, nucleic acids and fragments, variants and combinations thereof. The term "immunogen ^" is a substance that is able to induce an adaptive immune response.

The terms peptides, proteins and poh peptides are used interchangeably herein.

An "isolated ^" polypeptide is one that has been removed from its natural environment. For instance, an isolated polypeptide is a polypeptide that has been removed from the cytoplasm or from the membrane of a cell, and man}- of the poh peptides, nucleic acids, and other cellular material of its natural environment are no longer present. An "isolatable ^" polypeptide is a polypeptide that could be isolated from a particular source. A "purified ^" polypeptide is one that is at least 60% free, preferabh' at least 75% free, and most preferabh' at least 90% free from other components with which the}' are naturally associated. Poh peptides that are produced outside the organism in which the}' naturally occur, e.g. through chemical or recombinant means, are considered to be isolated and purified by definition, since the}' were never present in a natural environment.

As used herein, a "fragment ^" of a polypeptide preferabh' has at least about 40 residues, oi ^¬ 60 residues, and preferabh' at least about 100 residues in length. Fragments of S. pneumoniae poh peptides can be generated by methods known to those skilled in the art.

The term "antibody" or "antibodies ^" includes whole or fragmented antibodies in unpurified or partially purified form (i.e., hybridoma supernatant, ascites, polyclonal antisera) or in purified form. A "purified ^" antibody is one that is separated from at least about 50% of the proteins with which it is initially found (i.e., as part of a hybridoma supernatant or ascites preparation).

As used in the specification and the appended claims, the singular forms "a ^" , "an ^" , and "the ^" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a fragment ma}' include mixtures of fragments and reference to a pharmaceutical carrier or adjuvant ma}' include mixtures of two or more such carriers or adjuvants.

As used herein, a subject or a host is meant to be an individual.

Optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase, "optionally the composition can comprise a combination ^" means that the composition ma}' comprise a combination of different molecules or ma}' not include a combination such that the description includes both the combination and the absence of the combination (i.e., individual members of the combination). Ranges ma}' be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximateh', it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independent ' of the other endpoint.

When the terms prevent, preventing, and prevention are used herein in connection with a given treatment for a given condition (e.g., preventing S. pneumoniae infection), it is meant to convey that the treated subject either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment. These terms are not limited solely to a situation in which the subject experiences no aspect of the condition whatsoever. For example, a treatment will be said to have prevented the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the subject's experiencing fewer and/or milder symptoms of the condition than otherwise expected. A treatment can "prevent ^" infection by resulting in the subject's displaying only mild overt symptoms of the infection; it does not imph' that there must have been no penetration of an}' cell by the infecting microorganism.

Similarly, reduce, reducing, and reduction as used herein in connection with the risk of infection with a given treatment (e.g., reducing the risk of a S.pneumoniae infection) refers to a subject developing an infection more slowly or to a lesser degree as compared to a control or basal level of developing an infection in the absence of a treatment (e.g., administration of an immunogenic polypeptide). A reduction in the risk of infection ma}' result in the subject displaying only mild overt symptoms of the infection or delayed symptoms of infection; it does not imph' that there must have been no penetration of an}' cell by the infecting microorganism.

All references cited within this disclosure are hereby incorporated by reference in their entirety.

EXAMPLES The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described soleh' for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances ma}' suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, immunology and fermentation technology used, but not explicitly described in this disclosure and these Examples, are amply reported in the scientific literatures and are well within the ability of those skilled in the art.

EXAMPLE 1A

Recombinant PcpA and PhtD polypeptides

This Example describes the preparation of the PcpA protein and PhtD protein recombinantly. In brief, two recombinantly-derived protein antigens from Streptococcus pneumoniae (strain 14453 (a mouse-virulent capsule serotype 6B strain), deposited on June 27, 1997 as ATCC 55987), PhtD (WO2009/012588) and PcpA (WO 2008/022302) were recombinantly expressed in Kcoli, isolated and purified by serial column chromatography following conventional purification protocols.

The phtD gene (but excluding its native signal peptide) was PCR amplified from the S. pneumoniae 14453 genome, using the AccuPrime High Fidelity polymerase (Invitrogen) and primers Spn0211 and Spn0213. Spn0211 and Spn0213 introduced Noel and Xhol restriction sites into the 5' and 3' ends, respectively (see Table 2). The PCR product was purified using a QIAquick PCR purification kit (Qiagen) and run on an agarose gene to confirm the size. The PCT product and the pET28a(+) vector (Novagen) were both digested with Ncol and Xhol and subsequently purified from an agarose gel using the QIAEX gel extraction kit (Qiagen). The digested vector and gene were ligated together using T4 DNA ligase (Invitrogen). The ligation mixture was transformed into chemically competent E.coli DH5a and positive clones were selected by plating on Luria agar containing 5(^g/ml kanamycin. DNA from plasmid clone pBAC27 was isolated and was confirmed by sequencing to be correct.

The plasmid (pBAC27) was then introduced into E.coli BL21 (DE3) cells by

electroporation. Transformed strains were grown at approximately 37°C and protein expression was induced by the addition of ImM IPTG. Expression of gene product was verified by the presence of an induced protein band of the correct size (i.e, approximately 91.9 kDa) by SDS- PAGE analysis. Table 2

The predicted amino acid sequence of the polypeptide of pBAC27 is as follows:

MGSYELGRHQAGQVKKESNRVSYIDGDQAGQKAENLTPDEVSKREGINAEQIVIKITDQG YVTSHGDHYHYY NGKVPYDAIISEELLMKDPNYQLKDSDIVNEIKGGYVIKVDGKYYVYLKDAAHADNIRTK EEIKRQKQEHSH NHNSPADNAVAAARAQGRYTTDDGYIFNASDIIEDTGDAYIVPHGDHYHYIPKNELSASE LAAAEAYWNGKQ GSRPSSSSSYNANPVQPRLSENHNLTVTPTYHQNQGENISSLLRELYAKPLSERHVESDG LIFDPAQITSRT ARGVAVPHGNHYHFIPYEQMSELEKRIARIIPLRYRSNHWVPDSRPEQPSPQSTPEPSPS LQPAPNPQPAPS NPIDEKLVKEAVRKVGDGYVFEENGVSRYIPAKDLSAETAAGIDSKLAKQESLSHKLGAK KTDLPSSDREFY NKAYDLLARIHQDLLDNKGRQVDFEVLDNLLERLKDVSSDKVKLVDDILAFLAPIRHPER LGKPNAQITYTD DEIQVAKLAGKYTTEDGYIFDPRDITSDEGDAYVTPHMTHSHWIKKDSLSEAEPAAAQAY AKEKGLTPPSTD HQDSGNTEAKGAEAIYNRVKAAKKVPLDRMPYNLQYTVEVKNGSLIIPHYDHYHNIKFEW FDEGLYEAPKGY SLEDLLATVKYYVEHPNERPHSDNGFGNASDHVRKNKADQDSKPDEDKEHDEVSEPTHPE SDEKENHAGLNP SADNLYKPSTDTEETEEEAEDTTDEAEIPQVENSVINAKIADAEALLEKVTDPSIRQNAM ETLTGLKSSLLL GTKDNNTISAEVDSLLALLKESQPAPIQ (SEQ ID No: 5)

The pcpA gene (but excluding the signal sequence and the choline-binding domains) was PCR amplified from the S. pneumoniae 14453 genome using Accuprime Taq DNA polymerase (Invitrogen) and PCR primers (see Table 3) that incorporated restriction endonuclease sites designed for simplified cloning. Plasmid DNA of pET-30a(+) (Novagen) was purified as a lo - cop}' plasmid and prepared for use as the cloning vector by digesting with Ndel and Xhol, follow ed by gel purification. The resulting 1335 base pair fragment w as pcpA (without signal sequence and choline-binding domains) flanked by Xhol (3 '-end) and Ndel (5 'end) restriction sites. The amplified fragment was cleaned, digested with Ndel and Xhol and then gel purified and ligated into the pET-30a(+) vector. The insert was verified by sequencing and the new ^r plasmid was designated pJMS87.

Table 3 (Primers) The predicted amino acid sequence of the polypeptide of pJMS87 is as follows:

MADTPSSEVIKETKVGSIIQQNNIKYKVLTVEGNIGTVQVGNGVTPVEFEAGQDGKPFTI PTKITVGDKVFT VTEVASQAFSYYPDETGRIVYYPSSITIPSSIKKIQKKGFHGSKAKTIIFDKGSQLEKIE DRAFDFSELEEI ELPASLEYIGTSAFSFSQKLKKLTFSSSSKLELISHEAFANLSNLEKLTLPKSVKTLGSN LFRLTTSLKHVD VEEGNESFASVDGVLFSKDKTQLIYYPSQKNDESYKTPKETKELASYSFNKNSYLKKLEL NEGLEKIGTFAF ADAIKLEEISLPNSLETIERLAFYGNLELKELILPDNVKNFGKHVMNGLPKLKSLTIGNN INSLPSFFLSGV LDSLKEIHIKNKSTEFSVKKDTFAIPETVKFYVTSEHIKDVLKSNLSTSNDIIVEKVDNI KQETDVAKPKKN SNQGWGWVKDKG (SEQ ID No : 7 )

Chemically competent E. coli BL21 (DE3) cells were transformed with plasmid pJMS87 DNA. Expression of gene product w as verified by the presence of an induced protein band of the correct size (i.e, approximately 49.4 kDa) by SDS-PAGE analysis.

As the cloned PcpA polypeptide lacks the signal sequence and choline-binding domains, its amino acid sequence correlates with amino acids 27 to 470 of the full length PcpA protein. This region is extremely conserved among all surveyed strains with only 8 variable positions. The most diverged pair of sequences shares 98.7% identity.

The predicted isoelectric points by Vector NTi for the recombinant PcpA protein and the recombinant PhtD protein were 7.19 and 5.16, respectively.

The pcpA gene and phtD gene were each detected in the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7, 7F, 9N, 9V, 11A/B, 11A/D/F, 12F/B, 14, 15B, 15B/C, 16, 18C, 19A, 19F, 22, 23, 23B, 23F, 33F, 34, 35B. A number of these serotypes are not covered by the currently marketed pneumococcal conjugate vaccine PCV7.

The recombinant protein products were expressed, isolated and purified using standard methods.

Adjuvanted monovalent compositions of either recombinant protein w ere prepared by formulating isolated purified protein with adjuvant (e.g.. Aluminum hydroxide adjuvant (e.g. Alhydrogel 85 2%) or A1P0 ₄ ) in Tris buffered saline (pH 7.4) using standard methods.

Formulated materials were transferred to glass vials and stored at 2°C to 8°C. Adjuvanted bivalent compositions of both PhtD and PcpA w ere prepared by aliquoting the desired concentration of each adjuvanted monovalent formulation into a vessel and mixing on a nutator for approximately 0.5 hours at room temperature. Desired formulation volumes were then aliquoted into sterile 3 mL glass vials with rubber stopper closure and aluminum cap.

Alternative!}', bivalent compositions w ere prepared by mixing the desired concentration of each isolated purified protein together and then formulating mixture with adjuvant in Tris buffered saline (pH 7.4). EXAMPLE IB

This Example describes the preparation of a surface modified adjuvant and formulations with this adjvuant. A surface modified adjuvant was prepared by treating aluminum hydroxide adjuvant (Alhydrogel™, Brenntag) with phosphate. The aluminum hydroxide adjuvant used was a wet gel suspension which according to the manufacturer tolerates re-autoclavation but is destroyed if frozen. According to the manufacturer, when the pH is maintained at 5-7, the adjuvant has a positive charge and can adsorb negatively charged antigens (e.g., proteins with acidic isoelectric points when kept at neutral pH).

a) Phosphate treatment of AIO(OH) - An aqueous suspension of AIO(OH) (approximate 20 mg/mL) w as mixed with a stock solution of phosphate buffer (approximateh' 400 mol/L) and diluted with 10 mM Tris-HCL buffer (Sigma Aldrich) at about pH 7.4 to prepare a phosphate- treated AIO(OH) suspension (herein referred to as "PTH ^" ) having approximateh' 13 mg/mL A1OOH/200 mM P04. This suspension was then mixed for approximateh' 30 minutes to 24 hr at room temperature.

b) Antigen adsorption - Recombinantly -derived PcpA and PhtD antigens (expressed, isolated and purified as described in Example 1A) were individually adsorbed to the phosphate- treated AIO(OH).

A mixture was prepared containing about 0.2 -0.4 mg/mL of purified antigen (i.e., rPcpA or rPhtD) each antigen and 0.56 mg elemental aluminum /ml/P04 mM of the PTH suspension. Alternatively, mixtures were prepared containing purified antigen with aluminum hydroxide adjuvant (as Alhydrogel ® 85 2%) or A1P04 in Tris buffered saline (pH 7.4) using standard methods. The mixtures wereas mixed in an orbital shaker for about 30 minutes to 24 hours at room temperature to facilitate the association of antigen and adjuvant. Similar adsorptions were prepared a number of times and the typical pre-adsorbed composition was: protein (PhtD or PcpA): 0.2-0.4 mg/ml, phosphate: 2 to 20 80 mM (preferably, 2 to 20 mM) and AIO(OH): 1.25 mg/ml (0.56 mg of elemental Al/ml). Prepared antigen adsorbed samples were stored at about 2°C - 8°C until used. Alternatively, antigens were adjuvanted together (to prepare bivalent formulations) by using a stock solution of phosphate treated aluminum hydroxide adjuvant. c) Preparation of a bivalent formulation - The intermediate bulk lots (monovalent formulations) of PhtD adsorbed to PTH and PcpA adsorbed to PTH w ere blended and mixed together for about 30 minutes at room temperature in an orbital shaker to prepare a bivalent formulation. The typical pre -adsorbed formulation composition was: 0.05 mg/ml of each protein (rPhtD, rPcpA); phosphate: 2 to 20 mM and 1.25 mg/mL AIO(OH) (0.56 mg of elemental Al/ml).

EXAMPLE 2

Assessment of antigenic interference and humoral response with bivalent compositions formulated with varying doses of PcpA and PhtD

This Example describes the analysis of the immunogenicity of a multi-component composition in animals. Formulations were prepared (as described in Example 1) using purified PhtD and PcpA proteins, aluminum hydroxide adjuvant (AltrydrogeP 85 2%, 25.52mg/mL), Tris buffered saline ( lOmM Tris-HCl pH 7.4/ 150mM NaCl). The formulations were mixed on a Nutator for approximately 30 minutes and dispensed into glass vials.

Groups of 10 female mice Balb/c K-72 mice (Charles River), 6 to 8 w eeks of age, w ere immunized subcutaneously (SC) three times at 3 week intervals with the applicable formulation:

A. (5 μg/mL of PcpA + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

B. (12.5 μg/mL of PcpA + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

C. (25 μg/mL of PcpA + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

D. (5 μg/mL of PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

E. (12.5 μg/mL of PhtD+ 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

F. (25 of PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

G. (5 f PcpA + 5 PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4 H. (5 μg/mL of PcpA + 12.5 μg/mL PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

I. (5 μg/mL of PcpA + 25 μg/mL PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4 J. (12.5 μg/mL of PcpA + 5 μg/mL PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4 K. (12.5 μg/mL of PcpA + 12.5 μg/mL PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

L. (12.5 μg/mL of PcpA + 25 μg/mL PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4 M. (25 μg/mL of PcpA + 5 μ /ηιί PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4 N. (25 μg/mL of PcpA + 12.5 μg/mL PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4 O. (25 μg/mL of PcpA + 25 μg/mL PhtD + 1.3 mg/mL AIOOH) in Tris buffered saline pH=7.4

Sample bleeds were taken from all animals 2 days prior to first immunization and following the first, second and third immunizations. Blood samples from individual mice were centrifuged at 9,000 rpm for 5 minutes and the recovered sera were stored at -20°C. Total antigen-specific IgG titres were measured in pooled prebleeds and in sera collected following the first second and third immunizations by endpoint dilution ELISA and geometric mean titres for each group are shown in Figure 1. The antibody titers in the prebleeds were below ^r the limit of detection (< 100), while the final bleed titers for both PhtD and Pep A monovalent formulations w ere high for both antigens in all groups consistent with those observed from previous studies. PhtD and PcpA-specific antibody ELISA titers are summarized in Table 4.

Table 4

PcpA and PhtD-specific ELISA Titers for Groups of Mice Immunized with Monovalent or Bivalent Formulation

ELISA Titers

Formulation Bleed*

PcpA PhtD

Pre-immuiiization < 100 < 100

5 [ig PcpA+ 5 [ig PhtD

Final bleed 117627 764341

* Final bleed anti-PcpA and PhtD titers were determined from individual mice and are represented as the geometrical mean.

Statistical analysis of the ELISA data investigated the effect of PcpA concentration on the anti-PhtD responses that were elicited (following the third immunization) by the bivalent formulations in comparison to the anti-PhtD responses that w ere elicited by the monovalent PhtD formulations. Similarly, the effect of PhtD concentration on the anti-PcpA responses that w ere elicited (following the third immunization) by the bivalent formulations in comparison to the anti- PcpA responses that were elicited by the monovalent PcpA formulations was also assessed. With respect to the anti-PcpA IgG titers, no statistically significant differences were observed when comparing the responses elicited by the monovalent PcpA formulations to those elicited by the bivalent formulations (9/9 groups). Therefore, no statistical!}' significant interaction, either positive or negative, with PhtD was observed at any dose examined. In regards to the anti-PhtD titres, in most comparisons between the anti-PhtD titres (i.e., responses) elicited by the bivalent formulations and those elicited by the corresponding monovalent PhtD formulations, no statistical!}' significant inhibition was noted (7/9 groups). Two exceptions were observed, each showing a two-fold decrease in anti-PhtD titers: (i) the bivalent formulation containing PcpA at ^g/dose and PhtD at 2.5 μg/dose in comparison to the monovalent formulation of PhtD at 2.5 μg/dose (p=0.034); and (ii) the bivalent formulation containing PcpA at ^g/dose and PhtD at 5.0 μg/dose in comparison to the monovalent formulation of PhtD at 5.0 μg/dose (p=0.027).

Statistical significance was not observed for the 1 μg dose of PhtD, nor with the higher doses of PcpA (i.e., 2.5 μg and 5 μg). However, this two fold decrease is within the range of variability of the model and thus does not reflect significant levels of interference.

The optimum concentration of each antigen (PcpA, PhtD) in a bivalent composition as determined by statistical analysis was 25 μ^ηιΐ, (i.e. , 5 μg/dose). Monovalent compositions with this concentration of antigen (i.e. , 25 μg/mL of PcpA or PhtD) also elicited the highest antigen specific IgG titres. EXAMPLE 3

Immunogenicity Study in Rats Following 3 Intramuscular Injections of the Bivalent Vaccine

This Example describes the analysis of the safety and immunogenicity of a multi- component vaccine in another animal species {i.e. , rat).

Four groups of (20 per sex) Wistar Crl:WI (Han) rats were given 3 IM injections of either control, bivalent vaccine composition with or without adjuvant or adjuvanted monovalent PcpA vaccine composition at three weekly inten als on Days 0, 21 and 42 (see stud}' design in Table 5 below). Animals were necropsied on Days 2 or 15 after the last administration. Compositions were prepared as described in Example 1. The adjuvant used to prepare adjuvanted compositions was aluminum hydroxide (AlhydrogeP, Brenntag). See Table 5 for an outline summary of the stud}' design.

Table 5 (Stud}' Design)

Morbidity/mortality checks were performed at least twice daily and clinical examinations were performed daily. There were no premature deaths, adverse clinical signs, effects on body weight, food consumption, clinical chemistry or ophthalmology that were considered treatment related.

Sera were anal} zed for PhtD and PcpA specific IgG antibody titers by ELISA. The results are set out in Figures 2 a to d. All treated animals showed robust anti-PcpA and anti-PhtD responses, although the responses in the unadjuvanted group were more variable. Adjuvanted monovalent PcpA vaccine elicited an immune response that was equivalent to the adjuvanted bivalent vaccine, indicating the absence of immunological interference by PhtD in the bivalent formulation. The bivalent and PcpA monovalent vaccine compositions each induced an immune response in all animals. According to the results here, the bivalent and PcpA monovalent vaccine compositions are immunogenic in rats. Adjuvanted compositions were more immunogenic than unadjuvanted compositions.

EXAMPLE 4

Assessing Immunogenicity of bivalent composition formulated with different Aluminum - Based Adjuvants

This Example describes the analysis of the immunogenicity of a multi-component composition formulated with different aluminum-based adjuvants.

In one study, recombinant PhtD and PcpA (prepared and purified as described in Example 1) were formulated with either fresh aluminum hydroxide adjuvant (AlhydrogeP ^' ), aged aluminum hydroxide adjuvant (Altrydrogel*, Brenntag), which had been incubated at 2-8°C for approximately 6 months, aluminum hydroxide adjuvant (Alhydrogel*, Brenntag) treated with various concentrations of phosphate P0 ₄ (2 mM, 10 mM and 20 mM) or A1P0 ₄ (Adjuphos*, Brenntag). Formulations were prepared as described in Example 1. Groups of 5 (or 4) female Balb/c mice (Charles River), 6-8 w eeks of age upon arrival, w ere immunized intramuscularly (IM) three times at 3 week intervals with the applicable formulation. The specific formulations administered to each group is set out in Table 6.

The PhtD and PcpA-specific antibody ELISA titers following the final bleed are summarized in Table 6. Mice immunized with PcpA and/or PhtD proteins generated antigen- specific antibody responses after immunization. No significant differences in anti-PhtD and anti- PcpA titres were seen in animals immunized with bivalent formulations with either fresh or aged AIOOH or pre-treated with phosphate (at any of the three concentrations used). Immunization w ith the bivalent composition formulated w ith A1P0 ₄ (which is less immunogenic than AIOOH) gave rise to significantly low er anti-PhtD IgG titres when compared to formulations containing AIOOH or P0 ₄ -containing AIOOH adjuvants. These results were confirmed in other studies that compared bivalent compositions formulated with aluminum hydroxide adjuvant and A1P04 adjuvants.

In total, four studies were completed using both recombinant PcpA and PhtD as immunogens formulated with aluminum-based adjuvants (aluminum hydroxide adjuvant, aluminum hydroxide adjuvant treated with various concentrations of P0 , A1P0 ). Both antigens were given at various doses ranging from 1-5 μg/dose. Specific PcpA and PhtD antibody titers were determined in pooled prebleeds and in sera collected following three IM or SC

immunizations. The antibody titers in the prebleeds w ere below ^r the limit of detection (< 100), while the final bleed titers were ranged between 124827 to 204800 for anti-PcpA and 36204 to 97454 for anti-PhtD.

In sum, according the results here, compositions formulated with any of the adjuvants tested were immunogenic. Immunization with recombinant PhtD and PcpA proteins formulated with aluminum hydroxide adjuvants (i.e. aluminum hydroxide adjuvant and aluminum hydroxide adjuvant treated with phosphate) generated significantly higher antigen-specific antibody responses (IgG tiers) to both PcpA and PhtD in comparison to immunizations with A1P0 ₄ formulations.

Table 6

PcpA and PhtD-specific ELISA Titers for Groups of Mice Immunized w ith Placebo or Bivalent Vaccine Formulation

* Final bleed anti-PcpA and anti-PhtD titers were determined from individual mice and are represented as the geometrical mean. EXAMPLE 5

Survival following challenge with S. pneumonaie strains 14453, MD or 941192

This Example describes the protective ability of a multi-component vaccine against fatal pneumococcal challenge in the mouse intranasal challenge model.

A bivalent formulation of recombinant PhtD and PcpA was evaluated using an intranasal (IN) challenge model. In this model, groups of female CBA/j mice (N = 15 per group) w ere immunized intramuscularh' (IM) with a bivalent composition containing a 5 μg/dose of each of purified recombinant PhtD and PcpA proteins, formulated in TBS with adjuvant (AIOOH treated with 2 mM P0 ₄ (65 μg/dose)). The injection volume was 50 per dose. As a negative control, a PBS placebo-containing aluminum adjuvant was injected. Animals were immunized IM at 0, 3, and 6 w eeks following initiation of the stud}'. At 9 weeks, animals were administered a lethal dose (approximately 106 CFU) intranasally of an S. pneumoniae strain MD, strain 14453 or 941192 in PBS suspension (40 challenge volume per mouse). Sample bleeds were taken from all animals 4 days prior to the first injection (pre-immunization at 0 weeks) and 4 days prior to the challenge. Sera were analyzed for total PhtD and PcpA-specific IgG response by means of an antibody ELISA assay.

Following the challenge, mice were monitored daily for mortality. All sun iving mice were euthanized 11 days post-challenge. Protection was determined using Fisher's one-sided Exact test by comparing survival in the immunized group(s) to the placebo control (p values <0.05 were considered significant). The results of the stud}' (noted in % survival) are set out in Figure 3 and Table 7 below.

Table 7

Survival Results of Mice Immunized w ith Bivalent Vaccine or Placebo

7 86.7 93.3 40 6.7

8 86.7 93.3 40 6.7

9 86.7 93.3 40 6.7

10 86.7 93.3 40 6.7

11 86.7 93.3 40 6.7 p-value* 0.01 0.000

* p-value calculated using the Fisher exact test versus placebo group; difference from placebo group 11 days post-challenge

Immunization with combined recombinant PhtD and PcpA proteins generated protection against fatal IN challenge with three different strains of S. pneumoniae in the IN challenge model. The protection noted in groups that had been challenged with either the 14453 strain or the MD strain w as statisticalh' significant. The group challenged with the 941192 strain also had a high % survival, but the protection w as not considered statisticalh' significant in light of the percentage of survival noted in the negative control group (immunized with adjuvant alone).

EXAMPLE 6

Humoral response and survival following challenge using different routes of administration (subcutaneous or intramuscular)

This Example describes the protective ability of a multi-component vaccine against fatal pneumococcal challenge in the mouse intranasal challenge model.

Bivalent compositions of PhtD and PcpA were prepared (using two different lots of each of rPhtD and rPcpA) and w ere formulated w ith an aluminum hydroxide adjuvant (AIOOH) that was pre-treated with 2mM of phosphate (according to process described in a patent application filed concurrently with this application). The prepared formulations were evaluated in the mouse active immunization intranasal challenge model (based on a model described in Zhang Y.A. et. al.. Infect. Immunol. 69:3827-3836). More specifically, 16 groups of 6 female CBA/j mice (Charles River), 6-8 weeks of age upon arrival, were immunized intramuscularly or

subcutaneously three times at 3 week intervals with the applicable formulation:

A. PcpA Lot A, PhtD Lot C, Unadjuvanted, s.c. (25 μg/ml/protein)

B. PcpA Lot B, PhtD Lot C, Unadjuvanted, s.c. (25 μg /ml/protein)

C. PcpA Lot A, PhtD Lot D, Unadjuvanted, s.c. (25 μg /ml/protein)

D. PcpA Lot B, PhtD Lot D, Unadjuvanted, s.c (25 μg /ml/protein)

E. PcpA Lot A, PhtD Lot C + 2 mM phosphate treated AIOOH, s.c (25 μg /ml/protein)

F. PcpA Lot B, PhtD Lot C + 2 mM phosphate treated AIOOH, s.c (25 μg /ml/protein) G. PcpA Lot A, PhtD Lot D + 2 mM phosphate treated AIOOH, s.c (25 μg /ml/protein)

H. PcpA Lot B, PhtD Lot D + 2 mM phosphate treated AIOOH, s.c (25 μg /ml/protein)

I. PcpA Lot A, PhtD Lot C Unadjuvanted, i.m ( 100 μg /ml/protein)

J. PcpA Lot B, PhtD Lot C Unadjuvanted, i.m (100 μg /ml/protein)

K. PcpA Lot A, PhtD Lot D Unadjuvanted, i.m ( 100 μg /ml/protein)

L. PcpA Lot B, PhtD Lot D Unadjuvanted, i.m ( 100 μg /ml/protein)

M. PcpA Lot A, PhtD Lot C + 2 mM phosphate treated AIOOH, i.m ( 100 μg /ml/protein) N. PcpA Lot B, PhtD Lot C + 2 mM phosphate treated AIOOH, i.m ( 100 μg /ml/protein) O. PcpA Lot A, PhtD Lot D + 2 mM phosphate treated AIOOH, i.m ( 100 μg /ml/protein) P. PcpA Lot B, PhtD Lot D + 2 mM phosphate treated AIOOH, i.m. ( 100 μg /ml/protein)

The bivalent formulations administered each included 5 μg/dose of each antigen (i.e. , PhtD and PcpA) and were formulated with adjuvant in TBS pH 7.4 ( 1.3 mg/mL AIO(OH) pretreated with 2 mM phosphate). Mice were administered a lethal dose lxlO ⁶ CFU) of S.

pneumoniae strain MD, 4 days following the third (final) bleed.

Sample bleeds were taken from all animals one da}' prior to the first, second and third immunization and three weeks following the third immunization. Blood samples from individual mice were centrifuged at 9,000 rpm for 5 minutes and the recovered sera were stored at -20°C.

Total antigen-specific IgG titres were measured by endpoint dilution ELISA and by quantitative ELISA and geometric mean titres for each group are shown in Figures 4a to 4b. Sun ival results are summarized in Figure 5.

There was no statistical difference between anti-PcpA and anti-PhtD IgG titres elicited by the different lots of PcpA and PhtD. There w as an advantage noted in administering adjuvanted formulations subcutaneously; more specifically, formulations administered intramuscularly w ere less immunogenic than those administered subcutaneously. In addition, unadjuvanted formulations were less immunogenic than adjuvanted formulations.

In regards to sun ival, the formulations tested conferred protection against fatal S.

pneumoniae challenge ( 100% survival seen in groups immunized with formulations of 100 μg/mL of each of PhtD and PcpA and pretreated AIO(OH)). There w as no significant difference in % sun ival between the groups immunized intramuscularly and those immunized

subcutaneously. The % sun ival of groups immunized with the two PhtD lots did not differ significanth' whearas the % sun ival of groups immunized with the two PcpA lots did (with lot B providing a significanth' higher sun ival) . The PcpA lot B also gave significanth' higher % survival in adjuvanted versus unadjuvanted formulations. There were no other statistical advantages noted in adjuvanted versus unadjuvanted formulations.

In this stud}', the particular lot of bacteria used for challenging the mice w as found less virulent than a previously used lot of this bacterial strain. In a separate stud}' (also using the intranasal challenge model), approximate ' 80% (p value 0.011) of the mice immunized with a formulation of 100 ug.mL of each of PhtD and PcpA + 1.3 mg/mL AIO(OH) (Alhydroge "85 ^" 2%, 25.08 mg/mL) in Tris-HCl, saline, 150 mM, at pH=7.4, survived a lethal S. pneumoniae challenge.

EXAMPLE 7

This Example describes the preparation of rabbit PhtD and PcpA anti-sera. Antisera w ere raised in rabbits using both His-tagged PhtD, His-tagged PcpA and recombinant PhtD and PcpA by a standard methodology. Measurement of PhtD and PcpA specific antibody in sera was determined by ELISA. As shown in Table 8, as an example for PhtD, a high titer of PhtD specific antibody was detected in the sera of all immunized rabbits but not in prebleed (before vaccination) sera. Both His-tagged PhtD and PhtD proteins w ere immunogenic in rabbits and antisera have high titres of PhtD specific antibody. Similar results were obsen ed with His-PcpA and PcpA proteins (data not shown).

Table 8: Generation of PhtD Rabbit Antisera

EXAMPLE 8

This Example describes the preparation of human PhtD and PcpA specific antibodies. Human polyclonal antibodies were purified from normal pooled adult human serum using affinity chromatography. Affinity chromatography columns were prepared using CNBr-activated sepharose resin covalently coupled to the purified recombinant antigen protein (PhtD or Pep A). Human AB serum (Sigma) was bound to the affinity column, which was then washed and the specific antibody eluted with Gh cine-HQ buffer.

The final purified antibody w as obtained by concentrating the pooled elution fractions by ultrafiltration and buffer exchange into PBS. The antibody solution was sterilized by filtration through a 0.22-um syringe filter. The total protein concentration was determined using UV spectroscopy. The endotoxin level of the final antibody preparation was determined using an Endosafe PTS Reader from Charles River Laboratories. Purity, specificity and cross reactivity of the purified antibody was determined by SDS-PAGE, Western blot and antibody ELISA anah sis. Each lot was purified from 100 mL of human AB serum unless otherwise stated.

EXAMPLE 9

Surface Accessibility FACS Assay with Anti-PhtD and Anti-PcpA Antibodies

This Example describes the anah sis of the binding capacity of anti-PhtD and anti-PcpA antibodies. Cultures were grown from frozen stocks to OD450 0.4-0.6, in either complete oi ^¬ Mn2+-depleted medium. Bacteria were washed and incubated with varying concentrations of human affinity purified antibodies in PBS. Human purified monoclonal antibodies against PspA were used as a positive control. Antibody binding to the bacteria was detected using a secondary antibody, FITC-conjugated anti-human IgG, and evaluated using flow cytometry. Similarly, anti- PhtD and anti-PcpA specific rabbit sera were used. Antibody binding to the bacteria was detected using a secondary antibody, FITC-conjugated anti-rabbit IgG and evaluated using flow ⁷ cytometry.

As a qualitative assay read-out, bacteria were scored positive when a fluorescent signal was detected. Mean fluorescence intensity (MFI) was analyzed as a means of measuring the amount of antibodies bound to the surface of the bacteria.

Surface accessibility assays ("SASSY') were performed to determine the ability of antigen-specific rabbit sera and purified human antibodies to bind live, intact S. pneumoniae.

Purified human antibodies and rabbit PhtD- and PcpA- antisera (prepared as described in Example 7 and 8) bound protein on the surface of live S. pneumoniae. Both PhtD and PcpA rabbit antisera bound to all strains of S. pneumoniae tested, including laboratory and clinical isolates, with the exception of strain D39 which was negative for PcpA. However, this is consistent with the finding that strain D39 (a laboratory strain) was pep A -negative by PCR amplification of the pcpA gene. In the case of PcpA, recognition occurred particularly when the bacteria were grown in conditions of depleted Mn2+ and increased Zn2+. Together, the data provide evidence that antibodies raised against recombinant protein or generated by natural infection recognize native protein and that epitopes on a wide variety of clinical isolates are conserved. The data also suggest that both PcpA and PhtD are highly surface accessible (Figure 6, and data not shown). Rabbit preimmune sera were used as negative controls.

In order to determine whether human purified PhtD and PcpA antisera have any additive effects on binding to S. pneumoniae _^ 10 EU/ml anti-PhtD antibody w as spiked into each sample containing increasing amounts of anti-PcpA antisera. The amount of total antibodies bound to the bacteria was measured by MFI (Figure 7). Anti-PcpA antibodies were able to bind live S.

pneumoniae in a dose-dependent manner. The addition of anti-PhtD antibodies led to a consistent increase in the MFI of the sample, confirming that antibodies against multiple surface proteins can bind simultaneously and that this leads to an increase in the total amount of antibody bound on the surface of the bacteria.

Purified human anti-PcpA antibodies, with or without purified human anti-PhtD antibodies, were incubated at varying concentrations with live S. pneumoniae strain WU2 which had been cultured in Mn2+-deficient medium. Antibodies bound to the surface of the bacteria were detected using FITC -goat-anti-human IgG. Mean Fluorescence Intensity (MFI) is shown in Figure 7. Antibody titles are shown in anti-PcpA EU/ml (anti-PcpA and anti-PcpA + anti-PhtD samples) or anti-PhtD EU/ml (anti-PhtD sample).

Surface accessibility experiments with anti-PhtD and anti-PcpA rabbit sera and purified human antibodies indicated that both PcpA and PhtD are surface accessible. Furthermore, human anti-PcpA and anti-PhtD antibodies could bind simultaneously, and therefore, increase the total amount of antibodies bound to the bacteria.

EXAMPLE 10

This Example describes the analysis of the passive protection provided by a multivalent composition.

In this study, a bivalent composition of recombinant PhtD and PcpA formulated w ith AlP0 ₄ was used to immunize two New ^r Zealand White Rabbits (Charles River) intramuscularly (i.m.) to obtain anti-PcpA/ anti-PhtD polyclonal serum. Each rabbit was injected i.m. with 10 μg/dose of rPcpA and 10 μg/dose of rPhtD in A1P0 ₄ (3 mg/ml), (20 μg total protein, 500 μΐ total volume of injection/rabbit). Two subsequent immunizations w ere given at 3 w eek inten als with 10 μg/dose of rPcpA and 10 μg/dose of rPhtD in A1P0 ₄ . Sample bleeds were collected following the 1 ^st and 2 ^nd immunizations. Final bleeds were collected three weeks following the final immunization. The blood was collected in gel separator tubes, allowed to clot, and serum was obtained by centrifugation, pooled and stored at about -20 C. The PhtD and PcpA-specific total IgG antibody titers w ere assessed for both rabbits. The serum from one of the rabbits used in the experiment had the following titer by ELISA: PhtD 204,800 and PcpA 102,400.

Recombinant PhtD protein and/or recombinant PcpA protein were added to certain sera samples to competitively inhibit (block) the corresponding antibodies present in the sera. As a control, neither recombinant protein was added to certain sera samples. Using a mouse model of passive protection based on one published earlier (Briles DE et. a/. , J. Infect Pis. 2000 Dec), various dilutions of sera samples w ere then administered to mice challenged with S. pneumoniae. The % survival observed per log dilution of sera administered w as graphed in order to identify the Probit dose response curve (see Figure 8). For each sera sample, the ED50 (log dilution effective for 50% survival) was calculated. Differences at ED50 between blocked and unblocked sera samples were assessed using a statistical model (see Table 9 below).

Table 9

Statistical Comparisons between protein blocked groups to unblocked groups

* : Fisher's Exact Test

Competitively inhibiting the PcpA antibodies in the sera containing both PcpA and PhtD specific antibodies significantly decreased the ED50 (i.e., the log dilution of the sera effective for 50% survival) and this difference w as statistically significant in comparison to the ED50 of unblocked sera. Competitively inhibiting the PhtD antibodies in the sera containing both PcpA and PhtD specific antibodies also decreased the ED50 (albeit not statistically significant). In regards to the sera sample in which both PcpA and PhtD antibodies were competitively inhibited (by adding to the sera each of PhtD and PcpA protein at a protein to sera ratio of 1 : 10), a low ^r % survival was obtained with statistical significance by Fisher's Exact Test only with the highest dilution used and therefore ED50 was not determinable.

In sum, both the PhtD and PcpA antibodies contributed to the passive protection elicited by the sera raised to the bivalent formulation. The protection provided by the sera raised to the bivalent formulation was blocked by competitively inhibiting both PhtD and PcpA antibodies, and this result w as significantly different from that obtained w hen only one of the antibodies (PhtD or PcpA) was competitively inhibited. Similar results were obtained using PhtD and PcpA proteins with rabbit trivalent hyper-immune sera (raised using a trivalent composition comprising PhtD, PcpA and PlyD l) in the same passive protection model. In that study, PhtD and PcpA proteins together w ere able to block the protective potential of the trivalent hyperimmune sera. These results from this passive protection model imply that the contributions of each protein-specific antibody are additive.

EXAMPLE 1 1

Effects of aluminum concentration on Immunogenicity of Vaccine Formulation

This Example describes the analysis of the immunogenicity of a multi-component composition formulated with phosphate pretreated AIO(OH) and varying concentrations of elemental aluminum.

Female Balb/c mice were used to assess the immune response elicited by adjuvanted trivalent formulations. To prepare the trivalent formulations, recombinant PhtD, PcpA and an enzymatically inactive pneumolysin mutant (PlyD l, as described in PCT/CA/2009/001843, as SEQ ID NO:44 and herein as SEQ ID NO:9) were formulated with A10(OH)-containing P0 ₄ (2 mM) as described in Example 1. Samples of prepared formulations w ere stored at 2 to 8°C prior to the start of the stud}'. Groups of Balb/c mice w ere immunized intramuscularly (IM) three times at 3 week intervals with the applicable formulation:

A. Unadjuvanted (Trivalent 50 μg/mL of PcpA and PhtD and 100 μg/mL of PI}' mutant in TBS pH=7.4)

B. Trivalent 50 μg/mL of PcpA and PhtD and 100 μg/mL of PI}' mutant + 0.56 mg All mL PTH, P:A1 molar ratio= 0.1 (0.56 mg Al/mL AIO(OH) treated with 2mM P04) in Tris Saline pH=7.4. C. Trivalent 50 ug/mL of PcpA and PhtD and 100 ug/mL of Ply mutant+ 0.28 mg Al AnL PTH, P: Al molar ratio= 0.1 (0.28 mg Al AnL AIO(OH) treated with 1 mM P04) in Tris Saline pH=7.4.

D. Trivalent 50 ug/mL of PcpA and PhtD and 100 ug/mL of Ply mutant+ 1.12 mg Al AnL PTH, P: Al molar ratio= 0.1 ( 1.12 mg Al AnL AIO(OH) treated with 4 mM P04) in Tris Saline pH=7.4.

E. Trivalent 50 μgAnL of PcpA and PhtD and 100 μgAnL of Ply mutant + 1.68 mg Al AnL PTH, P:A1 molar ratio= 0.1 ( 1.68 mg Al AnL AIO(OH) treated with 6 mM P04) in Tris Saline pH=7.4.

Sera were collected following the 1st, second and third immunization. Total antigen- specific IgG titres were measured by quantitative ELISA and geometric mean titres (+/- SD) for each group were calculated . A summary of the total IgG titers obtained are set out in Figure 9.

All adjuvanted groups (B, C, D and E) produced significantly higher titres against all three antigens than the unadjuvanted group (A) (p<0.001). With respect to each antigen, titre levels peaked when adjuvanted with PTH with 0.56 mg elemental aluminum/mL (and, in the case of PhtD, the difference betw een titres elicited with aluminum 0.56 mg AnL and the two higher concentrations was statistical!}' significant). Similarly, with respect to each antigen, titre levels were lower when adjuvanted with PTH with 0.28 mg elemental aluminum/mL (and, in the case of PcpA, the difference was statistical!}' significant). These findings were surprising. Antibody (IgG) titers were expected to increase proportional to the concentration of aluminum (as reported in Little S.F. et. al.. Vaccine, 25 :2771-2777 (2007)). Surprisingly, even though the concentration of each of the antigens was kept constant, the titres decreased, rather than plateau, with increasing aluminum concentration (and with PhtD this was statistically significant).

EXAMPLE 12 This example describes the evaluation of the stability of an adjuvanted vaccine formulation under various conditions. A number of PTH adsorbed vaccine formulations were incubated for 5 days at 5°C, 25°C, 37°C (i.e., under thermal accelerated conditions).

To evaluate the stability of 4 different vaccine formulations of PcpA (formulated in AIO(OH) or PTH), the formulations were each incubated for 6 weeks at 37°C and then assessed by RP-HPLC. The stability results obtained are summarized in Table 10. The recover}' from untreated AIO(OH) decreased by almost 50% following the incubation period (at 37°C) whereas little to no degradation was observed in the PTH containing formulations.

Table 10

% Recover}- (RP-HPLC) of PcpA after 6 weeks incubation

To evaluate the stability of PcpA and PhtD in monovalent and bivalent formulations (formulated with AIO(OH) or PTH), formulations were prepared as described in Example 1 using AIO(OH) or phosphate-treated AIO(OH) w ith 2mM phosphate and samples were then incubated for about 16 weeks at various temperatures (i.e., 5°C, 25°C, 37°C or 45°C). Antigen

concentration was then assessed by RP-HPLC. The stability results obtained are set out in Figures 10a to f. As shown the figures, in comparison to the formulations adjuvanted with untreated AIO(OH), the degradation rate of PcpA and PhtD, particularly under accelerated (stress) conditions (e.g., 25, 37, 45°C) was significanth' decreased in formulations adjuvanted w ith phosphate treated AIO(OH).

To evaluate to the antigenicity stability of the antigenicity of PcpA and PhtD in multivalent formulations (formulated with AIO(OH) or PTH), bivalent formulations (at 100 μg/mL) were prepared as described in Example 1 and then samples were incubated at about 37°C for approximately 12 weeks. Antigenicity of each formulation was evaluated by a quantitative ELISA sandwich assay at time zero and following the 12 week incubation period. Results are set out in Figure 11. The antigenicity of both PcpA and PhtD following the 12 week incubation period at 37°C was significanth' higher when formulated with PTH in comparison to formulations with AIO(OH). EXAMPLE 13

This example describes the evaluation of the effect of various excipients on the stability of a number of formulations.

A screening of 18 GRAS (generally regarded as safe) compounds at various

concentrations was performed. An assay was used to screen for compounds that increase the thermal stability of each protein under evaluation (i.e. , PcpA, PhtD and a detoxified pneumolysin mutant (PlyDl, as described in PCT/CA/2009/001843: Modified PLY Nucleic Acids and Polypeptides, as SEQ ID NO: 44) .

Each of the protein antigens were recombinantly expressed in E.coli and purified by serial column chromatography following conventional purification protocols substantial!}' as described in Example 1, for PhtD and PcpA and as described in PCT/CA/2009/001843 (as SEQ ID NO: 44) for PlyDl (the sequence for which is noted herein as SEQ ID NO: 9). Protein purity for all three antigens was typically higher than 90% as evaluated by RP-HPLC and SDS-PAGE. Proteins bulks were supplied at approximately 1 mg/mL in 10 mM Tris, pH 7.4 containing 150 mM sodium chloride. Each protein was diluted to the desired concentration ( 100 μg/mL PcpA; 100 μg/mL PhtD; 200μg/mL PlyDl) with the appropriate excipient solution (in the concentration noted in Table 11) in 10 mM tris buffer saline, pH 7.5 (TBS), and PTH was added to the protein solutions to achieve a final concentration of 0.6 mg of elemental Al/mL. Control samples (lacking the applicable excipient) were also assayed. SYPRO* Orange, 5000X (Invitrogen, Inc., Carlsbad, CA), was diluted to 500X with DMSO (Sigma) and then added to the adjuvanted protein solutions. In all cases optimal dilution of SYPRO-Orange was 10X from a commercial stock solution of 5000X.

Assays were performed in a 96 well polypropylene plate (Stratagene, La Jolla, CA) using a real-time polymerase chain reaction (RT-PCR) instrument (Mx3005p QPCR Systems, Stratagene, La Jolla, CA). A sample volume of approximately 100 μΐ, was added to each well and the plate was then capped with optical cap strips (Stratagene, La Jolla, CA) to prevent sample evaporation. Plates were centrifuged at 200g for 1 min at room temperature in a Contifuge Stratos centrifuge (Heraeus Instruments, England) equipped with a 96 well plate rotor . The plates w ere then heated at 1°C per min from 25°C to 96 °C. Fluorescence excitation and emission filters were set at 492 nm and 610 nm, respectively. Fluorescence readings (emission at 610 nm, excitation at 492 nm) w ere taken for each sample at 25°C and then with each increase in 1 °C. Thermal transitions (melting temperatures, Tm) were obtained using the corresponding temperature of the first derivative of the minimum of fluorescence. The minimum of the negative first derivative trace from the melting curve (or dissociation curve) was calculated using MxPro software provided with RT-PCR system. Tm is defined as a midpoint in a thermal melt and represents a temperature at w hich the free energy of the native and non-native forms of a protein are equivalent. The effect of each excipient w as assessed as the ΔΤιη = Tm (sample with protein + compound) - Tm (protein control sample). A summary of the results obtained are noted in Table 11. The sensitivity of the assay w as +/- 0.5°C.

Polyols, monosaccharides and disaccharides increased the Tm of adjuvanted PlyD l in a concentration dependant manner with maximum stabilization (i.e. , an increase in Tm of about 4°C) observed at high concentration of sugars. Similar results w ere detected for each of PcpA and PhtD w ith the exception of arginine which decreased the Tm of PhtD by about 2°C. The follow ing excipients w ere found to efficiently increase the thermal stability of all three proteins: sorbitol (20%, 10%), trehalose (20%), dextrose (20%, 10%), sucrose (10%, 5%), and 10% lactose.

The effect of several excipients identified in the screening assays on the physical stability and antigenicity of PcpA stored under stress conditions w as also studied to note an}- correlation with the thermal stability effects noted earlier. PcpA protein was diluted to the desired concentration (e.g., about 100 μg/mL) with the appropriate excipient solution described in the figure (10% Sorbitol, 10% Sucrose, 10% Trehalose in lOmM Tris Buffer pH 7.4), and PTH was added to the protein solutions to achieve a final concentration of 0.6 mg of elemental Al/mL. A control sample (lacking excipient) was also included in the stud}'. Samples were stored at 50°C for a three day period. Protein degradation was evaluated by RP-HPLC and antigenicity was assessed by quantitative, sandwich ELISA. Results are set out in Figures 12A and 12B. The concentration of intact protein w as measured by RP-HPLC in an Agilent 1200 HPLC system equipped with a diode array UV detector. Samples were desorbed from the adjuvant in PBS/Zwittergent buffer for 5 h at 37 °C and separated using an ACE C4 column (Advanced Chromatography Technologies, Aberdeen, UK) and a mobile phase gradient of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in CAN) using a gradient of 0.75% of buffer B per minute over 30 min at a flow rate of 1 ml/min. Proteins were monitored by UV absorbance at 210nm and quantitated against a 5-point linear calibration curve produced with external standards. The quantitative antigen ELISA sandwich was used to evaluate antigenicity of PcpA formulations at time zero and after 3 days of incubation at 50 °C. A rabbit IgG anti-PcpA sera was used for antigen capture, and a well characterized monoclonal anti-PcpA for detection. Briefly, 96 well plates were coated with rabbit anti-PhtD IgG at a concentration of 2 μg/mL in 0.05M Na ₂ C0 ₃ /NaHC0 ₃ buffer for 18 hours at room temperature (RT), and blocked with 1% BSA/PBS for 1 hour at RT follow ed by 2 w ashes in a w ashing buffer of PBS/0.1% Tween 20 (WB). Two-fold dilutions of test samples, an internal control and a reference standard of purified PcpA of known concentration w ere prepared in 0.1% BSA/PBS/0.1% Tw een 20 (SB), added to wells and incubated at RT for 1 hour followed by 5 washes in WB. Detecting primary mAb was diluted in SB to a concentration of 0.1 μg/mL, and incubated for 1 hour at RT and followed by 5 washes in WB, and addition of F(ab')2 Donkey anti-mouse IgG (H+L) specific at 1/40K dilution in SB. Following 5 washes in WB, TMB/H ₂ 0 ₂ substrate is added to the wells, and incubated for 10 minutes at RT. The reaction is stopped by the addition of 1M H ₂ S0 ₄ . ELISA plates were read in a plate reader (SpectraMax, M5, Molecular Devices, Sunnyvale, CA) at A450/540 nm, and test sample data is calculated by extrapolation from a standard curve using 4-parameter logistic using the software SoftMax PRO.

As shown in Figure 12 A, data derived from RP-HPLC show ed that those excipients that increased the Tm of adjuvanted PcpA also decreased the protein's rate of degradation at 50°C over a three da}' period. The greatest stability as determined by percent recovery of the PcpA protein over time was provided by 10% sorbitol (as shown in Figure 12A). The antigenicity of adjuvanted PcpA was also preserved by these excipients (as shown in Figure 12B). In good correlation w ith RP-HPLC results, sorbitol appeared to preserve antigenicity to a higher degree than sucrose or trehalose.

The addition of 10% sorbitol, 10% sucrose, or 10 % trehalose significantly decreased the rate constant at 50°C and increased the half life of PcpA w hen compared to that of the control sample without excipients (Table 12). The buffer pH of 9.0 decreased the Tm of the protein, but accelerated degradation (i.e. , increased the rate constant) at 50 °C as compared to that of the control (Table 12). Altogether, these results suggest a good correlation between thermal stability- detected by the assay, physical stability detected by RP-HPLC and antigenicity detected by ELISA.

In view- of the results obtained in these studies, sorbitol, sucrose, dextrose, lactose and/or trehalose are examples of excipients that may be included in monovalent and multivalent (e.g.. bivalent, trivalent) formulations of PcpA, PhtD and detoxified pneumoh sin proteins (such as, PlyDl) to increase physical stability .

Table 11

Effect of GRAS excipieiits on Tm (as assessed by monitoring fluorescence emission over a temperature range). Compounds that increase thermal stability provide a positive Tm difference value.

Table 12

Rate constant values from stability data of formulations incubated at 50 °C.

Formulation Ar at 50°C Half life at 50°C R ²

^ig.mL ¹ .day ^"1 ) (days)

10% Sorbitol 7.5 7.3 0.99

10% Trehalose 9.8 5.6 0.95

10% Sucrose 10.9 5.1 0.98

Control (TBS pH 7.4) 13.4 4.1 0.94

TBS H9 16.2 3.4 0.93

Rate constant for formulations incubated at 50°C were calculated by fitting the RP-HPLC stability data presented in Figure 12A using zero order kinetics equation (1) [A _t ]=-kt + [A ₀ ]. where A, is the concentration of the antigen at a given time. A ₀ is the initial protein concentration in μ^ιηί and t is the time in days. R ² is reported for the linear fit of the data using equation (1).

EXAMPLE 14

The effect of pH on the stability of three different antigens formulated with or without an aluminum adjuvant was performed. An assay was used to evaluate the effect of pH on the thermal stability of each protein under evaluation (i.e. , PcpA, PhtD and a detoxified pneumolysin mutant (PlyD l, as described in PCT/CA2009/001843:Modified PLY Nucleic Acids and

Polypeptides, as SEQ ID NO:44 and noted in the Sequence Listing herein as SEQ ID NO:9) .

Each of the protein antigens were recombinantly expressed in E.coli and purified by serial column chromatography following conventional purification protocols substantial!}' as described in Example 1, for PhtD and PcpA and as described in PCT/CA2009/001843 for Ph D 1. Protein purity for all three antigens w as typically higher than 90% as evaluated by RP-HPLC and SDS-PAGE. Proteins bulks were supplied at approximately 1 mg/mL in 10 mM Tris, pH 7.4 containing 150 mM sodium chloride. Each protein was diluted to the desired concentration ( 100 μg/mL PcpA; 100 μg/mL PhtD; 200 μg/mL PlyD l) with the appropriate buffer solution (i.e. , 10 mM Tris buffer (pH 7.5-9.0), 10 mM phosphate buffer (pH 6.0-7.0) and 10 mM acetate buffer (pH 5.0 - 5.5)) and an aluminum adjuvant (i.e. , aluminum hy droxide (Alhydrogel, Brenntag Biosector, Denmark), or aluminum phosphate (Adju-Phos, Brenntag Biosector, Denmark) or aluminum hy droxide pre-treated with 2mM phosphate (PTH)) was added to the protein solutions to achieve a final concentration of 0.6 mg of elemental Al/mL. Control samples (lacking the applicable adjuvant) were also assayed. SYPRO* Orange, 5000X (Invitrogen, Inc., Carlsbad, CA), was diluted to 500X with DMSO (Sigma) and then added to the adjuvanted protein solutions. In all cases optimal dilution of SYPRO-Orange was 10X from a commercial stock solution of 5000X. Assays were performed in a 96 well polypropylene plate (Stratagene, La Jolla, CA) using a real-time polymerase chain reaction (RT-PCR) instrument (Mx3005p QPCR Systems, Stratagene, La Jolla, CA). A sample volume of approximately 100 μί was added to each well and the plate was then capped with optical cap strips (Stratagene, La Jolla, CA) to prevent sample evaporation. Plates were centrifuged at 200g for 1 min at room temperature in a Contifuge Stratos centrifuge (Heraeus Instruments, England) equipped with a 96 well plate rotor . The plates w ere then heated at 1°C per min from 25°C to 96 °C. Fluorescence excitation and emission filters were set at 492 nm and 610 nm, respectively. Fluorescence readings (emission at 610 nm, excitation at 492 nm) w ere taken for each sample at 25°C and then with each increase in 1 °C.

Thermal transitions (melting temperatures, Tm) were obtained using the corresponding temperature of the first derivative of the minimum of fluorescence. The minimum of the negative first derivative trace from the melting curve (or dissociation curve) was calculated using MxPro software provided with RT-PCR system. Tm is defined as a midpoint in a thermal melt and represents a temperature at w hich the free energy of the native and non-native forms of a protein are equivalent. A summary of the results obtained are noted in Figure 13. The sensitivity of the assay was +/- 0.5°C.

For most proteins, solution pH determines the type and total charge on the protein, and thus, ma}' affect electrostatic interactions and overall stability. For adjuvanted proteins the solution pH and buffer species have a strong effect on microenvironment pH at the surface of the aluminum adjuvants w hich could ultimateh' influence the degradation rate of proteins adsorbed to aluminum adjuvants.

All three proteins were 90 to 100 % adsorbed to aluminum hydroxide in the range of pH under stud}'. In aluminum phosphate, the adsorption of Pep A was higher than 80% while PhtD and PlyDl (each an acidic protein) were negligibly adsorbed to the adjuvant above pH 5 (data not shown).

Figure 13 show s the effect of pH on each of the 3 antigens when formulated with adjuvant and in unadjuvanted controls. The unadjuvanted antigens displayed their distinctive pH stability profile. PcpA showed stead}' Tm values on a broad pH range from 6.0 to 9.0 with decreasing Tm values as the pH was dropped from 6.0 to 5.0. On the other hand, the thermal stability of unadjuvanted PhtD and PlyDl appeared maximized under acidic pHs (see Figure 13). The thermal stability profiles of the unadjuvanted proteins were significant!}' modified as a result of the addition of an aluminum adjuvant. As compared to the unadjuvanted controls, aluminum hydroxide, appeared to decrease the stability of all three proteins at relative!}' high and low pH values showing a bell-shaped curve as the pH w as increased from 5 to 9 with a maximum stability at near neutral pH. These data show that pretreatment of AIOOH w ith 2mM phosphate significant ' improved the stability of all three antigens at high and low pH as compared to untreated AIOOH (Figure 13 A-C). No significant changes w ere observed in the range of pH 6.0- 7.5 by this method.

As compared to unadjuvanted controls, no major changes were observed on the Tm vs pH profile of PcpA and PlyD l when aluminum phosphate was used as the adjuvant (Figure 13A and 13C). In the case of PhtD adjuvanted with AP, as compared to the unadjvanted control, a significant decrease in the Tm was observed at pH low er than 6 (Figure 13B).

EXAMPLE 15

This example describes the evaluation of the effect of various antigen combinations in multi- component formulations.

Three separate S. pneumoniae antigens w ere formulated in monovalent, bivalent and trivalent form and evaluated using the IN challenge model (substantialh' as described in previous examples). Monovalent, bivalent and trivalent formulations were prepared using suboptimal doses of purified recombinant PcpA, PhtD and PlyD l (a detoxified pneumoh sin) in TBS with adjuvant (AIOOH treated with 2 mM P0 ₄ (0.56 μg Al/dose)) pH 7.4. Suboptimal doses of each antigen that had been shown to induce either limited or no protection were chosen so as to detect additive effects. Each of the protein antigens were recombinantly expressed in E.coli and purified by serial column chromatography following conventional purification protocols substantialh' as described earlier. Protein purity for all three antigens was typically higher than 90% as evaluated by RP-HPLC and SDS-PAGE. Groups (n=26) of female CBA/J mice (n=15/group) were immunized intramuscularly three times at 3 week intervals between each immunization with applicable formulations (50uL).

Mice were administered a lethal dose of S.pneumoniae strain 14453, serotype 6B

( 1.5xl0 ⁶ cfu/mouse 3 weeks post final immunization and observed for survival and health for 2 weeks. Survival results (summarized in Table 13 below) were calculated and statistically analyzed by Fisher Exact test. Total antigen-specific IgG titres (from sera that had been collected following each immunization) were measured by quantitative ELISA and geometric mean titres (+/- SD) for each group were calculated. A summary of the total IgG titers obtained are set out in Figure 14. Table 13

The PcpA monovalent formulations were protective even at very low doses (and despite low ⁷ antibody titres). In comparison to the PcpA monovalent formulation, the trivalent formulations provided similar levels of protection. In comparison to the PhtD and PlyD l monovalent formulations, the trivalent formulations provided significanth' higher protection. The trivalent formulations elicited higher survival percentages as compared to the bivalent formulations (and difference was statistically significant, p=0.043, in regards to two trivalent formulations (0.0067:0.027:0.5; 0.0067:0.027:0.166; PcpA: PhtD: PlyD l) in comparison to bivalent formulation (0.0067:0.027; PcpA:PhtD)). The bivalent formulation was not protective at 0.0067 and 0.027 μg for PcpA and PhtD, respectively, which for PcpA was a protective dose when administered as a monovalent formulation. How ever, as the difference in survival betw een these two groups was not statistical!}' significant, the observed difference between

monovalent/bivalent formulations was due to assay variability.

The median effective dose of each of PcpA and PhtD in protecting at least 60% of mice from lethal challenge (ED60) in a bivalent formulation (0.0067:0.027; PcpA:PhtD) and in the trivalent formulations w ere calculated (see Table 14 below ). For each of PcpA and PhtD, the ED60 was reduced in the trivalent formulations as compared to the corresponding bivalent formulation. By these results, the addition of Ph D 1 had on average a 2-fold dose sparing effect on the bivalent formulation (i.e., PcpA+PhtD).

These data show ^r that immunization with trivalent formulations elicits better protection as compared to bivalent formulations. The inclusion of Ph D 1 in the trivalent formulations does not have an inhibitory effect on overall protection.

Table 14

EXAMPLE 16 This example describes the evaluation of the minimum effective antigen dose that elicits the highest level of antibody responses.

From monovalent studies conducted total antigen-specific IgG titres (as measured by ELISA) per antigen dose were graphically plotted to evaluate the minimum effective antigen dose eliciting highest titre. Representative graphs are set out in Figures 15 A, B, C. For PcpA, the estimated minimum antigen dose was assessed as 0.196 μg/mouse (0.147, 95% low ; 0.245, 95% high), and for PhtD the estimated minimum antigen dose was assessed as 0.935 ug/mouse (0.533, 95% low ^r ; 1.337, 95% high) which provides a ratio of PcpA:PhtD of 1 :4. The minimum antigen dose for Ph Dl was estimated as > 5 μg/mouse. As no immunological interference between antigens w ere detected at any of the evaluated ratios in the bivalent and trivalent studies performed (such as, for example, in Example 15), a 1 : 1 : 1 ratio of Pep A: PhtD: P Dl may be used in a muli-component composition.

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<110> SANOFI PASTEUR, LTD <120> Immunogenic Compositions

<130> APL-10-03-PCT

<150> US 61/289236

<151> 2009-12-22

<150> US 61/325660

<151> 2010-04-19 <160> 10

<170> Patentln version 3.5

<210> 1

<211> 838

<212> PRT

<213> Streptococcus pneumoniae <400> 1

Met Lys He Asn Lys Lys Tyr Leu Ala Gly Ser Val Ala Val Leu Ala 1 5 10 15

Leu Ser Val Cys Ser Tyr Glu Leu Gly Arg His Gin Ala Gly Gin Val

20 25 30

Lys Lys Glu Ser Asn Arg Val Ser Tyr He Asp Gly Asp Gin Ala Gly

35 40 45

Gin Lys Ala Glu Asn Leu Thr Pro Asp Glu Val Ser Lys Arg Glu Gly 50 55 60

He Asn Ala Glu Gin He Val He Lys He Thr Asp Gin Gly Tyr Val 65 70 75 80

Thr Ser His Gly Asp His Tyr His Tyr Tyr Asn Gly Lys Val Pro Tyr

85 90 95

Asp Ala He He Ser Glu Glu Leu Leu Met Lys Asp Pro Asn Tyr Gin

100 105 110

Leu Lys Asp Ser Asp He Val Asn Glu He Lys Gly Gly Tyr Val He

115 120 125

Lys Val Asp Gly Lys Tyr Tyr Val Tyr Leu Lys Asp Ala Ala His Ala 130 135 140 Asp Asn He Arg Thr Lys Glu Glu He Lys Arg Gin Lys Gin Glu His 145 150 155 160

Ser His Asn His Asn Ser Arg Ala Asp Asn Ala Val Ala Ala Ala Arg

165 170 175

Ala Gin Gly Arg Tyr Thr Thr Asp Asp Gly Tyr He Phe Asn Ala Ser

180 185 190

Asp He He Glu Asp Thr Gly Asp Ala Tyr He Val Pro His Gly Asp

195 200 205

His Tyr His Tyr He Pro Lys Asn Glu Leu Ser Ala Ser Glu Leu Ala 210 215 220 Ala Ala Glu Ala Tyr Trp Asn Gly Lys Gin Gly Ser Arg Pro Ser Ser

225 230 235 240

Ser Ser Ser Tyr Asn Ala Asn Pro Val Gin Pro Arg Leu Ser Glu Asn

245 250 255

His Asn Leu Thr Val Thr Pro Thr Tyr His Gin Asn Gin Gly Glu Asn

260 265 270 lie Ser Ser Leu Leu Arg Glu Leu Tyr Ala Lys Pro Leu Ser Glu Arg

275 280 285 His Val Glu Ser Asp Gly Leu lie Phe Asp Pro Ala Gin lie Thr Ser

290 295 300

Arg Thr Ala Arg Gly Val Ala Val Pro His Gly Asn His Tyr His Phe

305 310 315 320 lie Pro Tyr Glu Gin Met Ser Glu Leu Glu Lys Arg lie Ala Arg lie

325 330 335 lie Pro Leu Arg Tyr Arg Ser Asn His Trp Val Pro Asp Ser Arg Pro

340 345 350

Glu Gin Pro Ser Pro Gin Ser Thr Pro Glu Pro Ser Pro Ser Leu Gin

355 360 365 Pro Ala Pro Asn Pro Gin Pro Ala Pro Ser Asn Pro lie Asp Glu Lys

370 375 380

Leu Val Lys Glu Ala Val Arg Lys Val Gly Asp Gly Tyr Val Phe Glu

385 390 395 400

Glu Asn Gly Val Ser Arg Tyr lie Pro Ala Lys Asp Leu Ser Ala Glu

405 410 415

Thr Ala Ala Gly lie Asp Ser Lys Leu Ala Lys Gin Glu Ser Leu Ser

420 425 430

His Lys Leu Gly Ala Lys Lys Thr Asp Leu Pro Ser Ser Asp Arg Glu

435 440 445 Phe Tyr Asn Lys Ala Tyr Asp Leu Leu Ala Arg lie His Gin Asp Leu 450 455 460 Leu Asp Asn Lys Gly Arg Gin Val Asp Phe Glu Val Leu Asp Asn Leu 465 470 475 480

Leu Glu Arg Leu Lys Asp Val Ser Ser Asp Lys Val Lys Leu Val Asp

485 490 495

Asp He Leu Ala Phe Leu Ala Pro He Arg His Pro Glu Arg Leu Gly

500 505 510

Lys Pro Asn Ala Gin He Thr Tyr Thr Asp Asp Glu He Gin Val Ala

515 520 525

Lys Leu Ala Gly Lys Tyr Thr Thr Glu Asp Gly Tyr He Phe Asp Pro 530 535 540

Arg Asp He Thr Ser Asp Glu Gly Asp Ala Tyr Val Thr Pro His Met 545 550 555 560

Thr His Ser His Trp He Lys Lys Asp Ser Leu Ser Glu Ala Glu Arg

565 570 575

Ala Ala Ala Gin Ala Tyr Ala Lys Glu Lys Gly Leu Thr Pro Pro Ser

580 585 590

Thr Asp His Gin Asp Ser Gly Asn Thr Glu Ala Lys Gly Ala Glu Ala

595 600 605

He Tyr Asn Arg Val Lys Ala Ala Lys Lys Val Pro Leu Asp Arg Met 610 615 620

Pro Tyr Asn Leu Gin Tyr Thr Val Glu Val Lys Asn Gly Ser Leu He

625 630 635 640

He Pro His Tyr Asp His Tyr His Asn He Lys Phe Glu Trp Phe Asp

645 650 655

Glu Gly Leu Tyr Glu Ala Pro Lys Gly Tyr Ser Leu Glu Asp Leu Leu 660 665 670

Ala Thr Val Lys Tyr Tyr Val Glu His Pro Asn Glu Arg Pro His Ser

675 680 685

Asp Asn Gly Phe Gly Asn Ala Ser Asp His Val Arg Lys Asn Lys Ala

690 695 700

Asp Gin Asp Ser Lys Pro Asp Glu Asp Lys Glu His Asp Glu Val Ser 705 710 715 720

Glu Pro Thr His Pro Glu Ser Asp Glu Lys Glu Asn His Ala Gly Leu

725 730 735 Asn Pro Ser Ala Asp Asn Leu Tyr Lys Pro Ser Thr Asp Thr Glu Glu

740 745 750

Thr Glu Glu Glu Ala Glu Asp Thr Thr Asp Glu Ala Glu lie Pro Gin

755 760 765

Val Glu Asn Ser Val lie Asn Ala Lys lie Ala Asp Ala Glu Ala Leu 770 775 780

Leu Glu Lys Val Thr Asp Pro Ser lie Arg Gin Asn Ala Met Glu Thr 785 790 795 800

Leu Thr Gly Leu Lys Ser Ser Leu Leu Leu Gly Thr Lys Asp Asn Asn

805 810 815 Thr lie Ser Ala Glu Val Asp Ser Leu Leu Ala Leu Leu Lys Glu Ser

820 825 830

Gin Pro Ala Pro lie Gin

835

<210> 2 <211> 641

<212> PRT

<213> Streptococcus pneumoniae <400> 2

Met Lys Lys Thr Thr lie Leu Ser Leu Thr Thr Ala Ala Val lie Leu 1 5 10 15

Ala Ala Tyr Val Pro Asn Glu Pro lie Leu Ala Asp Thr Pro Ser Ser

20 25 30

Glu Val lie Lys Glu Thr Lys Val Gly Ser lie lie Gin Gin Asn Asn

35 40 45 lie Lys Tyr Lys Val Leu Thr Val Glu Gly Asn lie Arg Thr Val Gin

50 55 60

Val Gly Asn Gly Val Thr Pro Val Glu Phe Glu Ala Gly Gin Asp Gly 65 70 75 80

Lys Pro Phe Thr lie Pro Thr Lys lie Thr Val Gly Asp Lys Val Phe

85 90 95

Thr Val Thr Glu Val Ala Ser Gin Ala Phe Ser Tyr Tyr Pro Asp Glu

100 105 110

Thr Gly Arg lie Val Tyr Tyr Pro Ser Ser lie Thr lie Pro Ser Ser

115 120 125 lie Lys Lys lie Gin Lys Lys Gly Phe His Gly Ser Lys Ala Lys Thr 130 135 140 lie lie Phe Asp Lys Gly Ser Gin Leu Glu Lys lie Glu Asp Arg Ala 145 150 155 160

Phe Asp Phe Ser Glu Leu Glu Glu lie Glu Leu Pro Ala Ser Leu Glu

165 170 175 Tyr lie Gly Thr Ser Ala Phe Ser Phe Ser Gin Lys Leu Lys Lys Leu 180 185 190 Thr Phe Ser Ser Ser Ser Lys Leu Glu Leu lie Ser His Glu Ala Phe

195 200 205

Ala Asn Leu Ser Asn Leu Glu Lys Leu Thr Leu Pro Lys Ser Val Lys 210 215 220

Thr Leu Gly Ser Asn Leu Phe Arg Leu Thr Thr Ser Leu Lys His Val

225 230 235 240

Asp Val Glu Glu Gly Asn Glu Ser Phe Ala Ser Val Asp Gly Val Leu

245 250 255

Phe Ser Lys Asp Lys Thr Gin Leu lie Tyr Tyr Pro Ser Gin Lys Asn

260 265 270 Asp Glu Ser Tyr Lys Thr Pro Lys Glu Thr Lys Glu Leu Ala Ser Tyr

275 280 285

Ser Phe Asn Lys Asn Ser Tyr Leu Lys Lys Leu Glu Leu Asn Glu Gly

290 295 300

Leu Glu Lys lie Gly Thr Phe Ala Phe Ala Asp Ala lie Lys Leu Glu

305 310 315 320

Glu lie Ser Leu Pro Asn Ser Leu Glu Thr lie Glu Arg Leu Ala Phe

325 330 335

Tyr Gly Asn Leu Glu Leu Lys Glu Leu lie Leu Pro Asp Asn Val Lys

340 345 350 Asn Phe Gly Lys His Val Met Asn Gly Leu Pro Lys Leu Lys Ser Leu

355 360 365 Thr lie Gly Asn Asn lie Asn Ser Leu Pro Ser Phe Phe Leu Ser Gly

370 375 380

Val Leu Asp Ser Leu Lys Glu lie His lie Lys Asn Lys Ser Thr Glu 385 390 395 400

Phe Ser Val Lys Lys Asp Thr Phe Ala lie Pro Glu Thr Val Lys Phe

405 410 415 Tyr Val Thr Ser Glu His lie Lys Asp Val Leu Lys Ser Asn Leu Ser

420 425 430

Thr Ser Asn Asp lie lie Val Glu Lys Val Asp Asn lie Lys Gin Glu

435 440 445

Thr Asp Val Ala Lys Pro Lys Lys Asn Ser Asn Gin Gly Val Val Gly 450 455 460

Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly Ser 465 470 475 480

Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu Asn

485 490 495 Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu Trp

500 505 510

Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys Asp

515 520 525

Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr Gly

530 535 540

Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly Ser 545 550 555 560

Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu Asn 565 570 575

Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu Trp

580 585 590

Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr Gly Trp Phe Thr Val

595 600 605

Ser Gly Lys Trp Tyr Tyr Thr Tyr Asn Ser Gly Asp Leu Leu Val Asn

610 615 620

Thr Thr Thr Pro Asp Gly Tyr Arg Val Asn Ala Asn Gly Glu Trp Val

625 630 635 640 Gly

<210> 3

<211> 2514

<212> DNA

<213> Streptococcus pneumoniae <400> 3

atgaaaatca ataaaaaata tctagcaggt tcagtggcag tccttgccct aagtgtttgt 60 tcctatgaac ttggtcgtca ccaagctggt caggttaaga aagagtctaa tcgagtttct 120 tatatagatg gtgatcaggc tggtcaaaag gcagaaaatt tgacaccaga tgaagtcagt 180 aagagagagg ggatcaacgc cgaacaaatt gttatcaaga ttacggatca aggttatgtg 240 acctctcatg gagaccatta tcattactat aatggcaagg ttccttatga tgccatcatc 300 agtgaagaac ttctcatgaa agatccgaat tatcagttga aggattcaga cattgtcaat 360 gaaatcaagg gtggctatgt gattaaggta gacggaaaat actatgttta ccttaaagat 420 gcggcccatg cggacaatat tcggacaaaa gaagagatta aacgtcagaa gcaggaacac 480 agtcataatc ataactcaag agcagataat gctgttgctg cagccagagc ccaaggacgt 540 tatacaacgg atgatgggta tatcttcaat gcatctgata tcattgagga cacgggtgat 600 gcttatatcg ttcctcacgg cgaccattac cattacattc ctaagaatga gttatcagct 660 agcgagttag ctgctgcaga agcctattgg aatgggaagc agggatctcg tccttcttca 720 agttctagtt ataatgcaaa tccagttcaa ccaagattgt cagagaacca caatctgact 780 gtcactccaa cttatcatca aaatcaaggg gaaaacattt caagcctttt acgtgaattg 840 tatgctaaac ccttatcaga acgccatgta gaatctgatg gccttatttt cgacccagcg 900 caaatcacaa gtcgaaccgc cagaggtgta gctgtccctc atggtaacca ttaccacttt 960 atcccttatg aacaaatgtc tgaattggaa aaacgaattg ctcgtattat tccccttcgt 1020 tatcgttcaa accattgggt accagattca agaccagaac aaccaagtcc acaatcgact 1080 ccggaaccta gtccaagtct gcaacctgca ccaaatcctc aaccagctcc aagcaatcca 1140 attgatgaga aattggtcaa agaagctgtt cgaaaagtag gcgatggtta tgtctttgag 1200 gagaatggag tttctcgtta tatcccagcc aaggatcttt cagcagaaac agcagcaggc 1260 attgatagca aactggccaa gcaggaaagt ttatctcata agctaggagc taagaaaact 1320 gacctcccat ctagtgatcg agaattttac aataaggctt atgacttact agcaagaatt 1380 caccaagatt tacttgataa taaaggtcga caagttgatt ttgaggtttt ggataacctg 1440 ttggaacgac tcaaggatgt ctcaagtgat aaagtcaagt tagtggatga tattcttgcc 1500 ttcttagctc cgattcgtca tccagaacgt ttaggaaaac caaatgcgca aattacctac 1560 actgatgatg agattcaagt agccaagttg gcaggcaagt acacaacaga agacggttat 1620 atctttgatc ctcgtgatat aaccagtgat gagggggatg cctatgtaac tccacatatg 1680 acccatagcc actggattaa aaaagatagt ttgtctgaag ctgagagagc ggcagcccag 1740 gcttatgcta aagagaaagg tttgacccct ccttcgacag accatcagga ttcaggaaat 1800 actgaggcaa aaggagcaga agctatctac aaccgcgtga aagcagctaa gaaggtgcca 1860 cttgatcgta tgccttacaa tcttcaatat actgtagaag tcaaaaacgg tagtttaatc 1920 atacctcatt atgaccatta ccataacatc aaatttgagt ggtttgacga aggcctttat 1980 gaggcaccta aggggtatag tcttgaggat cttttggcga ctgtcaagta ctatgtcgaa 2040 catccaaacg aacgtccgca ttcagataat ggttttggta acgctagtga ccatgttcgt 2100 aaaaataagg cagaccaaga tagtaaacct gatgaagata aggaacatga tgaagtaagt 2160 gagccaactc accctgaatc tgatgaaaaa gagaatcacg ctggtttaaa tccttcagca 2220 gataatcttt ataaaccaag cactgatacg gaagagacag aggaagaagc tgaagatacc 2280 acagatgagg ctgaaattcc tcaagtagag aattctgtta ttaacgctaa gatagcagat 2340 gcggaggcct tgctagaaaa agtaacagat cctagtatta gacaaaatgc tatggagaca 2400 ttgactggtc taaaaagtag tcttcttctc ggaacgaaag ataataacac tatttcagca 2460 gaagtagata gtctcttggc tttgttaaaa gaaagtcaac cggctcctat acag 2514

<210>

<211> 1923

<212> DNA

<213> Streptococcus pneumoniae

<400> 4

atgaaaaaaa ctacaatatt atcattaact acagctgcgg ttattttagc agcatatgtc 60 cctaatgaac caatcctagc agatactcct agttcggaag taatcaaaga gactaaagtt 120 ggaagtatta ttcaacaaaa taatatcaaa tataaggttc taactgtaga aggtaacata 180 agaactgttc aagtgggtaa tggagttact cctgtagagt ttgaagctgg tcaagatgga 240 aaaccattca cgattcctac aaaaatcaca gtaggtgata aagtatttac cgttactgaa 300 gtagctagtc aagcttttag ttattatcca gatgaaacag gtagaattgt ctactatcct 360 agctctatta ctatcccatc aagcataaaa aaaatacaaa aaaaaggctt ccatggaagt 420 aaagctaaaa ctattatttt tgacaaaggc agtcagctgg agaaaattga agatagagct 480 tttgattttt ctgaattaga agagattgaa ttgcctgcat ctctagaata tattggaaca 540 agtgcatttt cttttagtca aaaattgaaa aagctaacct tttcctcaag ttcaaaatta 600 gaattaatat cacatgaggc ttttgctaat ttatcaaatt tagagaaact aacattacca 660 aaatcggtta aaacattagg aagtaatcta tttagactca ctactagctt aaaacatgtt 720 gatgttgaag aaggaaatga atcgtttgcc tcagttgatg gtgttttgtt ttcaaaagat 780 aaaactcaat taatttatta tccaagtcaa aaaaatgacg aaagttataa aacgcctaag 840 gagacaaaag aacttgcatc atattcgttt aataaaaatt cttacttgaa aaaactcgaa 900 ttgaatgaag gtttagaaaa aatcggtact tttgcatttg cggatgcgat taaacttgaa 960 gaaattagct taccaaatag tttagaaact attgaacgtt tagcctttta cggtaattta 1020 gaattaaaag aacttatatt accagataat gttaaaaatt ttggtaaaca cgttatgaac 1080 ggtttaccaa aattaaaaag tttaacaatt ggtaataata tcaactcatt gccgtccttc 1140 ttcctaagtg gcgtcttaga ttcattaaag gaaattcata ttaagaataa aagtacagag 1200 ttttctgtga aaaaagatac atttgcaatt cctgaaactg ttaagttcta tgtaacatca 1260 gaacatataa aagatgttct taaatcaaat ttatctacta gtaatgatat cattgttgaa 1320 aaagtagata atataaaaca agaaactgat gtagctaaac ctaaaaagaa ttctaatcag 1380 ggagtagttg gttgggttaa agacaaaggt ttatggtatt acttaaacga atcaggttca 1440 atggctactg gttgggttaa agacaaaggt ttatggtatt acttaaacga atcaggttca 1500 atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaacga atcaggttca 1560 atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaatga atcaggttca 1620 atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaacga atcaggttca 1680 atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaacga atcaggttca 1740 atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaatga atcaggttca 1800 atggctactg gttggtttac agtttctggt aaatggtact atacctataa ttcaggagat 1860 ttattagtaa acacgactac acccgatggc tatcgagtca atgctaacgg tgagtgggta 1920 gga 1923

<210> 5

<211> 820

<212> PRT

<213> Streptococcus pneumoniae <400> 5

Met Gly Ser Tyr Glu Leu Gly Arg Hi s Gin Ala Gly Gin Val Lys Lys 1 5 10 15

Glu Ser Asn Arg Val Ser Tyr He Asp Gly Asp Gin Ala Gly Gin Lys

20 25 30

Ala Glu Asn Leu Thr Pro Asp Glu Val Ser Lys Arg Glu Gly He Asn

35 40 45

Ala Glu Gin He Val He Lys He Thr Asp Gin Gly Tyr Val Thr Ser 50 55 60 Hi s Gly Asp Hi s Tyr Hi s Tyr Tyr Asn Gly Lys Val Pro Tyr Asp Ala

65 70 75 80

He He Ser Glu Glu Leu Leu Met Lys Asp Pro Asn Tyr Gin Leu Lys

85 90 95

Asp Ser Asp He Val Asn Glu He Lys Gly Gly Tyr Val He Lys Val

100 105 110

Asp Gly Lys Tyr Tyr Val Tyr Leu Lys Asp Ala Ala Hi s Ala Asp Asn

115 120 125

He Arg Thr Lys Glu Glu He Lys Arg Gin Lys Gin Glu Hi s Ser Hi s 130 135 140 Asn Hi s Asn Ser Arg Ala Asp Asn Ala Val Ala Ala Ala Arg Ala Gin 145 150 155 160

Gly Arg Tyr Thr Thr Asp Asp Gly Tyr He Phe Asn Ala Ser Asp He

165 170 175

He Glu Asp Thr Gly Asp Ala Tyr He Val Pro Hi s Gly Asp Hi s Tyr

180 185 190 His Tyr lie Pro Lys Asn Glu Leu Ser Ala Ser Glu Leu Ala Ala Ala 195 200 205 Glu Ala Tyr Trp Asn Gly Lys Gin Gly Ser Arg Pro Ser Ser Ser Ser 210 215 220

Ser Tyr Asn Ala Asn Pro Val Gin Pro Arg Leu Ser Glu Asn His Asn 225 230 235 240

Leu Thr Val Thr Pro Thr Tyr His Gin Asn Gin Gly Glu Asn lie Ser

245 250 255

Ser Leu Leu Arg Glu Leu Tyr Ala Lys Pro Leu Ser Glu Arg His Val

260 265 270

Glu Ser Asp Gly Leu lie Phe Asp Pro Ala Gin lie Thr Ser Arg Thr

275 280 285 Ala Arg Gly Val Ala Val Pro His Gly Asn His Tyr His Phe lie Pro

290 295 300

Tyr Glu Gin Met Ser Glu Leu Glu Lys Arg lie Ala Arg lie lie Pro

305 310 315 320

Leu Arg Tyr Arg Ser Asn His Trp Val Pro Asp Ser Arg Pro Glu Gin

325 330 335

Pro Ser Pro Gin Ser Thr Pro Glu Pro Ser Pro Ser Leu Gin Pro Ala

340 345 350

Pro Asn Pro Gin Pro Ala Pro Ser Asn Pro lie Asp Glu Lys Leu Val

355 360 365 Lys Glu Ala Val Arg Lys Val Gly Asp Gly Tyr Val Phe Glu Glu Asn

370 375 380 Gly Val Ser Arg Tyr lie Pro Ala Lys Asp Leu Ser Ala Glu Thr Ala

385 390 395 400

Ala Gly lie Asp Ser Lys Leu Ala Lys Gin Glu Ser Leu Ser His Lys

405 410 415

Leu Gly Ala Lys Lys Thr Asp Leu Pro Ser Ser Asp Arg Glu Phe Tyr

420 425 430 Asn Lys Ala Tyr Asp Leu Leu Ala Arg lie His Gin Asp Leu Leu Asp

435 440 445

Asn Lys Gly Arg Gin Val Asp Phe Glu Val Leu Asp Asn Leu Leu Glu 450 455 460

Arg Leu Lys Asp Val Ser Ser Asp Lys Val Lys Leu Val Asp Asp lie 465 470 475 480

Leu Ala Phe Leu Ala Pro lie Arg His Pro Glu Arg Leu Gly Lys Pro

485 490 495

Asn Ala Gin lie Thr Tyr Thr Asp Asp Glu lie Gin Val Ala Lys Leu

500 505 510 Ala Gly Lys Tyr Thr Thr Glu Asp Gly Tyr lie Phe Asp Pro Arg Asp

515 520 525 lie Thr Ser Asp Glu Gly Asp Ala Tyr Val Thr Pro His Met Thr His

530 535 540

Ser His Trp lie Lys Lys Asp Ser Leu Ser Glu Ala Glu Arg Ala Ala

545 550 555 560

Ala Gin Ala Tyr Ala Lys Glu Lys Gly Leu Thr Pro Pro Ser Thr Asp

565 570 575

His Gin Asp Ser Gly Asn Thr Glu Ala Lys Gly Ala Glu Ala lie Tyr 580 585 590

Asn Arg Val Lys Ala Ala Lys Lys Val Pro Leu Asp Arg Met Pro Tyr

595 600 605

Asn Leu Gin Tyr Thr Val Glu Val Lys Asn Gly Ser Leu lie lie Pro

610 615 620

His Tyr Asp His Tyr His Asn lie Lys Phe Glu Trp Phe Asp Glu Gly 625 630 635 640

Leu Tyr Glu Ala Pro Lys Gly Tyr Ser Leu Glu Asp Leu Leu Ala Thr

645 650 655 Val Lys Tyr Tyr Val Glu His Pro Asn Glu Arg Pro His Ser Asp Asn

660 665 670

Gly Phe Gly Asn Ala Ser Asp His Val Arg Lys Asn Lys Ala Asp Gin

675 680 685

Asp Ser Lys Pro Asp Glu Asp Lys Glu His Asp Glu Val Ser Glu Pro

690 695 700

Thr His Pro Glu Ser Asp Glu Lys Glu Asn His Ala Gly Leu Asn Pro 705 710 715 720

Ser Ala Asp Asn Leu Tyr Lys Pro Ser Thr Asp Thr Glu Glu Thr Glu

725 730 735

Glu Glu Ala Glu Asp Thr Thr Asp Glu Ala Glu lie Pro Gin Val Gl

740 745 750

Asn Ser Val lie Asn Ala Lys lie Ala Asp Ala Glu Ala Leu Leu Glu

755 760 765

Lys Val Thr Asp Pro Ser lie Arg Gin Asn Ala Met Glu Thr Leu Thr 770 775 780 Gly Leu Lys Ser Ser Leu Leu Leu Gly Thr Lys Asp Asn Asn Thr lie

785 790 795 800 Ser Ala Glu Val Asp Ser Leu Leu Ala Leu Leu Lys Glu Ser Gin Pro

805 810 815

Ala Pro lie Gin

820

<210> 6

<211> 2463

<212> DNA

<213> Streptococcus pneumoniae

<400> 6

atgggatcct atgaacttgg tcgtcaccaa gctggtcagg ttaagaaaga gtctaatcga 60 gtttcttata tagatggtga tcaggctggt caaaaggcag aaaatttgac accagatgaa 120 gtcagtaaga gagaggggat caacgccgaa caaattgtta tcaagattac ggatcaaggt 180 tatgtgacct ctcatggaga ccattatcat tactataatg gcaaggttcc ttatgatgcc 240 atcatcagtg aagaacttct catgaaagat ccgaattatc agttgaagga ttcagacatt 300 gtcaatgaaa tcaagggtgg ctatgtgatt aaggtagacg gaaaatacta tgtttacctt 360 aaagatgcgg cccatgcgga caatattcgg acaaaagaag agattaaacg tcagaagcag 420 gaacacagtc ataatcataa ctcaagagca gataatgctg ttgctgcagc cagagcccaa 480 ggacgttata caacggatga tgggtatatc ttcaatgcat ctgatatcat tgaggacacg 540 ggtgatgctt atatcgttcc tcacggcgac cattaccatt acattcctaa gaatgagtta 600 tcagctagcg agttagctgc tgcagaagcc tattggaatg ggaagcaggg atctcgtcct 660 tcttcaagtt ctagttataa tgcaaatcca gttcaaccaa gattgtcaga gaaccacaat 720 ctgactgtca ctccaactta tcatcaaaat caaggggaaa acatttcaag ccttttacgt 780 gaattgtatg ctaaaccctt atcagaacgc catgtagaat ctgatggcct tattttcgac 840 ccagcgcaaa tcacaagtcg aaccgccaga ggtgtagctg tccctcatgg taaccattac 900 cactttatcc cttatgaaca aatgtctgaa ttggaaaaac gaattgctcg tattattccc 960 cttcgttatc gttcaaacca ttgggtacca gattcaagac cagaacaacc aagtccacaa 1020 tcgactccgg aacctagtcc aagtctgcaa cctgcaccaa atcctcaacc agctccaagc 1080 aatccaattg atgagaaatt ggtcaaagaa gctgttcgaa aagtaggcga tggttatgtc 1140 tttgaggaga atggagtttc tcgttatatc ccagccaagg atctttcagc agaaacagca 1200 gcaggcattg atagcaaact ggccaagcag gaaagtttat ctcataagct aggagctaag 1260 aaaactgacc tcccatctag tgatcgagaa ttttacaata aggcttatga cttactagca 1320 agaattcacc aagatttact tgataataaa ggtcgacaag ttgattttga ggttttggat 1380 aacctgttgg aacgactcaa ggatgtctca agtgataaag tcaagttagt ggatgatatt 1440 cttgccttct tagctccgat tcgtcatcca gaacgtttag gaaaaccaaa tgcgcaaatt 1500 acctacactg atgatgagat tcaagtagcc aagttggcag gcaagtacac aacagaagac 1560 ggttatatct ttgatcctcg tgatataacc agtgatgagg gggatgccta tgtaactcca 1620 catatgaccc atagccactg gattaaaaaa gatagtttgt ctgaagctga gagagcggca 1680 gcccaggctt atgctaaaga gaaaggtttg acccctcctt cgacagacca tcaggattca 1740 ggaaatactg aggcaaaagg agcagaagct atctacaacc gcgtgaaagc agctaagaag 1800 gtgccacttg atcgtatgcc ttacaatctt caatatactg tagaagtcaa aaacggtagt 1860 ttaatcatac ctcattatga ccattaccat aacatcaaat ttgagtggtt tgacgaaggc 1920 ctttatgagg cacctaaggg gtatagtctt gaggatcttt tggcgactgt caagtactat 1980 gtcgaacatc caaacgaacg tccgcattca gataatggtt ttggtaacgc tagtgaccat 2040 gttcgtaaaa ataaggcaga ccaagatagt aaacctgatg aagataagga acatgatgaa 2100 gtaagtgagc caactcaccc tgaatctgat gaaaaagaga atcacgctgg tttaaatcct 2160 tcagcagata atctttataa accaagcact gatacggaag agacagagga agaagctgaa 2220 gataccacag atgaggctga aattcctcaa gtagagaatt ctgttattaa cgctaagata 2280 gcagatgcgg aggccttgct agaaaaagta acagatccta gtattagaca aaatgctatg 2340 gagacattga ctggtctaaa aagtagtctt cttctcggaa cgaaagataa taacactatt 2400 tcagcagaag tagatagtct cttggctttg ttaaaagaaa gtcaaccggc tcctatacag 2460 tag 2463

<210> 7

<211> 445

<212> PRT

<213> Streptococcus pneumoniae

<400> 7

Met Ala Asp Thr Pro Ser Ser Glu Val lie Lys Glu Thr Lys Val Gly

Ser lie lie Gin Gin Asn Asn lie Lys Tyr Lys Val Leu Thr Val Glu

20 25 30 Gly Asn lie Gly Thr Val Gin Val Gly Asn Gly Val Thr Pro Val Glu

35 40 45 Phe Glu Ala Gly Gin Asp Gly Lys Pro Phe Thr lie Pro Thr Lys lie 50 55 60

Thr Val Gly Asp Lys Val Phe Thr Val Thr Glu Val Ala Ser Gin Ala

65 70 75 80

Phe Ser Tyr Tyr Pro Asp Glu Thr Gly Arg lie Val Tyr Tyr Pro Ser

85 90 95

Ser lie Thr lie Pro Ser Ser lie Lys Lys lie Gin Lys Lys Gly Phe

100 105 110

His Gly Ser Lys Ala Lys Thr lie lie Phe Asp Lys Gly Ser Gin Leu

115 120 125

Glu Lys lie Glu Asp Arg Ala Phe Asp Phe Ser Glu Leu Glu Glu lie

130 135 140

Glu Leu Pro Ala Ser Leu Glu Tyr lie Gly Thr Ser Ala Phe Ser Phe 145 150 155 160

Ser Gin Lys Leu Lys Lys Leu Thr Phe Ser Ser Ser Ser Lys Leu Glu

165 170 175

Leu lie Ser His Glu Ala Phe Ala Asn Leu Ser Asn Leu Glu Lys Leu

180 185 190

Thr Leu Pro Lys Ser Val Lys Thr Leu Gly Ser Asn Leu Phe Arg Leu

195 200 205

Thr Thr Ser Leu Lys His Val Asp Val Glu Glu Gly Asn Glu Ser Phe

210 215 220

Ala Ser Val Asp Gly Val Leu Phe Ser Lys Asp Lys Thr Gin Leu lie 225 230 235 240

Tyr Tyr Pro Ser Gin Lys Asn Asp Glu Ser Tyr Lys Thr Pro Lys Glu 245 250 255

Thr Lys Glu Leu Ala Ser Tyr Ser Phe Asn Lys Asn Ser Tyr Leu Lys

260 265 270

Lys Leu Glu Leu Asn Glu Gly Leu Glu Lys lie Gly Thr Phe Ala Phe

275 280 285

Ala Asp Ala lie Lys Leu Glu Glu lie Ser Leu Pro Asn Ser Leu Glu 290 295 300

Thr lie Glu Arg Leu Ala Phe Tyr Gly Asn Leu Glu Leu Lys Glu Leu

305 310 315 320 lie Leu Pro Asp Asn Val Lys Asn Phe Gly Lys His Val Met Asn Gly

325 330 335

Leu Pro Lys Leu Lys Ser Leu Thr lie Gly Asn Asn lie Asn Ser Leu

340 345 350

Pro Ser Phe Phe Leu Ser Gly Val Leu Asp Ser Leu Lys Glu lie His

355 360 365 lie Lys Asn Lys Ser Thr Glu Phe Ser Val Lys Lys Asp Thr Phe Ala 370 375 380 lie Pro Glu Thr Val Lys Phe Tyr Val Thr Ser Glu His lie Lys Asp

385 390 395 400 Val Leu Lys Ser Asn Leu Ser Thr Ser Asn Asp lie lie Val Glu Lys

405 410 415

Val Asp Asn lie Lys Gin Glu Thr Asp Val Ala Lys Pro Lys Lys Asn

420 425 430

Ser Asn Gin Gly Val Val Gly Trp Val Lys Asp Lys Gly

435 440 445 <210> 8

<211> 1338

<212> DNA

<213> Streptococcus pneumoniae

<400> 8

atggcagata ctcctagttc ggaagtaatc aaagagacta aagttggaag tattattcaa 60 caaaataata tcaaatataa ggttctaact gtagaaggta acataggaac tgttcaagtg 120 ggtaatggag ttactcctgt agagtttgaa gctggtcaag atggaaaacc attcacgatt 180 cctacaaaaa tcacagtagg tgataaagta tttaccgtta ctgaagtagc tagtcaagct 240 tttagttatt atccagatga aacaggtaga attgtctact atcctagctc tattactatc 300 ccatcaagca taaaaaaaat acaaaaaaaa ggcttccatg gaagtaaagc taaaactatt 360 atttttgaca aaggcagtca gctggagaaa attgaagata gagcttttga tttttctgaa 420 ttagaagaga ttgaattgcc tgcatctcta gaatatattg gaacaagtgc attttctttt 480 agtcaaaaat tgaaaaagct aaccttttcc tcaagttcaa aattagaatt aatatcacat 540 gaggcttttg ctaatttatc aaatttagag aaactaacat taccaaaatc ggttaaaaca 600 ttaggaagta atctatttag actcactact agcttaaaac atgttgatgt tgaagaagga 660 aatgaatcgt ttgcctcagt tgatggtgtt ttgttttcaa aagataaaac tcaattaatt 720 tattatccaa gtcaaaaaaa tgacgaaagt tataaaacgc ctaaggagac aaaagaactt 780 gcatcatatt cgtttaataa aaattcttac ttgaaaaaac tcgaattgaa tgaaggttta 840 gaaaaaatcg gtacttttgc atttgcggat gcgattaaac ttgaagaaat tagcttacca 900 aatagtttag aaactattga acgtttagcc ttttacggta atttagaatt aaaagaactt 960 atattaccag ataatgttaa aaattttggt aaacacgtta tgaacggttt accaaaatta 1020 aaaagtttaa caattggtaa taatatcaac tcattgccgt ccttcttcct aagtggcgtc 1080 ttagattcat taaaggaaat tcatattaag aataaaagta cagagttttc tgtgaaaaaa 1140 gatacatttg caattcctga aactgttaag ttctatgtaa catcagaaca tataaaagat 1200 gttcttaaat caaatttatc tactagtaat gatatcattg ttgaaaaagt agataatata 1260 aaacaagaaa ctgatgtagc taaacctaaa aagaattcta atcagggagt agttggttgg 1320 gttaaagaca aaggttaa 1338

<210> 9

<211> 471

<212> PRT

<213> Streptococcus pneumoniae <400> 9

Met Ala Asn Lys Ala Val Asn Asp Phe He Leu Ala Met Asn Tyr Asp 1 5 10 15 Lys Lys Lys Leu Leu Thr Hi s Gin Gly Glu Ser He Glu Asn Arg Phe

20 25 30

He Lys Glu Gly Asn Gin Leu Pro Asp Glu Phe Val Val He Glu Arg

35 40 45

Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp He Ser Val Thr Ala 50 55 60

Cys Asn Asp Ser Arg Leu Tyr Pro Gly Ala Leu Leu Val Val Asp Glu 65 70 75 80

Thr Leu Leu Glu Asn Asn Pro Thr Leu Leu Ala Val Asp Arg Ala Pro

85 90 95 Met Thr Tyr Ser He Asp Leu Pro Gly Leu Ala Ser Ser Asp Ser Phe

100 105 110

Leu Gin Val Glu Asp Pro Ser Asn Ser Ser Val Arg Gly Ala Val Asn

115 120 125

Asp Leu Leu Ala Lys Trp Hi s Gin Asp Tyr Gly Gin Val Asn Asn Val 130 135 140

Pro Ala Arg Met Gin Tyr Glu Lys He Thr Ala Hi s Ser Met Glu Gin 145 150 155 160

Leu Lys Val Lys Phe Gly Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu

165 170 175 Asp He Asp Phe Asn Ser Val Hi s Ser Gly Glu Lys Gin He Gin He

180 185 190 Val Asn Phe Lys Gin lie Tyr Tyr Thr Val Ser Val Asp Ala Val Lys 195 200 205

Asn Pro Gly Asp Val Phe Gin Asp Thr Val Thr Val Glu Asp Leu Lys 210 215 220

Gin Arg Gly lie Ser Ala Glu Arg Pro Leu Val Tyr lie Ser Ser Val

225 230 235 240 Ala Tyr Gly Arg Gin Val Tyr Leu Lys Leu Glu Thr Thr Ser Lys Ser

245 250 255

Asp Glu Val Glu Ala Ala Phe Glu Ala Leu lie Lys Gly Val Lys Val

260 265 270

Ala Pro Gin Thr Glu Trp Lys Gin lie Leu Asp Asn Thr Glu Val Lys

275 280 285

Ala Val lie Leu Cys Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr 290 295 300

Gly Lys Val Asp Met Val Glu Asp Leu lie Gin Glu Gly Ser Arg Phe 305 310 315 320 Thr Ala Asp His Pro Gly Leu Pro lie Ser Tyr Thr Thr Ser Phe Leu

325 330 335

Arg Asp Asn Val Val Ala Thr Phe Gin Asn Ser Thr Asp Tyr Val Glu

340 345 350

Thr Lys Val Thr Ala Tyr Arg Asn Gly Asp Leu Leu Leu Asp His Ser

355 360 365

Gly Ala Tyr Val Ala Gin Tyr Tyr lie Thr Trp Asp Glu Leu Ser Tyr 370 375 380

Asp His Gin Gly Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn 385 390 395 400

Gly Gin Asp Leu Thr Ala Hi s Phe Thr Thr Ser He Pro Leu Lys Gly

405 410 415

Asn Val Arg Asn Leu Ser Val Lys He Arg Glu Ala Thr Gly Leu Ala

420 425 430

Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu Val

435 440 445

Arg Lys Arg Thr He Ser He Trp Gly Thr Thr Leu Tyr Pro Gin Val 450 455 460

Glu Asp Lys Val Glu Asn Asp

465 470

<210> 10

<211> 471

<212 > PRT

<213> Streptococcus pneumoniae <400> 10

Met Ala Asn Lys Ala Val Asn Asp Phe He Leu Ala Met Asn Tyr Asp 1 5 10 15

Lys Lys Lys Leu Leu Thr Hi s Gin Gly Glu Ser He Glu Asn Arg Phe

20 25 30

He Lys Glu Gly Asn Gin Leu Pro Asp Glu Phe Val Val He Glu Arg

35 40 45

Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp He Ser Val Thr Ala 50 55 60 Thr Asn Asp Ser Arg Leu Tyr Pro Gly Ala Leu Leu Val Val Asp Glu 65 70 75 80

Thr Leu Leu Glu Asn Asn Pro Thr Leu Leu Ala Val Asp Arg Ala Pro

85 90 95

Met Thr Tyr Ser lie Asp Leu Pro Gly Leu Ala Ser Ser Asp Ser Phe

100 105 110

Leu Gin Val Glu Asp Pro Ser Asn Ser Ser Val Arg Gly Ala Val Asn

115 120 125

Asp Leu Leu Ala Lys Trp His Gin Asp Tyr Gly Gin Val Asn Asn Val 130 135 140

Pro Ala Arg Met Gin Tyr Glu Lys lie Thr Ala His Ser Met Glu Gin 145 150 155 160

Leu Lys Val Lys Phe Gly Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu

165 170 175

Asp lie Asp Phe Asn Ser Val His Ser Gly Glu Lys Gin lie Gin lie

180 185 190

Val Asn Phe Lys Gin lie Tyr Tyr Thr Val Ser Val Asp Ala Val Lys

195 200 205

Asn Pro Gly Asp Val Phe Gin Asp Thr Val Thr Val Glu Asp Leu Lys 210 215 220

Gin Arg Gly lie Ser Ala Glu Arg Pro Leu Val Tyr lie Ser Ser Val

225 230 235 240

Ala Tyr Gly Arg Gin Val Tyr Leu Lys Leu Glu Thr Thr Ser Lys Ser

245 250 255

Asp Glu Val Glu Ala Ala Phe Glu Ala Leu lie Lys Gly Val Lys Val 260 265 270

Ala Pro Gin Thr Glu Trp Lys Gin He Leu Asp Asn Thr Glu Val Lys

275 280 285

Ala Val He Leu Gly Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr

290 295 300

Gly Lys Val Asp Met Val Glu Asp Leu He Gin Glu Gly Ser Arg Phe 305 310 315 320

Thr Ala Asp His Pro Gly Leu Pro He Ser Tyr Thr Thr Ser Phe Leu

325 330 335 Arg Asp Asn Val Val Ala Thr Phe Gin Asn Ser Thr Asp Tyr Val Glu

340 345 350

Thr Lys Val Thr Ala Tyr Arg Asn Gly Asp Leu Leu Leu Asp His Ser

355 360 365

Gly Ala Tyr Val Ala Gin Tyr Tyr He Thr Trp Asp Glu Leu Ser Tyr

370 375 380

Asp His Gin Gly Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn 385 390 395 400

Gly Gin Asp Leu Thr Ala His Phe Thr Thr Ser He Pro Leu Lys Gly

405 410 415

Asn Val Arg Asn Leu Ser Val Lys He Arg Glu Cys Thr Gly Leu Ala

420 425 430

Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu Val

435 440 445

Arg Lys Arg Thr He Ser He Trp Gly Thr Thr Leu Tyr Pro Gin Val 450 455 460 Glu Asp Lys Val Glu Asn Asp 465 470