Patent application title: Immunogenic PcpA Polypeptides and Uses Thereof
David E. Briles (Birmingham, AL, US)
Susan K. Hollingshead (Birmingham, AL, US)
Jeremy Yethon (Toronto, CA)
Joe Wang (Toronto, CA)
THE UAB RESEARCH FOUNDATION
Sanofi Pasteur, Ltd.
IPC8 Class: AA61K3909FI
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) bacterium or component thereof or substance produced by said bacterium (e.g., legionella, borrelia, anaplasma, shigella, etc.) streptococcus (e.g., group b streptococcus, pneumococcus or streptococcus pneumoniae, etc.)
Publication date: 2011-01-06
Patent application number: 20110002962
Provided herein are compositions and methods for eliciting an immune
response against Streptococcus pneumoniae. More particularly, the
compositions and methods relate to immunogenic polypeptides, including
fragments of PcpA and variants thereof, and nucleic acids, vectors and
transfected cells that encode or express the polypeptides. Methods of
making and using the immunogenic polypeptides are also described.
24. An immunogenic fragment of PcpA comprising one or more leucine rich regions of naturally occurring PcpA.
25. The immunogenic fragment of claim 24, wherein the fragment lacks a choline binding anchor sequence.
26. The immunogenic fragment of claim 24, wherein the leucine rich region comprises an N-terminal region of naturally occurring PcpA.
27. The immunogenic fragment of claim 26, wherein the N-terminal region comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:41 with one or more conservative amino acid substitutions.
28. The immunogenic fragment of claim 26, wherein the N-terminal region comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:41.
29. The immunogenic fragment of claim 24, wherein the naturally occurring PcpA is from Streptococcus pneumoniae serotype 6.
30. The immunogenic fragment of claim 24, wherein the fragment comprises SEQ ID NO:45 with one or more conservative amino acid substitutions.
31. The immunogenic fragment of claim 24, wherein the fragment comprises SEQ ID NO:45.
32. A composition comprising the immunogenic fragment of claim 24 and a pharmaceutically acceptable carrier.
33. The composition of claim 32, further comprising an adjuvant.
34. The composition of claim 32, further comprising an immunogenic Staphylococcus polypeptide.
35. The composition of claim 32, further comprising an immunogenic fragment of PspA, pneumolysin, or a combination thereof.
36. The composition of claim 32, further comprising an immunogenic fragment of pneumolysin.
37. The composition of claim 32, wherein the composition is suitable for administration to a mucosal surface.
38. The composition of claim 37, wherein the composition is a nasal spray.
39. The composition of claim 37, wherein the composition is a nebulizer solution.
40. The composition of claim 37, wherein the composition is an aerosol inhalant.
41. A container comprising the composition of claim 32.
42. The container of claim 41, wherein the container is a nasal sprayer.
43. The container of claim 41, wherein the container is a nebulizer.
44. The container of claim 41, wherein the container is an inhaler.
45. A method of generating antibodies specific to PcpA in a subject comprising administering to the subject the immunogenic fragment of claim 1.
46. A method of preventing or reducing pneumococcal nasal carriage in a subject comprising administering to the subject the immunogenic fragment of claim 1.
47. A method of reducing the risk of a pneumococcal infection in a subject comprising administering to the subject the immunogenic fragment of claim 1.
48. The method of claim 47, wherein the pneumococcal infection is meningitis.
49. The method of claim 47, wherein the pneumococcal infection is otitis media.
50. The method of claim 47, wherein the pneumococcal infection is pneumonia.
51. The method of claim 47, wherein the pneumococcal infection is hemolytic uremia.
52. The method of claim 47, wherein the administration of the immunogenic fragment does not eliminate pneumococcal nasal carriage.
53. The method of claim 47, further comprising administering a second immunogenic fragment selected from the group consisting of an immunogenic fragment of PspA, pneumolysin, or a combination thereof.
54. The method of claim 47, further comprising administering an immunogenic fragment of pneumolysin.
55. An isolated polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:41.
56. An isolated polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:45.
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Ser. No. 60/822,715, filed Aug. 17, 2006; U.S. Ser. No. 60/827,348, filed Sep. 28, 2006; and U.S. Ser. No. 60/917,178, filed May 10, 2007, which are incorporated by reference herein in their entireties.
Streptococcus pneumoniae is a rather ubiquitous human pathogen, which can infect several organs including lungs, the central nervous system (CNS), the middle ear, and the nasal tract. Infection results in various symptoms such as bronchitis, pneumonia, meningitis, sinus infection, and sepsis. S. pneumoniae is a major cause of bacterial meningitis in humans and is associated with significant mortality and morbidity despite antibiotic treatment (Quagliarello et al., (1992) N. Eng. J. Med. 327: 864-872).
There are two currently available pneumococcal vaccines. One is a vaccine for adults composed of 23 different capsular polysaccharides, which together represent the capsular types of about 90% of strains causing pneumococcal infection. This vaccine, however, is not immunogenic in children, an age group with high susceptibility to pneumococcal infection. 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. Plotkin and W. A. Orenstein (eds.), W. B. Saunders and Co., Philadelphia, Pa.; Shapiro et al., N. Engl. J. Med. 325:1453-1460 (1991)). This vaccine has not been shown to be effective against non-bacteremic pneumococcal pneumonia, the most common form of infection.
The second available vaccine is a 7-valent conjugate vaccine that is efficacious against bacteremic pneumococcal infections in children less than 2 years of age. It has also demonstrated efficacy against pneumonia (Black et al., Pediatr. Infect. Dis. 21:810-5 (2002); Black et al., Arch. Pediatr 11 (7):843-53 (2004)). The production of this vaccine is complicated because of the need to produce 7 different conjugates, which leads to the vaccine being expensive (about $200/child). Moreover, the vaccine does not do a good job of covering infections in the developing world where non-vaccine types of Streptococcus pneumoniae are very common (Di Fabio et al., Pediatr. Infect. Dis. J. 20:959-967 (2001); Mulholland, Trop. Med. Int. Health 10:497-500 (2005)). This vaccine does not work as well against otitis media and colonization as it does against invasive disease. It has also been shown that the use of the 7-valent conjugate vaccine has led to an increase in colonization and disease with 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, 180:1171-1176 (1999)). Therefore, a need remains for effective treatments for Streptococcus pneumoniae.
Compositions and methods for eliciting an immune response against Streptococcus pneumoniae are described. More particularly, the present disclosure relates to immunogenic PcpA polypeptides, including fragments of PcpA and variants thereof, and nucleic acids that encode the polypeptides. The present disclosure further relates to methods of making and using the immunogenic polypeptides. These compositions and methods offer improved efficacy and efficiency and reduced cost as compared to presently available compositions and methods designed to reduce or prevent pneumococcal infection.
DESCRIPTION OF DRAWINGS
FIG. 1 shows PCR confirmation of pcpA. PCR analysis of genomic DNA of various S. pneumoniae strains. Primer pair (BGP1 (SEQ ID NO:50) and BGP2 (SEQ ID NO:51)) were used to amplify the nucleic acid encoding the N-terminal portion of PcpA (including the LRR region). Lane 1, TIGR4; Lane 2, L82013; Lane 3, D1091B; Lane 4, BG12730; Lane 5, TJ0893; Lane 6, R6; Lane 7, BG10752; Lane 8, V175; Lane 9, EF3030; Lane 10, negative control (no template DNA).
FIG. 2 shows Western blot analysis of PcpA presence under low Mn2+ conditions. Bacteria were cultured in low Mn2+ medium until mid-log phase and total cellular protein samples prepared. Samples were separated by SDS-PAGE, transferred to nitrocellulose and probe with a rPcpA polyclonal antiserum. Lane 1, JEN11 (pcpA- mutant); Lane 2, JEN7 (pcpA constitutive mutant); Lane 3, D1091B; Lane 4, EF5668; Lane 5, BG10752; Lane 6, V175; Lane 7, L82013; Lane 8, BG12730; Lane 9, TJ0893.
FIG. 3 shows that protection conferred by rPcpA immunization compared to adjuvant alone was statistically significant in a murine model of pneumonia. CBA/N mice were subcutaneously immunized with rPcpA adsorbed to aluminum hydroxide or aluminum hydroxide alone. Mice were challenged intranasally under light anesthesia, with 5×106 CFUs of EF3030. Mice were sacrificed 7 days post-infection and bacterial counts determined from lung homogenates (FIG. 3A) and nasal washes (FIG. 3B). Horizontal line denotes median Log 10 CFUs. (**:p=0.0019, Mann-Whitney).
FIG. 4 shows that protection conferred against other S. pneumoniae capsular serotypes by rPcpA immunization versus adjuvant alone was statistically significant in a murine model of pneumonia. Mice were challenged with strains (FIG. 4A) TJ0893, serotype 14 (**:p=0.0209); (FIG. 4B) L82016, serotype 6B (**:p=0.0193); or (FIG. 4C) EF9303, serotype 23F (**:p=0.0388, Mann-Whitney). Horizontal line denotes median Log 10 CFUs.
FIG. 5 shows the effect of pcpA inactivation on intranasal colonization of S. pneumoniae. Mice were challenged intranasally with 106 CFUs of EF3030 or its derivative JEN18. Mice were sacrificed 7 days post-infection and bacterial counts determined from nasal washes. Horizontal line denotes median Log 10 CFUs/Nose.
FIG. 6 shows that protection conferred by rPcpA immunization versus adjuvant alone was statistically significant in a murine model of fatal sepsis. CBA/N mice were subcutaneously immunized with rPcpA adsorbed to aluminum hydroxide or aluminum hydroxide alone. Mice were challenged intravenously with 300 CFUs of TIGR4 and survival time was monitored for 21 days. Horizontal line denotes median survival time. (**:p=0.0067, Mann-Whitney). Surviving mice were euthanized and, upon examination, none had detectable S. pneumoniae in their blood.
FIG. 7 shows virulence of TIGR4 and its pcpA inactivated derivative JEN11 in a murine model of sepsis. Mice were challenged intravenously with 300 CFUs of TIGR4 or JEN11 and survival time was monitored for 21 days. Horizontal line denotes median survival time. (**:p=0.0299, Mann-Whitney).
FIG. 8 shows that protection was conferred by rPcpA mucosal immunization compared to adjuvant alone in a murine model of pneumonia. CBA/N mice were intranasally immunized with rPcpA and cholera toxin B subunit (CTB) or CTB alone. Mice were challenged intranasally under light anesthesia, with 5×106 CFUs of EF3030. Mice were sacrificed 7 days post-infection and bacterial counts in the homogenized lungs were determined. Horizontal line denotes median Log 10 CFUs. (*:p=0.0001, Mann-Whitney).
FIG. 9 shows adherence of pcpA+ and pcpA- TIGR4 strains (TIGR4 and JEN11, respectively) to human lung epithelial cells. A549 human lung epithelial cell monolayers were incubated for 150 minutes with 106 CFU of pcpA+ and pcpA- TIGR4 strains that had been grown under high manganese (High Mn2+) or low manganese (Low Mn2+) growth conditions. The epithelial cells were washed and lysed. Numbers of adherent pneumococci in each lysate were determined by quantitative plating on blood agar plates. Log 10 CFU recovered refers to the number of pneumococci associated with the lung epithelial cells at the end of the experiment. (**:p=0.0022, Mann-Whitney).
FIG. 10 shows pcpA+ and pcpA- TIGR4 strains did not adhere to human nasal epithelial cells. Detroit562 human nasal epithelial cell monolayers were incubated for 150 minutes with 106 CFU of pcpA+ and pcpA- TIGR4 strains that had been grown under high manganese (High Mn2+) or low manganese (Low Mn2+) growth conditions. The cells were then washed and lysed. Numbers of pneumococci in the lysate were determined by quantitative plating on blood agar plates. Log 10 CFU recovered refers to the number of pneumococci at the end of the experiment.
FIG. 11 shows inhibition of adherence of pneumococci to A549 cells by an antibody to PcpA. A549 human lung epithelial cell monolayers were incubated with 106 CFU of pcpA+ and pcpA- TIGR4 strains grown in high manganese (High Mn2+) or low manganese (Low Mn2+) without antibody, with 1/100 dilution, or with 1/50 dilution of PcpA antibody. The cells were washed and lysed. Numbers of pneumococci in the lysate were determined by quantitative plating on blood agar plates.
FIG. 12 shows protection against sepsis using rabbit sera to PcpA. Rabbit serum was prepared by immunizing a rabbit with 100 μg rPcpA in complete Freund's adjuvant followed two and four weeks later by 100 μg rPcpA in complete Freund's adjuvant. Sera was collected two weeks after the final boost and was shown to contain antibody to PcpA by dot blot assay. Pre-immune sera was also collected before the start of the immunizations. Mice were tested in groups of two for the ability of dilutions of the rabbit anti-sera to protect against fatal pneumococcal infection. Three groups of mice received 0.1 mL of 1/10, 1/100 or 1/1000 dilutions of the immune sera intraperitoneally one hour prior to i.v. challenge with TIGR4. Two mice received 1/10 pre-immune (non-immune) rabbit serum and two mice received the diluent, PBS, only. Mice were observed for 500 hours or until time of death. The two mice receiving 1/10 immune sera lived throughout the experiment. All other mice died between 40 and 60 hours post challenge.
FIG. 13 shows protection against lung infection with PcpA and pneumolysin (Ply). Mice were immunized three times with 5 μg of rPcpA, 5 μg of pneumolysin (Ply), or 5 μg of rPcpA plus 5 μg Ply. The first two injections were with alum and the third injection was with protein alone. The Ply used was wild-type Ply. Mice were anethesized with isoflurane (MinRAD, Buffalo, N.Y.) and challenged i.n. with 5×106 CFU of capsular type 19F strain EF3030 in 40 μL volume. This procedure results in lung infection and nasal colonization. Seven days later mice were sacrificed and homogenized lungs were plated. The CFU observed indicated that immunization with either PcpA or Ply resulted in similar levels of protection. Mice immunized with PcpA and Ply resulted in over 100-fold more protection than control mice and 10 times more protection than Ply or PcpA alone.
FIG. 14 is a schematic showing the construction of the plasmid pJMS87 formed by ligation of the plasmid pET30a and a nucleic acid encoding a fragment of PcpA (ΔSPΔCBD PcpA from Streptococcus pneumoniae strain B6).
FIGS. 15A and 15B are graphical representations of the protection conferred by immunizing mice with the recombinant PcpA (rPcpA) of Example 8 (10 to 0.625 μg/dose) in a murine sepsis model. Mice were challenged intraperitoneally with 300 CFU of strain WUBM3. FIG. 15A shows that rPcpA immunized mice at each dose were significantly protected when compared to the adjuvant control group (PBS) (Fisher Exact Test) over a period of time. FIG. 15B shows the level of protection in each group at day 7 post challenge.
FIG. 16 is a graphical representation of the protection conferred by immunization with the rPcpA of Example 8 in a mouse pneumonia model. Groups 1 to 6 were immunized with placebo (Group 1), PspA (Group 2) or rPcpA (Groups 3 to 6). Approximately 14 CBA/N mice per group were immunized at day 0 subcutaneously (s.c.) with a primary dose of immunogen. A second immunization was performed at day 21 and a third immunization at day 43. On day 63 the mice were challenged intranasally with 5.6×106 CFU of S. pneumoniae strain EF3030. Five days post infection CFU were determined in homogenized lung tissue.
Immunogenic fragments and variants of PcpA are described herein along with methods of making and using the fragments and variants. PcpA, which was initially identified as a choline binding protein (CBP) of Streptococcus pneumoniae, differs from the CBP proteins PspA and PspC (Sanchez-Beato et al., FEMS Microbiol. Lett. 164:207-214 (1998)), and mutations in pcpA have been shown to cause (1) reduced virulence in the lung, in bacteremia, and in the nasopharynx of mice in competition models in which a mutant strain and a wild type strain are allowed to compete (Hava and Camilli, Mol. Microbiol. 45:1389-1406 (2002)); (2) reduced virulence and bacterial load in a non-competition comparison of lung colonization (Johnston et al., Infect. Immun. 74:1171-1180 (2006)); (3) reduced ability of the invasive strain TIGR4 (capsular type 4) S. pneumoniae to cause sepsis in CBA/CaHN-Btkxid/J mice; and (4) reduced lung colonization in competition with wild type strains. The present disclosure provides the first evidence that PcpA is immunogenic and, in particular, that fragments and variants of PcpA are immunogenic.
Immunogenic polypeptides comprise the full-length PcpA amino acid sequence (in the presence or absence of the signal sequence), fragments thereof, and variants thereof. Full-length PcpA includes GenBank Accession No. CAB04758 from Streptococcus pneumoniae strain B6, GenBank Accession No. NP--346554 from S. pneumoniae strain TIGR4 and GenBank Accession No. NP--359536 from S. pneumoniae strain R6.
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 within SEQ ID NOs: 1, 2, 41 or 45.
An immunogenic polypeptide of PcpA optionally lacks the choline binding anchor sequence typically present in the naturally occurring mature PcpA protein. The naturally occurring sequence of the choline binding anchor is SEQ ID NO:52 of the mature PcpA protein. 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 any identity in between, e.g., 80, 85, 90 and 95% identity, to the naturally occurring PcpA. The N-terminal region may comprise the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 41 or 45, 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 may comprise an amino acid sequence having about 60 to about 99% sequence identity (or any identity in between 80 to 99% identity) to SEQ ID NOs: 1, 2, 3, 4, 41 or 45.
Immunogenic fragments of SEQ ID NOs:1, 2, 3, 4, 41 or 45 comprise 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 191 amino acid residues of SEQ ID NOs: 1, 2, 3, 4, 41 or 45 or any number of amino acid residues between 5 and 191. Examples of such fragments include, by way of example, amino acids comprising LEKIEDRAFD (SEQ ID NO:5), FSELEEIELP (SEQ ID NO:6), ASLEYIGTSA (SEQ ID NO:7), FSFSQKLKKL (SEQ ID NO:8), TFSSSSKLEL (SEQ ID NO:9), ISHEAFANLS (SEQ ID NO:10), NLEKLTLPKS (SEQ ID NO:11), VKTLGSNLFR (SEQ ID NO:12), LTTSLNMLML (SEQ ID NO:13), LTTSLKHVDV (SEQ ID NO:14), RGMIVASVDG (SEQ ID NO:15), EEGNESFASVDG (SEQ ID NO:16), VSFQSKTQLI (SEQ ID NO:17), VLFSKDKTQLI (SEQ ID NO:18), YYPSQKNDES (SEQ ID NO:19), YKTPKETKEL (SEQ ID NO:20), ASYSFNKNSY (SEQ ID NO:21), LKKLELNEGL (SEQ ID NO:22), QKIGTFAFAD (SEQ ID NO:23), EKIGTFAFAD (SEQ ID NO:24), ATKLEEISLP (SEQ ID NO:25), AIKLEEISLP (SEQ ID NO:26), NSLETIERLA (SEQ ID NO:27), FYGNLELKELIL (SEQ ID NO:28).
Optionally, immunogenic polypeptides of PcpA lack the LRRs. Examples of immunogenic polypeptides lacking the LRR include SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31 or any immunogenic fragment of either SEQ ID NOs:29, 30 or 31 comprising 5 or more amino acid residues. SEQ ID NOs:30 and 31 comprise the residues C-terminal to the leucine-rich region of PcpA.
Variants of the immunogenic polypeptides described herein may comprise one or more conservative amino acid substitutions. Variants of the immunogenic polypeptides include amino acid sequence having about 60 to about 99% sequence identity (or any identity in between 60 and 99% identity) to SEQ ID NOs:1 to 31, 41 and 45 or any fragment thereof. Variants are selected for their immunogenic capacity using methods taught herein.
The immunogenic polypeptides of PcpA described herein include fragments of PcpA and variants of such fragments. Variants of PcpA fragments may comprise amino acid sequence modifications. For example, amino acid sequence modifications include substitutional, insertional or deletional changes. Substitutions, deletions, insertions or any combination thereof may be combined in a single variant so long as the variant is an immunogenic polypeptide. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily 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 any one site within the protein molecule. These variants ordinarily 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 recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known and include, but are not limited to, M13 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 1 and are referred to as conservative substitutions. However, others are well known to those of skill in the art.
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions Exemplary Original Residue Substitutions Arg Lys Asn Gln Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
Variants as used herein may also include naturally occurring pcpA alleles from alternate strains that exhibit polymorphisms at one or more sites within the homologous pcpA gene. Variants can be produced by conventional molecular biology techniques. The variants are described herein relative to sequence identity as compared to the naturally occurring pcpA. Those of skill in the art readily understand how to determine the sequence identity of two polypeptides or nucleic acids. For example, the sequence identity can be calculated after aligning the two sequences so that the identity is at its highest level. Alignments are dependent to some extent upon the use of the specific algorithm in alignment programs. This could include, for example, the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, PNAS USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), BLAST and BLAST 2.0 and algorithms described by Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977; Altschul, et al., J. Mol. Biol. 215:403-410, 1990; Zuker, M. Science 244:48-52, 1989; Jaeger et al. PNAS USA 86:7706-7710, 1989 and Jaeger et al. Methods Enzymol. 183:281-306, 1989. Each of these references is incorporated by reference at least for the material related to alignment and calculation of identity. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ. Where sequence identity is provided as, for example, 95%, then such identity must be detectable with at least one of the accepted methods of calculation.
The immunogenic polypeptides described herein can include one or more amino acid analogs or non-naturally occurring stereoisomers. These amino acid analogs and stereoisomers can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic Engineering Reviews 13:1.97-216 (1995), Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs). Immunogenic fragments can be produced that resemble peptides but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH--, --CH2S--, --CH2-CH2-, --CH═CH-- (cis and trans), --COCH2-, --CH(OH)CH2-, and --CHH2SO-- (These and others can be found in Spatola, A. F. "Peptide backbone modifications: A structure-activity analysis of peptides containing amide bond surrogates, conformational constraints, and related backbone modifications." In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, pp. 267-357. Weinstein, B. editor, Marcel Dekker, New York, N.Y. (1983); Morley, Trends in Pharm. Sci. 1(2):463-468 (1980); Hudson, et al., Int J Pept Prot Res 14:177-185 (1979) (--CH2NH--, CH2CH2-); Spatola et al. Life Sci 38:1243-1249 (1986) (--CH H2-S); Hann, Journal of the Chemical Society. Perkin Transactioins I pp. 307-314 (1982) (--CH--CH--, cis and trans); Almquist et al., J. Med. Chem. 23:1392-1398 (1980) (--COCH2-); Jennings-White et al., Tetrahedron Lett 23:2533 (1982) (--COCH2-); European Publication No. EP0045665 to Szelke, et al. (1982) (--CH(OH)CH2-); Holladay et al., Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH2-); and Hruby Life Sci 31:189-199 (1982) (--CH2-S--); each of which is incorporated herein by reference at least for the material regarding linkages).
Amino acid analogs and stereoisomers often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), and others. For example, D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by naturally occurring peptidases. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference).
A composition comprising an immunogenic polypeptide of PcpA and a pharmaceutically acceptable carrier are described herein. Optionally, the composition further comprises an adjuvant. Compositions comprising the immunogenic polypeptide may contain combinations of other immunogenic polypeptides, including, for example, an immunogenic Staphylococcus polypeptide or immunogenic fragments of PspA, pneumolysin, or a combination thereof.
Optionally, the compositions described herein are suitable for administration to a mucosal surface. The composition can be a nasal spray, a nebulizer solution, or an aerosol inhalant, for example. Thus the composition may be present in a container and the container may be a nasal sprayer, a nebulizer, or an inhaler.
By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the immunogenic fragment of PcpA, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to about 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic PcpA polypeptides. Matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the PcpA immunogenic fragments to humans or other subjects.
Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents, adjuvants, immunostimulants, in addition to the immunogenic polypeptide. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents and anesthetics.
Adjuvants include metallic salts, such as aluminium salts, and are well known in the art as providing a safe excipient with adjuvant activity. The mechanism of action of these adjuvants are thought to include the formation of an antigen depot such that antigen may stay at the site of injection for up to 3 weeks after administration, and also the formation of antigen/metallic salt complexes which are more easily taken up by antigen presenting cells. In addition to aluminium, other metallic salts have been used to adsorb antigens, including salts of zinc, calcium, cerium, chromium, iron, and berilium. The hydroxide and phosphate salts of aluminium are the most common. Formulations or compositions containing aluminium salts, antigen, and an additional immunostimulant are known in the art. An example of an immunostimulant is 3-de-O-acylated monophosphoryl lipid A (3D-MPL).
The adjuvant and/or immunostimulant can be administered concomitantly with the polypeptide composition, immediately prior to, or after administration of the composition. Optionally, the composition further comprises the adjuvant. Adjuvant formulations include, for example, an agent that targets mucosal inductive sites. The adjuvant may optionally be selected from the group including, but not limited to, cytokines, chemokines, growth factors, angiogenic factors, apoptosis inhibitors, and combinations thereof. When a cytokine is chosen as an adjuvant, the cytokine may be selected from the group including, but not limited to, interleukins including IL-1, IL-3, IL-2, IL-5, IL-6, IL-12, IL-15 and IL-18; transforming growth factor-beta (TGF-β); granulocyte macrophage colony stimulating factor (GM-CSF); interferon-gamma (IFN-γ); or any other cytokine that has adjuvant activity. Portions of cytokines, or mutants or mimics of cytokines (or combinations thereof), having adjuvant activity or other biological activity can also be used in the compositions and methods of the present invention.
When a chemokine is chosen as an adjuvant, the chemokine may optionally be selected from a group including, but not limited to, Lymphotactin, RANTES, LARC, PARC, MDC, TAR C, SLC and FKN. When an apoptosis inhibitor is chosen as an adjuvant, the apoptosis inhibitor may optionally be selected from the group including, but not limited to, inhibitors of caspase-8, and combinations thereof. When an angiogenic factor is chosen as an adjuvant, the angiogenic factor may optionally be selected from the group including, but not limited to, a basic fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), a hyaluronan (HA) fragment, and combinations thereof.
Other examples of substantially non-toxic, biologically active adjuvants include hormones, enzymes, growth factors, or biologically active portions thereof. Such hormones, enzymes, growth factors, or biologically active portions thereof can be of human, bovine, porcine, ovine, canine, feline, equine, or avian origin, for example, and can be tumor necrosis factor (TNF), prolactin, epidermal growth factor (EGF), granulocyte colony stimulating factor (GCSF), insulin-like growth factor (IGF-1), somatotropin (growth hormone) or insulin, or any other hormone or growth factor whose receptor is expressed on cells of the immune system.
Adjuvants also include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, chimera, or mutants thereof. For example, a purified preparation of native cholera toxin subunit B (CTB) can be used. Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Suitable mutants or variants of adjuvants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that can be used in the methods and compositions include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants, such as RH3-ligand; CpG-motif oligonucleotide; a bacterial monophosphoryl lipid A(MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella exseri; saponins (e.g., QS21), or polylactide glycolide (PLGA) microspheres, can also be used. Possible other adjuvants are defensins and CpG motifs.
Provided are methods of making and using the immunogenic polypeptides described herein and compositions useful in such methods. The polypeptides can be generated 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 the pcpA gene that encodes an immunogenic polypeptide may be isolated and the polynucleotide encoding the immunogenic polypeptide may be cloned into any commercially available expression vector (such as pBR322 and pUC vectors (New England Biolabs, Inc., Ipswich, Mass.)) or expression/purification vectors (such as GST fusion vectors (Pfizer, Inc., Piscataway, N.J.)) and then expressed in a suitable prokaryotic, viral or eukaryotic host. Purification may then be achieved by conventional means or, in the case of a commercial expression/purification system, in accordance with a manufacturer's instructions.
Provided herein are nucleic acids comprising a sequence that encodes any one of SEQ ID NOs:1 to 31, 41 and 45. Provided herein is a nucleic acid comprising SEQ ID NOs:32, 33 and 47, which encode full length PcpA proteins or fragments thereof. Also provided are degenerate variants and fragments of these degenerate variants of SEQ ID NOs:32, 33 and 47.
Nucleic acids that encode SEQ ID NOs:1 and 2 or fragments thereof are described, including SEQ ID NO:34 and SEQ ID NO:35, respectively, or degenerate variants or fragments thereof.
Nucleic acids that encode SEQ ID NOs:3 and 4 or fragments thereof include, but are not limited to, SEQ ID NOs:36 and 37, respectively, or degenerate variants or fragments thereof.
Nucleic acids that encode SEQ ID NO:41 or fragments thereof are described, including SEQ ID NO:42 or degenerate variants or fragments thereof.
Nucleic acids that encode SEQ ID NO:45 or fragments thereof are described, including SEQ ID NO:46 or degenerate variants or fragments thereof.
Exemplary nucleic acids that encode SEQ ID NO:29 or fragments thereof include SEQ ID NO:38 or degenerate variants or fragments thereof.
More specifically, provided herein is a nucleic acid comprising any one of the sequences designated as SEQ ID NOs:32 to 38, 42, 46 and 47 or degenerate variants thereof.
Also provided are isolated nucleic acids comprising a sequence that hybridizes under highly stringent conditions to all or any portion of a hybridization probe having a nucleotide sequence that comprises SEQ ID NOs:32 to 38, 42, 46 and 47 or the complement of SEQ ID NOs:32 to 38, 42, 46 and 47 or any fragment of the sequence or complement thereof. The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 15, 20, 25, 30, 40, or more) nucleotides in length. The hybridizing portion is at least 80% (e.g., 85%, 90% or 95%) identical to the a portion of the sequence to which it hybridizes. Hybridizing nucleic acids are useful, for example, as cloning probes, primers (e.g., PCR primer), or a diagnostic probe. Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a probe dissociates from a target DNA. This melting temperature is used to define the required stringency conditions. If sequences are identified that are related and substantially identical to the probe, rather than identical, then it is useful to first establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt (e.g., SSC or SSPE). Assuming that a 1% mismatching results in a 1° C. decrease in Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having more than 95% identity are sought, the final wash temperature is decreased by 5° C.). In practice, the change in Tm can be between 0.5 and 1.5° C. per 1% mismatch. Highly stringent conditions involve hybridizing at 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature. Moderately stringent conditions include washing in 3×SSC at 42° C. Salt concentrations and temperatures can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Additional guidance regarding such conditions is readily available in the art, for example, in Molecular Cloning: A laboratory Manual, Third Edition by Sambrook et al., Cold Spring Harbor Press, 2001.
Thus, it is understood that the nucleic acids that can encode the aforementioned peptide sequences, variants and fragments thereof are disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e., all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.
Also disclosed are vectors comprising the nucleic acids described herein. Thus, provided is a vector that comprises a nucleic acid that encodes an immunogenic polypeptide (e.g., SEQ ID NOs:1 to 31, 41 or 45 or fragments or variants thereof). The vector can comprise any of the nucleic acid sequences SEQ ID NOs:32 to 38, 42 and 47 or degenerate variants or fragments thereof. Optionally, the nucleic acid of the vector is operably linked to an expression control sequence (e.g., a promoter or enhancer or both). Suitable expression vectors are well known to those of skill in the art and commercially available from a variety of sources such as Novagen, Inc., Madison, Wis.; Invitrogen Corporation, Carlsbad, Calif.; and Promega Corporation, Madison, Wis.
A cultured cell comprising the vector is also provided. The cultured cell can be a cultured cell transfected with the vector or a progeny of the cell, wherein the cell expresses the immunogenic polypeptide. Suitable cell lines are known to those of skill in the art and are commercially available, for example, through the American Type Culture Collection (ATCC).
The transfected cells can be used in a method of producing an immunogenic polypeptide. The method comprises culturing a cell comprising the vector under conditions that allow expression of the immunogenic polypeptide, optionally under the control of an expression sequence. The immunogenic polypeptide can be isolated from the cell or the culture medium using standard protein purification methods.
The immunogenic polypeptides can be made using standard enzymatic cleavage of larger polypeptides or proteins or can be generated by linking two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry. (Applied Biosystems, Inc., Foster City, Calif.). By peptide condensation reactions, native chemical ligation, solid phase chemistry, or enzymatic ligation, two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini to form an immunogenic PcpA polypeptide. (Synthetic Peptides: A User Guide., Grant, ed., W.H. Freeman and Co., New York, N.Y. (1992); Principles of Peptide Synthesis., Bodansky and Trost, eds. Springer-Verlag Inc., New York, N.Y. (1993); Abrahmsen L, et al., Biochemistry, 30:4151 (1991); Dawson et al. Science, 266:776-779 (1994); Solid Phase Peptide Synthesis, 2nd Edition, Stewart, ed., Pierce Chemical Company, Rockford, Ill., (1984), all of which are incorporated herein by reference for the methods described therein).
The immunogenic polypeptides and compositions comprising one or more polypeptides may be used to generate antibodies. Thus, a method of generating antibodies specific to PcpA in a subject comprises administering to the subject a immunogenic PcpA fragment described herein. Also provided herein are antibodies that bind the PcpA polypeptides as well as antibody fragments that bind the PcpA polypeptides.
Antibodies may be polyclonal or monoclonal, may be fully human or humanized, and include naturally occurring antibodies and single-chain antibodies. Antibodies can be made in vivo by administering to a subject an immunogenic PcpA polypeptide. Antibody production includes making monoclonal antibodies using hybridoma methods. Hybridoma methods are well known in the art and are described by Kohler and Milstein, Nature, 256:495 (1975) and Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), which are incorporated by reference in their entirety for the methods described therein.
Methods for the production of single-chain antibodies are well known to those of skill in the art. See, for example, U.S. Pat. No. 5,359,046, (incorporated herein by reference in its entirety for such methods). A single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation. See, for example, Huston, J. S., et al., Methods in Enzym. 203:46-121 (1991), which is incorporated herein by reference for its material regarding linkers.
Fully human and humanized antibodies to the PcpA polypeptides may be used in the methods described herein. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies (i.e., fully human antibodies) may be employed. The homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., PNAS USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993)). Human antibodies can also be produced in phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). The techniques of Cote et al. and Boerner et al. also describe methods for the preparation of human monoclonal antibodies (Cole, et al., "The EBV-hybridoma technique and its application to human lung cancer." In, Monoclonal Antibodies and Cancer Therapy, Volume 27, Reisfeld and Sell, eds., pp. 77-96, Alan R. Liss, Inc., New York, N.Y., (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991)). These references are incorporated by reference in their entirety for the methods described therein.
Antibody fragment as used herein includes F(ab')2, Fab', and Fab fragments, including hybrid fragments. Such fragments of the antibodies retain the ability to bind a specific PcpA polypeptide. Methods can be used to construct (ab) expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F(ab) fragments with the desired specificity for a PcpA polypeptide. Antibody fragments that contain the idiotypes to the polypeptide may be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an F(ab) fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F(v) fragments.
Described herein is a method of reducing the risk of a pneumococcal infection in a subject comprising administering to the subject the immunogenic fragment of PcpA or a composition thereof. Pneumococcal infections include, for example, meningitis, otitis media, pneumonia, sepsis, or hemolytic uremia. Thus, the risk of any one or more of these infections are reduced by the methods described herein. The method can further comprise the step of administering a second immunogenic fragment. The second immunogenic fragment can be from PspA, pneumolysin, or a combination thereof. The second immunogenic fragment can be administered at the same time, before or after the immunogenic fragment of PcpA.
The compositions comprising a PcpA polypeptide or fragments thereof may be administered orally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally or topically, including intranasal administration or administration to any part of the respiratory system. As used herein, administration to the respiratory system means delivery of the compositions into the nose and nasal passages through one or both of the nares or through the mouth, including delivery by a spraying mechanism or droplet mechanism, through aerosolization or intubation.
The exact amount of the compositions and PcpA polypeptides or fragments required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the polypeptide used, and its mode of administration. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art given the description herein. Furthermore, multiple doses of the PcpA polypeptide or fragment may be used including, for example, in a prime and boost regimen.
Combinations of PspA and pneumolysin are more efficacious that either protein alone at eliciting protective immunity against pneumonia and sepsis (Briles et al., J. Infect. Immun. 188:339-48 (2003); Ogunniyi et al., Infect. Immun. 68:3028-33 (2000)). Thus, the compositions comprising PcpA or immunogenic fragments can optionally comprise a second immunogenic fragment of PcpA, PspA, or pneumolysin, or a combination thereof. These references are incorporated herein by reference in their entireties for methods of combining and methods of administration for the proteins taught therein.
Any of the aforementioned treatments can be used in any combination with the compositions described herein. Combinations may be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term combination is used to refer to either concomitant, simultaneous, or sequential administration of two or more agents.
It must be noted that, 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 an antigenic fragment includes mixtures of antigenic fragments, reference to a pharmaceutical carrier or adjuvant includes mixtures of two or more such carriers or adjuvants.
As used herein, a subject is meant an individual. Thus, the subject can include domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs) and birds. In one aspect, the subject is a mammal such as a primate or a human.
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 may comprise a combination of different molecules or may 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 may 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, 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 independently 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 pneumococcal infection), they mean that the treated patient 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 patient 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 patient's experiencing fewer and/or milder symptoms of the condition than otherwise expected. A treatment can prevent infection by resulting in the patient's displaying only mild overt symptoms of the infection; it does not imply that there must have been no penetration of any 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 pneumococcal 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 may result the patient's displaying only mild overt symptoms of the infection or delayed symptoms of infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made. Furthermore, when one characteristic or step is described it can be combined with any other characteristic or step herein even if the combination is not explicitly stated. Accordingly, other embodiments are within the scope of the claims.
PcpA Elicits Protection Against Lung Infection and Fatal Sepsis
Materials and Methods.
Bacterial strains, medium, and growth conditions. S. pneumoniae strains TIGR4 and EF3030, and their derivatives, were used in this study. Pneumococci were grown at 37° C. in Todd-Hewitt broth with 0.5% yeast extract (THY) or on blood agar plates unless otherwise indicated. When appropriate, erythromycin was added to the medium at a concentration of 0.3 μg/ml. Clinical isolates of S. pneumoniae (Table 2) and isolates of major clonal groups (Table 3) were used.
TABLE-US-00002 TABLE 2 Clinical Isolates of Streptococcus pneumoniae Capsular PspA Strain type family Origin Reference R6 Non- 1 New York (Belanger et al., J. Bacteriol. encapsulated 186: 8164-71 (2004); variant of Ottolenghi and Hotchkiss, D39 (type 2) Science 132: 1257-8 (1960) TIGR4* 4 2 Norway Ren et al., Infect. Immun. 71: 75-85 (2003); Roach et al., PNAS 102: 9577-82 (2005) BG12730* 6 2/3 Gambia Shaper et al., Infect. Immun. 72: 5031-40 (2004) BG10752* 9 1 Alabama This Study TJ0893* 14 2 Mississippi Wu et al., J. Infect. Dis. 175: 839-46 (1997) V175* 18 2 Tennessee Robinson et al., J. Infect. Dis. 183: 1501-7 (2001) L82013* 19 2 Alaska Briles et al., Infect. Immun. 188: 339-48 (2003) EF3030* 19F 1 Sweden Briles et al., Infect. Immun. 188: 339-48 (2003); Briles et al., Infect. Immun. 73: 6945-51 (2005); Johnston et al., Infect. Immun. 74: 1171-80 (2006) EF9303* 23F Unknown Sweden Wu et al., Microb. Pathog. 23: 127-37 (1997) L82016* 6B 1 U.S.A. Briles et al., Infect. Immun. 60: 111-6 (1992); Briles et al., Infect. Immun. 188: 339-48 (2003) D-1091B* 23 1 Unknown This Study *clinical strains that are not separated by more than 10 passages from the original patient isolate. R6 was derived from strain D39, which was a patient isolate in the 1920's.
TABLE-US-00003 TABLE 3 Streptococcus pneumoniae of major clonal groups Capsular Strain type Origin Characteristics Reference MA-14 14 UK Worldwide Ermr clone; (1) MLST sequence type 9 MB-23F 23F UK Unknown disease; MLST (1) sequence type 81 MC-6B 6B Spain Unknown disease; MLST (3, 4) sequence type 90 MD-6B 6B Alaska Unknown disease; MLST (2) sequence type 138 ME-19 19 Tennessee Carriage clone; MLST (2) sequence type 236 MF-6A 6A Tennessee Carriage clone; Unknown (2) MLST sequence type MG-1 1 UK Major invasive clone; (1) MLST sequence type 227 MI-7F 7F Norway Major invasive clone; (1) MLST sequence type 191 MJ-35 35 Tennessee Carriage clone; MLST (2) sequence type 65 MK-22 22 Tennessee Major invasive clone; (2) Unknown MLST sequence type ML-11 11 Tennessee Carriage clone; MLST (2) sequence type 62 MM-14 14 Tennessee Major invasive clone; (2) MLST sequence type 124 MN-23F 23 Tennessee Carriage clone; MLST (2) sequence type 37 (1) Enright, M. C., and B. G. Spratt. 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144: 3049-60. (2) Robinson, D. A., K. M. Edwards, K. B. Waites, D. E. Briles, M. J. Crain, and S. K. Hollingshead. 2001. Clones of Streptococcus pneumoniae Isolated from Nasopharyngeal Carriage and Invasive Disease in Young Children in Central Tennessee. J Infect Dis 183: 1501-7. (3) Hakenbeck, R., T. Briese, L. Chalkley, H. Ellerbrok, R. Kalliokoski, C. Latorre, M. Leinonen, and C. Martin. 1991. Antigenic variation of penicillin-binding proteins from penicillin-resistant clinical strains of Streptococcus pneumoniae. J Infect Dis 164: 313-319. (4) Hakenbeck, R., T. Briese, L. Chalkley, H. Ellerbrok, R. Kalliokoski, C. Latorre, M. Leinonen, and C. Martin. 1991. Variability of penicillin-binding proteins from penicillin-sensitive Streptococcus pneumoniae. J Infect Dis 164: 307-312.
The clinical strains used in these studies were isolated within the last 25 years. To examine the possible diversity of PcpA, isolates were selected from the group of strains utilized in the Streptococcus pneumonia Genome Diversity Project (http://genome.microbio.uab.edu/strep/info).
During strain construction, plasmids were maintained in Escherichia coli TOP10 cells (Invitrogen, Carlsbad, Calif.) grown in Luria-Bertani (LB) broth or LB plates with 1.5% agar. Ampicillin (50 μg/ml) for pCR2.1, pCR4 and pET-20b-based plasmids or erythromycin (400 μg/ml) for pJY4164-based plasmids was added to the growth medium.
THY medium was used for growth of bacteria in high manganese medium. For growth in low manganese conditions, a manganese depleted form of THY was prepared. THY medium was prepared according to the manufacturer's directions, with Chelex-100 (2% w/v) (Sigma Aldrich, St. Louis, Mo.) added prior to autoclaving. After autoclaving, the THY/Chelex mixture was stirred overnight at room temperature, followed by filter sterilization. ZnCl2, MgCl2, CaCl2, and FeSO4 were added to concentrations of 1 mM each, and MnSO4 was add to a concentration of 0.1 μM prior to use. Growth was monitored by optical density at 600 nm.
Strain construction. The E. Coli strains, plasmids, and primers used in this study are listed (Table 4). Mutagenesis was used to inactivate pcpA in the parental strains TIGR4 and EF3030. The construction of mutant strains was previously carried out and described (Johnston, et al., Infect. Immun. 74:1171-80 (2006)).
TABLE-US-00004 TABLE 4 Additional bacterial strains, and plasmids used in this study Strain, plasmid, or primer Relevant characteristics or sequence and gene Reference Strains S. pneumoniae JEN7 TIGR4 psaR::Erm (pcpA constituative mutant) Johnston, et al., Infect. Immun. 74: 1171-80 (2006) Jen11 TIGR4 pcpA::Erm Johnston, et al., Infect. Immun. 74: 1171-80 (2006) E. coli TOP10 General cloning strain Invitrogen, Carlsbad, CA Rosetta (DE3) pLysS Expression strain Novagen, Madison, WI Plasmids pCR2.1 3.9 kb, Ampr, Kanr Invitrogen, Carlsbad, CA pCR4 3.9 kb, Ampr, Kanr Invitrogen, Carlsbad, CA pET-20b 3.7 kb, Ampr, C-term his-tag Novagen, Madison, WI pDG-1 pCR4 with pcpA fragmnet; Ampr This study pJM-1 pET-20b with pcpA fragment; Ampr This study pJJ035 pCR2.1 with 412 bp internal pcpA fragment: This study Ampr Primersa DTG-16 cgcggatccATATGTCCCTAATGAACC (SEQ This study ID NO: 39); pcpA F DTG-12 gcgctcgagTTCCTTTAATGAATCTAAGACGC This study CACTTAGGAAGAAGGAC (SEQ ID NO: 40); pcpA R JWJ28 AAC TGT TCA AGT GGG TAA TGG (SEQ Johnston, et al., Infect. ID NO: 48); pcpA F Immun. 74: 1171-80 (2006) JWJ29 TGA ACT TGA GGA AAA GGT TAG C (SEQ Johnston, et al., Infect. ID NO: 49); pcpA R Immun. 74: 1171-80 (2006) BGP1 ATGAAAAAACTACAATATTATCATTAAC This study TACAGCTGCG (SEQ ID NO: 50); pcpA F BGP2 CCATAAACCTTTGTCTTTAACCCAACCA This study ACTAC (SEQ ID NO: 51); pcpA R aPrimers were based on the complete genome sequence of S. pneumoniae TIGR4 (2). Lowercase denotes mismatches used to create restriction endonuclease sites. All sequences are expressed 5' to 3'.
Recombinant PcpA expression and purification. The strains, plasmids, and primers used in this study are listed in Table 2. Primers DTG-16 (5'-CGCGGATCCATATGTCCCTAATGAACC-3' (SEQ ID NO:39)) and DTG-12 (5'-GCGCTCGAGTTCCTTTAATGAATCTAAGACGCCACTTAGGAAGAAGGA C-3' (SEQ ID NO:40)) were designed to amplify a 1126 bp fragment of pcpA in strain TIGR4. The primers contain engineered restriction endonuclease sites, BamHI and XhoI respectively. Reactions were carried out for 30 cycles in a total volume of 50 μl in a cocktail containing 3.0 mM MgCl2, 125 μM dNTPs, 50 picomole of each primer, and 2.5 units of Taq DNA Polymerase. The cycle was 94° C., 1 min.; 55° C., 1 min; 72° C., 5 minutes. This amplified gene fragment was initially cloned into pTOPO4 (Invitrogen, Inc., Carlsbad, Calif.) by a T-tailed method forming plasmid pLMG.
This fragment was cloned into pCR4 with the TOPO TA cloning kit (Invitrogen, Carlsbad, Calif.). Purified plasmids were screened by endonuclease digestion with BamHI and XhoI (Promega, Madison, Wis.). Agarose gel electrophoresis, PCR analysis, and DNA sequencing were all used to confirm insertion of the pcpA fragment in the resulting plasmid, pDG-1. The insert from pDG-1 was subcloned into the pET-20b expression vector (Novagen, Madison, Wis.). The resulting plasmid, pJM-1, was transformed into the E. coli strain RosettaBlue (DE3) pLysS (Novagen, Madison, Wis.) for protein production. This strain contains a chromosomal copy of the T7 promoter under control of the inducible UV5 promoter. Upon IPTG induction a truncated protein, containing amino acids 19-391, was expressed. The over-expressed truncated protein was purified using the Novagen HIS-BIND® Purification Kit (Novagen, Madison, Wis.), which utilized a C-carboxy terminal histidine tag to facilitate purification. Subsequent SDS-PAGE analysis with Comassie Blue staining yielded a single band of approximately 41-kDa.
Below is the complete sequence of the rPcpA protein that has been cloned and expressed. Underlined portions are from the cloning vector.
TABLE-US-00005 (SEQ ID NO: 41) MDIGNSDPYVPNEPILADTPSSEVIKETKVGSIIQQNNIKYKVLTVEGNI GTVQVGNGVTPVEFEAGQDGKPFTIPTKITVGDKVFTVTEVASQAFSYYP DETGRIVYYPSSITIPSSIKKIQKKGFHGSKAKTIIFDKGSQLEKIEDRA FDFSELEEIELPASLEYIGTSAFSFSQKLKKLTFSSSSKLELISHEAFAN LSNLEKLTLPKSVKTLGSNLFRLTTSLKHVDVEEGNESFASVDGVLFSKD KTQLIYYPSQKNDESYKTPKETKELASYSFNKNSYLKKLELNEGLEKIGT FAFADAIKLEEISLPNSLETIERLAFYGNLELKELILPNNVKNFGKHVMN GLPKLKSLTIGNNINSLPSFFLSGVLDSLKELEHHHHHH
Anti-PcpA polyclonal antibody production. Purified rPcpA was used to immunize a New Zealand White Rabbit (Myrtle's Rabbity, Thompson Station, Tenn.) rabbit subcutaneously to obtain anti-PcpA polyclonal serum. The rabbit was injected subcutaneously with 100 μg of rPcpA in 1 ml of Freund's complete adjuvant, 2 ml total volume. A second boost, with 100 μg of rPcpA in Freund's incomplete adjuvant, was given 2 weeks later and a third boost of 100 μg of PcpA in Freund's incomplete adjuvant was given 2 weeks after the second boost. Two weeks following the final boost the rabbit was bled by cardiac puncture, under anesthesia. The blood was allowed to clot, and serum was obtained by centrifugation and stored at -80° C.
PCR confirmation of pcpA in S. pneumoniae strains. The presence or absence of pcpA in various S. pneumoniae strains was checked using PCR primer pair BGP-1 and BGP-2. The primer pair was designed to amplify a 1416 bp N-terminal fragment of pcpA in strain TIGR4. The PCR products were then separated on a T.A.E. agarose gel, stained with ethidium bromide, and examined for the correct size amplified band.
S. pneumoniae cell fractionation. Protoplasts were produced with the method described by Yother and White (Yother and White, J. Bacteriol. 176:2976-85 (1994)), with slight modification. Log-phase cells, grown in MTHY, were pelleted and washed in PBS. The cells were then resuspended in 0.5 ml of 2% choline chloride and the tube inverted several times. The cells were then pelleted and the supernatant drawn off and stored at -20° C. (choline elution fraction). Cells were pelleted and washed once with 300 μl of protoplast buffer (20% sucrose, 5 mM Tris [pH 7.4], 2.5 mM MgSO4). The pellet was then resuspended in 1 ml protoplast buffer, and Mutanolysin (Sigma Aldrich, St. Louis, Mo.) was then added at 5 U per ml of culture pelleted. The suspension was incubated overnight at room temperature. Cells were pelleted by centrifugation at 6000 rpm for 10 min, supernatant is stored at -20° C. (Cell Wall Fraction). The protoplast were then washed in 1 ml of protoplast buffer. The formation of protoplasts was confirmed by microscopic examination. The protoplast were pelleted and lysed in 0.3-1 ml of dH2O, this is stored at -20° C. (Cell Membrane/Cytosolic Fraction). Samples of each fraction are examined for the presence of PcpA by Western blot analysis.
Antibody staining of S. pneumoniae. Mid-log-phase cells, OD6000.6, grown in high or low manganese medium, were pelleted, washed with PBS, resuspended in PBS with 1% bovine serum albumin (PBSB), and incubated at room temperature 20 min. Cells were pelleted and resuspended in PBSB or anti-PcpA serum diluted 1:100 in PBSB and incubated at 37° C. for 30 min. Incubation was followed by two washes with PBS. Cells were then incubated with goat anti-rabbit immunoglobulin G (heavy and light chains)-fluorescein isothiocyanate (Southern Biotechnology Associates, Inc., Birmingham, Ala.) diluted in PBSB at 4° C. for 30 min. The cells were then washed twice with PBS and resuspended in 4% formaldehyde in PBS containing 0.01 mM of the lipophylic membrane dye TMA-DPH (Invitrogen, Carlsbad, Calif.). Bacterial cells were then inspected by epifluorescence using the Olympus IX 70 microscope.
Western blot. Bacterial cultures were grown in THY and MTHY to mid-log phase, OD6000.6. Equivalent amounts of each strain were washed twice with phosphate-buffered saline (PBS), resuspended in PBS with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and boiled for 5 min. Samples and a pre-stained protein standard (Invitrogen, Carlsbad, Calif.) were loaded onto a NuPAGE 10% Bis-Tris gel (Invitrogen, Carlsbad, Calif.) and separated by electrophoresis in morpholinoethanesulfonic acid (MES)-SDS running buffer (Invitrogen, Carlsbad, Calif.) in accordance to the manufacturer's instructions. Proteins were then transferred to a nitrocellulose membrane with the Trans-Blot SD semidry transfer cell (Bio-Rad, Hercules, Calif.). The blot was probed with anti-PcpA polyclonal antibody diluted 1:1000 in PBSB. Goat anti-rabbit immunoglobulin G (heavy and light chains)-alkaline phosphatase and streptavidin-alkaline phosphatase (Southern Biotechnology Associates, Inc., Birmingham, Ala.) were used as the secondary antibody. Colorimetric detection was performed with Sigma Fast nitrobluetetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) tablets (Sigma Aldrich, Switzerland).
Systemic immunization of nice. 6-8 week old CBA/CaHNBtkxid/J (CBA/N) mice (JacksonLabs, Bar Harbor, Me.) were initially injected subcutaneously with 10 μg of rPcpA with 2 μg of Aluminum hydroxide as an adjuvant, 200 μl total volume. A second boost with 10 μg of rPcpA with Aluminum hydroxide was given 2 weeks later. A third boost containing 10 μg of rPcpA without Aluminum hydroxide was given 2 weeks following. The mice were then allowed to rest 2 weeks prior to challenge with S. pneumoniae. Mice were bled 24 hrs prior to infection.
Murine model of sepsis. The virulence of pneumococci was examined in a systemic model of infection previously described (Coats, et al., Vaccine 23:4257-62 (2005); Ren et al., Infect. Immun. 71:75-85 (2003)). 6-8 week old CBA/N mice were injected intravenously with 300 CFUs of bacteria diluted in lactated ringers. Mice were monitored for 21 days. When they become unresponsive to touch and their body temperature decreased to below normal they were scored as moribund and the date and time were recorded. All moribund mice were euthanized with CO2 narcosis.
Murine model of pneumonia. Lung infections were performed as previously described (Balachandran et al., Infect. Immun. 70:2526-34 (2002); Briles et al., J. Infect. Dis. 188:339-48 (2003); Takashima et al., Infect. Immun. 65:257-260 (1997)). 6-8 week old CBA/N mice were anesthetized with Isoflurane (MinRAD, Buffalo, N.Y.), and suspensions of 40 μl of lactated ringers solution containing 5×106 bacteria were introduced into the nares of the mice to induce aspiration pneumonia. After 7 days the mice were sacrificed. The nasal cavities of sacrificed mice were washed with 50 μl of lactated ringers, as previously described (Wu et al., J Infect. Dis. 175:839-46 (997)). The nasal wash was serially diluted and plated onto blood agar with gentamicin (4 μg/ml). The lungs were harvested and placed into 2 ml of lactated ringers in a stomacher bag, homogenized, serially diluted, and plated onto blood agar with gentamicin in serial 3-fold dilutions.
Murine model of nasopharyngeal colonization: Intranasal inoculations were performed as previously described (Balachandran et al., Infect. Immun. 70:2526-34 (2002); Wu et al., J. Infect. Dis. 175:839-46 (1997)). 6-8 week old CBA/N mice were infected intranasally with 106 bacteria in 10 μl of lactated Ringer's solution without anesthesia. Infected mice were then sacrificed, and their nasal cavities were washed with 50 μl of Ringer's solution. The nasal washes were serially diluted and plated on blood agar with gentamicin. Visible counts from blood agar plates were determined after overnight incubation at 37° C. in candle jars.
Statistical analysis. Statistically analysis was carried out using Instat (GraphPad Software Inc., San Diego, Calif.). Comparisons of time to moribund or numbers of recovered CFU between the control and experimental groups were conducted using the Mann-Whitney two sample rank test. P-values less than 0.05 were considered to be statistically significant.
pcpA is present in clinically relevant strains of S. pneumoniae. The presence of pcpA was examined by PCR, with primers (BGP1 and BGP2) spanning the LRR region of the pcpA. Each of the 23 strains examined (Tables 2 and 3) yielded a roughly 1500-bp fragment. Eight of these strains are clinical strains isolated within the last 25 years that are representative strains of the seven common capsular types covered by the 7-valent conjugate vaccine (FIG. 1). The remaining 12 strains are a set of S. pneumoniae that were selected from a set of strains assembled as part of the Genome Diversity Project (http://genome.microbio.uab.edu/strep/info/) which includes a set of strains chosen to span the breadth of diversity in S. pneumoniae. These 12 strains were selected as highly divergent based on MLST data. Four strains were from patients with serious invasive disease, five were from asymptomatic carriage, for 2 strains disease/colonization was not know, and one strain was from a worldwide antibiotic resistant clone. These strains represent 12 different capsule types from different world regions.
To test for expression of PcpA in all strains they were grown in low (≦0.1 μM) manganese. Total cellular protein samples were prepared from mid-log phase cells cultured in the low manganese medium. All strains listed in (Tables 2 and 3) were examined, but only those representing capsular types included in the heptavalent vaccine are depicted (FIG. 2). Total cellular protein samples were separated by SDS-PAGE and transferred to nitrocellulose. The blot was probed with anti-PcpA polyclonal antiserum, identifying a band of approximately 62-kDa in each of these wild-type strains of capsulare serotypes 4, 6, 9, 14, 18, 19, 23 (FIG. 2). This 62-kDa band was absent in the pcpA-inactivated mutant JEN11 but was present in seven representative strains. Total cellular protein samples were also prepared from strains grown in high manganese medium for the same strains, but no bands were identified with the anti-PcpA antiserum. The PCR analysis in combination with the Western blot data, demonstrated that pcpA is present in all S. pneumoniae strains listed in Tables 2 and 3.
PcpA is exposed on the surface of S. pneumoniae under low manganese conditions. Studies have shown that through the action of the regulator PsaR, manganese controls the transcription of the pcpA gene (Johnston et al., Infect. Immun. 74:1171-80 (2006)). As described herein, manganese dependent regulation directly affects the presence of PcpA on surface of S. pneumoniae and surface PcpA is accessible to antibody even on encapsulated pneumococci.
Cell fractionation was performed to determine if PcpA was associated with the cell wall or cell membrane/cytosol of S. pneumonia. Western blot analysis of these cellular fractions revealed that PcpA was present predominantly in the cell wall of S. pneumoniae, in bacteria grown in low manganese medium. A small fraction of the PcpA was associated with the cell membrane/cytosol, and probably represents PcpA yet to be exported to the surface of the bacteria.
In addition to the cell fractionation, log-phase cells from wild type S. pneumoniae strain TIGR4 were grown in high or low manganese medium, stained with anti-PcpA polyclonal antiserum followed by fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin. Specifically, TIGR4 was cultured in high or low Mn2+ medium until mid-log phase. Bacteria were incubated with anti-PcpA rabbit serum, followed by incubation with FITC-conjugated anti-rabbit Ig antibodies. Cells were then fixed in 4% formaldehyde containing the membrane dye TMA-DPH. The labeled bacteria were then examined by immunofluorescence microscopy. The antibodies to PcpA were able to mediate staining of the bacteria grown in low manganese, but not those grown in high manganese.
These results indicate that PcpA is surface exposed on wild-type S. pneumoniae cultured under low manganese conditions in vitro. This indicates that PcpA is expressed and surfaced exposed on bacteria infecting low manganese sites inside the host, such as the lungs and blood. This exposure of PcpA facilitates PcpA-ligand interactions between the bacterium and the host epithelium during infection. These results also indicate that regulation of PcpA production by manganese concentration is generalizable to most pneumococci.
Immunization with rPcpA elicits antibody and provides protection against lung and systemic injection, but does not significantly affect nasopharyngeal colonization. Mice were immunized with rPcpA with aluminum hydroxide or received aluminum hydroxide alone, prior to use in infection studies. Total Ig(H+L) was quantified for both groups of mice by ELISA. The geometric mean level of antibody specific PcpA in the serum of the immunized mice was 0.465 (±0.119) μg/ml, versus a mean of 0.002 (±0.002) μg/ml for mice receiving the adjuvant alone, (±SEM). This indicates the route of immunization was successful at eliciting an immune response to rPcpA.
To see if the immunization protected mice from pneumonia, the immunized and alum-only mice were lightly anesthetized and inoculated in the nares with 5×106 CFU of strain EF3030. This procedure resulted in focal pneumonia without bacteremia. Protection in this model can thus be associated with pneumonia per se and not sepsis in general. Seven days post infection all mice were sacrificed. Bacterial counts were determined from homogenized lung tissue and nasal wash. Based on the median CFU recovered, there were less than 1/100 as many pneumococci recovered from the lung homogenates of mice immunized with rPcpA versus those receiving adjuvant alone (FIG. 3A)(P=0.002). These results indicate that immunization with rPcpA is able to elicit protection against pulmonary infection with S. pneumoniae. There was no significant difference in the bacterial counts recovered from nasal washes of mice immunized with rPcpA versus those receiving adjuvant alone (FIG. 3B). Recombinant PcpA ΔSPΔCBD (rPcpA ΔSPΔCBD), described in detail below, from S. pneumoniae strain serotype 6 also protected against lung infection but not colonization in a mouse model of pneumonia.
Next it was determined whether subcutaneous immunization would confer protection against focal lung infection with other strains of S. pneumoniae (TJ0893, serotype 14; EF9303, serotype 23F; and L82016, serotype 6B). Subcutaneous immunization with rPcpA elicited significant protection against each strain compared to mice receiving immunizations of just the adjuvant alone (FIG. 5).
Expression of PcpA is not required for optimal nasal colonization. Since immunization did not affect the number of bacteria recovered from the nasal washes of mice used for the pneumonia model, the effect of pcpA inactivation was examined in a model of nasopharyngeal carriage. This model allowed a direct view of any effects of PcpA on nasal carriage, as opposed to the indirect observations gathered from the nasal washes of mice in the pneumonia model. Mice were inoculated without prior anesthesia with 106 CFU of either strain EF3030 or its pcpA-inactivated mutant JEN18. Seven days post infection the mice were sacrificed and nasal washes were collected and plated to detect pneumococci. There was no significant difference in the number of bacteria recovered from the nasal washes of mice inoculated with either EF3030 or JEN18 (FIG. 5).
The failure of either the presence of an intact pcpA gene or subcutaneous immunization with rPcpA to have an effect on numbers of pneumococci recovered in the nasal washes of mice is consistent with the fact that the manganese concentration in the nasopharynx (≧36 μM) is high enough to suppress pcpA transcription. Under these conditions pcpA transcription would be repressed, by psaR, in the nasopharynx. Thus, immunity to PcpA would be expected to have little effect on bacteria in this host site.
PcpA and immunity to PcpA effects virulence in the murine model of systemic infection. To evaluate the ability of immunity to PcpA to protect against sepsis, CBA/N mice were subcutaneously immunized with PcpA in aluminum hydroxide or aluminum hydroxide alone as a control and challenged intravenously with capsular type 4, TIGR4 S. pneumoniae. This strain was used rather than EF3030 since this strain can readily cause bacteremia and sepsis in mice. The immunized animals were injected IV with 300 CFU of TIGR4 strain S. pneumoniae. Survival was monitored for 21 days. Mice receiving rPcpA immunizations had a median time to become moribund that was extended by 43.5 hours compared to mice receiving adjuvant alone (FIG. 6). Twenty six percent of mice immunized with rPcpA lived, whereas no mice immunized with aluminum hydroxide alone lived; this difference in survival was statistically significant (P=0.007).
Effect of inactivation of pcpA on the ability of pneumococci to cause mice to become moribund following intravenous inoculation. Inactivation of pcpA results in reduced virulence in the murine model of pneumonia and in a lung-sepsis model. As described herein, the effect of pcpA inactivation on systemic infection following intravenous challenge was examined by infecting naive mice with 300 CFU of either TIGR4 or its pcpA inactivated mutant JEN11. The median time to become moribund for mice infected with the pcpA.sup.- mutant was extended by 31.5 hours (P=0.0299) compared to those infected with wild-type bacteria (FIG. 7). This demonstrates that there is a role for PcpA in the ability of S. pneumoniae to cause systemic diseases.
Mucosal Immunization with PcpA Protects Against Lung Infection
As shown in FIG. 8, mucosal immunization with PcpA protects against pulmonary infection with strain EF3030. CBA/N mice were immunized intranasally with 5 μg of PcpA plus cholera toxin B sub-unit (CTB) as the adjuvant. Post-immunization mice were bled and then challenged intranasally with 5×106 CFU of strain EF3030. FIG. 8 shows log CFU of bacteria in lung homogenate at 7 days post-infection.
Mucosal immunization protection was observed to be slightly better than with immunization. These data and Example 1 indicate that protection against pneumonia and sepsis can be conferred using at least mucosal or subcutaneous routes of administration. Mucosal immunization with PcpA does not protect against nasal colonization with this strain. This is expected since PcpA is not expressed during colonization.
Antibody Elicited by Subcutaneous or Intranasal Immunization with PcpA
Sera obtained from mice immunized with PcpA were examined for the level of antibody to PcpA. CBA/N mice were immunized either subcutaneously (SC) with aluminum hydroxide or cholera toxin B subunit (CTB) as the adjuvant on days 0 and 14, and with PcpA alone on day 21. On day 35 mice were bled and the antibody levels in the serum were determined by using as a standard the OD observed with a known concentrations of PspA antibodies reacting with PspA-coated microtitration plates. As controls, additional groups of mice were immunized with diluent and adjuvant alone. A 1.3-fold higher IgG antibody response was observed with SC rather than intranasal (IN) immunization (Table 5).
TABLE-US-00006 TABLE 5 Antibodies to PcpA in mice immunized with PcpA rPcpA Route of Ig(H + L) IgG1 IgG2a IgG2b IgA Admin. Group Mean μg/ml (±SEM) S.C. rPcpA + 0.465 (0.159) 1.768 (0.378) 0.123 (0.041) 0.125 (0.048) <0.001 Adjuvant (n = 10) S.C. Adjuvant 0.002 (0.002) 0.007 (0.007) <0.001 0.002 (0.001) <0.001 alone (n = 10) I.N. rPcpA + 0.356 (0.159) 0.151 (0.085) 0.118 (0.057) 0.093 (0.033) <0.001 Adjuvant (n = 10) I.N. Adjuvant <0.001 <0.001 <0.001 <0.001 <0.001 alone (n = 10)
As is common with this type of assay, the amounts of the subclasses did not add up to the amount of total Ig. This is an indication that anti-IgG serum does not recognize all IgG subclasses equally.
PcpA is Necessary for Adherence to Lung Cells
PcpA is necessary for adherence to the A549 cell line of transformed lung epithelial cells (FIG. 9) but not to the Detroit562 line of transformed human nasal epithelial cell (FIG. 10). It was observed that adherence to the A549 lung epithelial cells also required that the pneumococci be grown in low Mn2+ so that they would produce PcpA. The pneumococci for these studies were grown in Todd-Hewitt and Yeast medium (high Mn2+) or Todd-Hewitt and Yeast Medium that had been passed over Chelex-100 (Sigma) and reconstituted with 0.1 μm MnSO4 and 1 mM ZnCl2, MgCl2, CaCl2, and FeSO4. (low Mn2+) (Briles et al., J. Infect. Dis. 188:339-48 (2003)). The Detroit 562 or A549 cells monolayers were incubated for 150 minutes with 106 CFU of TIGR4 (pcpA+) or JEN11 (pcpA- TIGR4 strain). The epithelial cells with adherent bacteria were washed and lysed with 0.5% Tween 20. The numbers of pneumococci in the lysate were determined by quantitative plating on blood agar plates.
Adherence of pneumococci to A549 cells is inhibited with antibody to PcpA (FIG. 11). These data demonstrate PcpA-dependent adherence of pneumococci to lung epithelial cells.
Passive Protection Model
Based on the ability of active immunization with PcpA to elicit protection against lung infection, it was determined whether antibody to PcpA would be able to passively protect mice from lung infection. However, passive protection has not yet been observed in a pneumonia model. In a second passive immunization study, passive protection against IV sepsis with the TIGR4 strain was determined using immune rabbit sera to PcpA. It was observed that the highest concentration of sera tested (1/10) was able to protect two of mice from death (FIG. 12). A non-immune serum was not able to protect at the same concentration. These data suggest that passive immunization can protect against TIGR4 strain, which can be a difficult strain to protect against (Roche et al., Infect. Immun. 71:4498-505 (2003)).
Protection by PcpA and Pneumolysin
Pneumolysin (Ply) is another protein that can elicit some protection against lung infection (Briles et al., J. Infect. Dis. 188:339-48 (2003)). Since pneumolysin and PcpA are both candidates for use in protein-based pneumococcal vaccines, it was determined whether the two proteins produce better protection against lung infection when both are used as immunogens than when either one is used alone. Mice were immunized three times with 5 μg of PcpA, 5 μg pneumolysin, or 5 μg of PcpA plus 5 μg of pneumolysin. The first two injections were with alum and the third injections were with protein alone. The pneumolysin used here was wild-type pneumolysin. FIG. 13 shows that pneumolysin elicits similar protection against lung infection to that elicited by PcpA. The combination of PcpA and pneumolysin was significantly more protective than pneumolysin alone. These data indicate that protection can be conferred using both PcpA and pneumolysin.
Cross-Protection Against Other Pneumococci
To determine whether PcpA elicits cross protection, strains in addition to those described in Examples 1-2 can be tested using the methods described above. For studies of sepsis, strains such as WU2, A66, BG7322, EF6796, D39 in addition to TIGR4 are tested. These strains are of capsular types 3, 3, 6B, 6A, and 2. To examine lung infection, strains that work well in a mouse model of focal lung infection are used. These strains include EF9309, TG0893, L82016, BG7322 and EF6796. These are capsular types 23F, 14, 6B, 6B, and 6A.
Cloning and Expression of Recombinant PcpA from Streptococcus pneumoniae Serotype 6
A fragment of the pcpA gene from Streptococcus pneumoniae serotype 6 strain, 14453, ATCC Designation No. 55987 was cloned as follows. The pcpA gene lacking the portion encoding the PcpA C-terminal choline-binding domain (CBD) repeats and lacking the portion encoding the native signal peptide (SP) sequence was cloned into pET-30a (Novagen, Inc., Madison, Wis.) between the NdeI and XhoI cloning sites as shown in FIG. 14. An internal gene fragment of the pcpA gene (ΔSPΔCBD1335 bp) was amplified by polymerase chain reaction (PCR) from Streptococcus pneumoniae serotype 6 strain chromosomal DNA using the oligonucleotide primers 5'-TAGCCTCGAGTTAACCTTTGTCTTTAACCCAACCAACTACTCCCTGATTA G-3 (SEQ ID NO:43) and 5'-CTAATGAACCACATATGGCAGATACTCCTAGTTCGGAAGTAATC-3' (SEQ ID NO:44). PCR reactions were carried out as described in Example 1. The PCR primers incorporated the restriction endonuclease sites NdeI and XhoI. The resulting 1335 base pair fragment encoding PcpA ΔSPΔCBD, contained the NdeI and XhoI sites at either end. The amplified fragment was gel purified and digested with NdeI and XhoI, the pcpA gene fragment was then ligated between NdeI and XhoI sites of the pET-30a vector (Novagen, Inc., Madison, Wis.) with a strong T7 promoter and translation signals (FIG. 14). DNA sequencing confirmed that the recombinant plasmid pJMS87 contained the pcpA gene fragment ΔSPΔCBD1335 bp. Plasmid pJMS87 was transformed into the E. coli strain BL21 (DE3) for protein production. This E. coli strain upon induction with IPTG, expressed the PcpA protein lacking the native signal peptide (ΔSP) and the c-terminal choline-binding domain (ΔCBD). The expressed protein was identified by SDS-PAGE analysis.
The sequence of the rPcpA protein (also known as PcpA ΔSPΔCBD) is as follows. The underlined residue (M) is from the cloning vector.
TABLE-US-00007 (SEQ ID NO: 45) MADTPSSEVI KETKVGSIIQ QNNIKYKVLT VEGNIGTVQV GNGVTPVEFE AGQDGKPFTI PTKITVGDKV FTVTEVASQA FSYYPDETGR IVYYPSSITI PSSIKKIQKK GFHGSKAKTI IFDKGSQLEK IEDRAFDFSE LEEIELPASL EYIGTSAFSF SQKLKKLTFS SSSKLELISH EAFANLSNLE KLTLPKSVKT LGSNLFRLTT SLKHVDVEEG NESFASVDGV LFSKDKTQLI YYPSQKNDES YKTPKETKEL ASYSFNKNSY LKKLELNEGL EKIGTFAFAD AIKLEEISLP NSLETIERLA FYGNLELKEL ILPDNVKNFG KHVMNGLPKL KSLTIGNNIN SLPSFFLSGV LDSLKEIHIK NKSTEFSVKK DTFAIPETVK FYVTSEHIKD VLKSNLSTSN DIIVEKVDNI KQETDVAKPK KNSNQGVVGW VKDKG
Immunization with PcpA ΔSPΔCBD Elicits Protection Against Pneumonia in a Sepsis Model
To determine whether PcpA ΔSPΔCBD protects against infection in a murine sepsis model, mice were immunized with 10, 5, 2.5, 1.25, and 0.625 μg per dose of purified recombinant PcpA ΔSPΔCBD (rPcpA ΔSPΔCBD) and challenged with approximately 300 CFU of S. pneumoniae strain WUBM3 per mouse. The rPcpA ΔSPΔCBD was formulated with aluminum phosphate adjuvant.
Briefly, mice were immunized with a PBS adjuvant control, S. pneumoniae PspA protein containing 30 μg of trivalent recombinant PspA protein, or 10, 5, 2.5, 1.25, or 0.625 μg per dose rPcpA ΔSPΔCBD. Healthy female BALB/c K-72 mice (Charles River Laboratories, Wilmington, Mass.), approximately 14 per group, were immunized at day 0 subcutaneously (s.c.). A second immunization was performed at day 21 and a third immunization at day 43. On day 63, the mice were challenged intraperitoneally (IP) with a 0.4 ml dose of about 300 CFU of S. pneumoniae strain WU2BM3 bacteria. The percent survival plotted against time (days) is shown in FIG. 15A. The percent survival at day 7 post challenge is shown in FIG. 15B.
These results show that rPcpA is protective from at least about 0.625 μg per dose to at least about 10 μg per dose. A statistically significant protection was conferred by rPcpA compared to the adjuvant control group (Fisher Exact Test 1-sided or 2-sided).
TABLE-US-00008 TABLE 6 Statistical Analysis of rPcpA Protection Compared to Control. Stats by Fisher Exact Test PcpA PcpA PcpA PcpA PcpA SPN222 PBS PspA 10 μg 5 μg 2.5 μg 1.25 μg 0.625 μg % Survival 0 80 67 64 53 60 40 1 sided p value -- 0.000 0.000 0.000 0.001 0.000 0.008 2 sided p value -- 0.000 0.000 0.000 0.002 0.001 0.017
Immunization with PcpA ΔSPΔCBD Elicits Protection Against Pneumonia
The rPcpA protein of Example 5 was also used to test the protective efficacy of this protein against challenge with S. pneumoniae strain EF3030 in a mouse pneumonia model. Groups of 10 CBA/N mice were immunized subcutaneously with 200 μl of an immunogen formulation as shown in Table 7, three (3) times at 3 week intervals (day 0, 21 and 42). Three weeks post the 3rd immunization (day 63), the mice were challenged, under anesthesia, intranasally with 5.6×106 CFUs of strain EF3030. Five days post challenge (day 68) mice were sacrificed and lung tissue and blood were harvested and plated for CFU recovery. The immunization groups were formulated in aluminum phosphate adjuvant at 3 mg/ml.
TABLE-US-00009 TABLE 7 Immunogen Formulations. Group Immunogen formulation 1 Placebo (3 mg/mL AlPO4) 2 Trivalent PspA (50 μg/mL each + 3 mg/mL AlPO4) 3 PcpA - (100 μg/mL + 3 mg/mL AlPO4) 4 PcpA - (50 μg/mL + 3 mg/mL AlPO4) 5 PcpA - (25 μg/mL + 3 mg/mL AlPO4) 6 PcpA - (12.5 μg/mL + 3 mg/mL AlPO4) Trivalent PspA immunogen consisted of PspA from S. pneumoniae Rx1-M1, EF3296 and EF5668.
The results are shown in FIG. 16. rPcpA protein conferred significant protection (groups 3-6) when compared to the control group (Adjuvant alone, group 1) and similar levels of protection to the positive control (group 2 PspA). The p values from the Mann-Whitney test are indicated in Table 8.
TABLE-US-00010 TABLE 8 Mann-Whitney Test of rPcpA Protection Compared to Control. Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 P values 0.0848 0.0407 0.0106 0.0637 0.0087 Mean 3.987 2.360 2.223 1.823 2.375 1.876 Median 3.390 1.590 1.900 1.590 2.590 1.590 Std error 0.6527 0.3397 0.6527 0.2009 0.1819 0.1332
Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.
521192PRTStreptococcus pneumoniae 1Leu Glu Lys Ile Glu Asp Arg Ala Phe Asp Phe Ser Glu Leu Glu Glu1 5 10 15Ile Glu Leu Pro Ala Ser Leu Glu Tyr Ile Gly Thr Ser Ala Phe Ser 20 25 30Phe Ser Gln Lys Leu Lys Lys Leu Thr Phe Ser Ser Ser Ser Lys Leu 35 40 45Glu Leu Ile Ser His Glu Ala Phe Ala Asn Leu Ser Asn Leu Glu Lys 50 55 60Leu Thr Leu Pro Lys Ser Val Lys Thr Leu Gly Ser Asn Leu Phe Arg65 70 75 80Leu Thr Thr Ser Leu Asn Met Leu Met Leu Arg Gly Met Ile Val Ala 85 90 95Ser Val Asp Gly Val Ser Phe Gln Ser Lys Thr Gln Leu Ile Tyr Tyr 100 105 110Pro Ser Gln Lys Asn Asp Glu Ser Tyr Lys Thr Pro Lys Glu Thr Lys 115 120 125Glu Leu Ala Ser Tyr Ser Phe Asn Lys Asn Ser Tyr Leu Lys Lys Leu 130 135 140Glu Leu Asn Glu Gly Leu Gln Lys Ile Gly Thr Phe Ala Phe Ala Asp145 150 155 160Ala Thr Lys Leu Glu Glu Ile Ser Leu Pro Asn Ser Leu Glu Thr Ile 165 170 175Glu Arg Leu Ala Phe Tyr Gly Asn Leu Glu Leu Lys Glu Leu Ile Leu 180 185 1902195PRTStreptococcus pneumoniae 2Leu Glu Lys Ile Glu Asp Arg Ala Phe Asp Phe Ser Glu Leu Glu Glu1 5 10 15Ile Glu Leu Pro Ala Ser Leu Glu Tyr Ile Gly Thr Ser Ala Phe Ser 20 25 30Phe Ser Gln Lys Leu Lys Lys Leu Thr Phe Ser Ser Ser Ser Lys Leu 35 40 45Glu Leu Ile Ser His Glu Ala Phe Ala Asn Leu Ser Asn Leu Glu Lys 50 55 60Leu Thr Leu Pro Lys Ser Val Lys Thr Leu Gly Ser Asn Leu Phe Arg65 70 75 80Leu Thr Thr Ser Leu Lys His Val Asp Val Glu Glu Gly Asn Glu Ser 85 90 95Phe Ala Ser Val Asp Gly Val Leu Phe Ser Lys Asp Lys Thr Gln Leu 100 105 110Ile Tyr Tyr Pro Ser Gln Lys Asn Asp Glu Ser Tyr Lys Thr Pro Lys 115 120 125Glu Thr Lys Glu Leu Ala Ser Tyr Ser Phe Asn Lys Asn Ser Tyr Leu 130 135 140Lys Lys Leu Glu Leu Asn Glu Gly Leu Glu Lys Ile Gly Thr Phe Ala145 150 155 160Phe Ala Asp Ala Ile Lys Leu Glu Glu Ile Ser Leu Pro Asn Ser Leu 165 170 175Glu Thr Ile Glu Arg Leu Ala Phe Tyr Gly Asn Leu Glu Leu Lys Glu 180 185 190Leu Ile Leu 1953379PRTStreptococcus pneumoniae 3Tyr Val Pro Asn Glu Pro Ile Leu Ala Ala Tyr Val Pro Asn Glu Pro1 5 10 15Ile Leu Ala Asp Thr Pro Ser Ser Glu Val Ile Lys Glu Thr Lys Val 20 25 30Gly Ser Ile Ile Gln Gln Asn Asn Ile Lys Tyr Lys Val Leu Thr Val 35 40 45Glu Gly Asn Ile Gly Thr Val Gln Val Gly Asn Gly Val Thr Pro Val 50 55 60Glu Phe Glu Ala Gly Gln Asp Gly Lys Pro Phe Thr Ile Pro Thr Lys65 70 75 80Ile Thr Val Gly Asp Lys Val Phe Thr Val Thr Glu Val Ala Ser Gln 85 90 95Ala Phe Ser Tyr Tyr Pro Asp Glu Thr Gly Arg Ile Val Tyr Tyr Pro 100 105 110Ser Ser Ile Thr Ile Pro Ser Ser Ile Lys Lys Ile Gln Lys Lys Gly 115 120 125Phe His Gly Ser Lys Ala Lys Thr Ile Ile Phe Asp Lys Gly Ser Gln 130 135 140Leu Glu Lys Ile Glu Asp Arg Ala Phe Asp Phe Ser Glu Leu Glu Glu145 150 155 160Ile Glu Leu Pro Ala Ser Leu Glu Tyr Ile Gly Thr Ser Ala Phe Ser 165 170 175Phe Ser Gln Lys Leu Lys Lys Leu Thr Phe Ser Ser Ser Ser Lys Leu 180 185 190Glu Leu Ile Ser His Glu Ala Phe Ala Asn Leu Ser Asn Leu Glu Lys 195 200 205Leu Thr Leu Pro Lys Ser Val Lys Thr Leu Gly Ser Asn Leu Phe Arg 210 215 220Leu Thr Thr Ser Leu Asn Met Leu Met Leu Arg Gly Met Ile Val Ala225 230 235 240Ser Val Asp Gly Val Ser Phe Gln Ser Lys Thr Gln Leu Ile Tyr Tyr 245 250 255Pro Ser Gln Lys Asn Asp Glu Ser Tyr Lys Thr Pro Lys Glu Thr Lys 260 265 270Glu Leu Ala Ser Tyr Ser Phe Asn Lys Asn Ser Tyr Leu Lys Lys Leu 275 280 285Glu Leu Asn Glu Gly Leu Gln Lys Ile Gly Thr Phe Ala Phe Ala Asp 290 295 300Ala Thr Lys Leu Glu Glu Ile Ser Leu Pro Asn Ser Leu Glu Thr Ile305 310 315 320Glu Arg Leu Ala Phe Tyr Gly Asn Leu Glu Leu Lys Glu Leu Ile Leu 325 330 335Pro Asp Asn Val Lys Asn Phe Gly Lys His Val Met Asn Gly Leu Pro 340 345 350Lys Phe Leu Thr Leu Ser Gly Asn Asn Ile Asn Ser Leu Pro Ser Phe 355 360 365Phe Leu Ser Gly Val Leu Asp Ser Leu Lys Glu 370 3754373PRTStreptococcus pneumoniae 4Tyr Val Pro Asn Glu Pro Ile Leu Ala Asp Thr Pro Ser Ser Glu Val1 5 10 15Ile Lys Glu Thr Lys Val Gly Ser Ile Ile Gln Gln Asn Asn Ile Lys 20 25 30Tyr Lys Val Leu Thr Val Glu Gly Asn Ile Gly Thr Val Gln Val Gly 35 40 45Asn Gly Val Thr Pro Val Glu Phe Glu Ala Gly Gln Asp Gly Lys Pro 50 55 60Phe Thr Ile Pro Thr Lys Ile Thr Val Gly Asp Lys Val Phe Thr Val65 70 75 80Thr Glu Val Ala Ser Gln Ala Phe Ser Tyr Tyr Pro Asp Glu Thr Gly 85 90 95Arg Ile Val Tyr Tyr Pro Ser Ser Ile Thr Ile Pro Ser Ser Ile Lys 100 105 110Lys Ile Gln Lys Lys Gly Phe His Gly Ser Lys Ala Lys Thr Ile Ile 115 120 125Phe Asp Lys Gly Ser Gln Leu Glu Lys Ile Glu Asp Arg Ala Phe Asp 130 135 140Phe Ser Glu Leu Glu Glu Ile Glu Leu Pro Ala Ser Leu Glu Tyr Ile145 150 155 160Gly Thr Ser Ala Phe Ser Phe Ser Gln Lys Leu Lys Lys Leu Thr Phe 165 170 175Ser Ser Ser Ser Lys Leu Glu Leu Ile Ser His Glu Ala Phe Ala Asn 180 185 190Leu Ser Asn Leu Glu Lys Leu Thr Leu Pro Lys Ser Val Lys Thr Leu 195 200 205Gly Ser Asn Leu Phe Arg Leu Thr Thr Ser Leu Lys His Val Asp Val 210 215 220Glu Glu Gly Asn Glu Ser Phe Ala Ser Val Asp Gly Val Leu Phe Ser225 230 235 240Lys Asp Lys Thr Gln Leu Ile Tyr Tyr Pro Ser Gln Lys Asn Asp Glu 245 250 255Ser Tyr Lys Thr Pro Lys Glu Thr Lys Glu Leu Ala Ser Tyr Ser Phe 260 265 270Asn Lys Asn Ser Tyr Leu Lys Lys Leu Glu Leu Asn Glu Gly Leu Glu 275 280 285Lys Ile Gly Thr Phe Ala Phe Ala Asp Ala Ile Lys Leu Glu Glu Ile 290 295 300Ser Leu Pro Asn Ser Leu Glu Thr Ile Glu Arg Leu Ala Phe Tyr Gly305 310 315 320Asn Leu Glu Leu Lys Glu Leu Ile Leu Pro Asn Asn Val Lys Asn Phe 325 330 335Gly Lys His Val Met Asn Gly Leu Pro Lys Leu Lys Ser Leu Thr Ile 340 345 350Gly Asn Asn Ile Asn Ser Leu Pro Ser Phe Phe Leu Ser Gly Val Leu 355 360 365Asp Ser Leu Lys Glu 370510PRTStreptococcus pneumoniae 5Leu Glu Lys Ile Glu Asp Arg Ala Phe Asp1 5 10610PRTStreptococcus pneumoniae 6Phe Ser Glu Leu Glu Glu Ile Glu Leu Pro1 5 10710PRTStreptococcus pneumoniae 7Ala Ser Leu Glu Tyr Ile Gly Thr Ser Ala1 5 10810PRTStreptococcus pneumoniae 8Phe Ser Phe Ser Gln Lys Leu Lys Lys Leu1 5 10910PRTStreptococcus pneumoniae 9Thr Phe Ser Ser Ser Ser Lys Leu Glu Leu1 5 101010PRTStreptococcus pneumoniae 10Ile Ser His Glu Ala Phe Ala Asn Leu Ser1 5 101110PRTStreptococcus pneumoniae 11Asn Leu Glu Lys Leu Thr Leu Pro Lys Ser1 5 101210PRTStreptococcus pneumoniae 12Val Lys Thr Leu Gly Ser Asn Leu Phe Arg1 5 101310PRTStreptococcus pneumoniae 13Leu Thr Thr Ser Leu Asn Met Leu Met Leu1 5 101410PRTStreptococcus pneumoniae 14Leu Thr Thr Ser Leu Lys His Val Asp Val1 5 101510PRTStreptococcus pneumoniae 15Arg Gly Met Ile Val Ala Ser Val Asp Gly1 5 101612PRTStreptococcus pneumoniae 16Glu Glu Gly Asn Glu Ser Phe Ala Ser Val Asp Gly1 5 101710PRTStreptococcus pneumoniae 17Val Ser Phe Gln Ser Lys Thr Gln Leu Ile1 5 101811PRTStreptococcus pneumoniae 18Val Leu Phe Ser Lys Asp Lys Thr Gln Leu Ile1 5 101910PRTStreptococcus pneumoniae 19Tyr Tyr Pro Ser Gln Lys Asn Asp Glu Ser1 5 102010PRTStreptococcus pneumoniae 20Tyr Lys Thr Pro Lys Glu Thr Lys Glu Leu1 5 102110PRTStreptococcus pneumoniae 21Ala Ser Tyr Ser Phe Asn Lys Asn Ser Tyr1 5 102210PRTStreptococcus pneumoniae 22Leu Lys Lys Leu Glu Leu Asn Glu Gly Leu1 5 102310PRTStreptococcus pneumoniae 23Gln Lys Ile Gly Thr Phe Ala Phe Ala Asp1 5 102410PRTStreptococcus pneumoniae 24Glu Lys Ile Gly Thr Phe Ala Phe Ala Asp1 5 102510PRTStreptococcus pneumoniae 25Ala Thr Lys Leu Glu Glu Ile Ser Leu Pro1 5 102610PRTStreptococcus pneumoniae 26Ala Ile Lys Leu Glu Glu Ile Ser Leu Pro1 5 102710PRTStreptococcus pneumoniae 27Asn Ser Leu Glu Thr Ile Glu Arg Leu Ala1 5 102812PRTStreptococcus pneumoniae 28Phe Tyr Gly Asn Leu Glu Leu Lys Glu Leu Ile Leu1 5 1029122PRTStreptococcus pneumoniae 29Ser Ser Glu Val Ile Lys Glu Thr Lys Val Gly Ser Ile Ile Gln Gln1 5 10 15Asn Asn Ile Lys Tyr Lys Val Leu Thr Val Glu Gly Asn Ile Gly Thr 20 25 30Val Gln Val Gly Asn Gly Val Thr Pro Val Glu Phe Glu Ala Gly Gln 35 40 45Asp Gly Lys Pro Phe Thr Ile Pro Thr Lys Ile Thr Val Gly Asp Lys 50 55 60Val Phe Thr Val Thr Glu Val Ala Ser Gln Ala Phe Ser Tyr Tyr Pro65 70 75 80Asp Glu Thr Gly Arg Ile Val Tyr Tyr Pro Ser Ser Ile Thr Ile Pro 85 90 95Ser Ser Ile Lys Lys Ile Gln Lys Lys Gly Phe His Gly Ser Lys Ala 100 105 110Lys Thr Ile Ile Phe Asp Lys Gly Ser Gln 115 1203043PRTStreptococcus pneumoniae 30Pro Asp Asn Val Lys Asn Phe Gly Lys His Val Met Asn Gly Leu Pro1 5 10 15Lys Phe Leu Thr Leu Ser Gly Asn Asn Ile Asn Ser Leu Pro Ser Phe 20 25 30Phe Leu Ser Gly Val Leu Asp Ser Leu Lys Glu 35 403144PRTStreptococcus pneumoniae 31Pro Asp Asn Val Lys Asn Phe Gly Lys His Val Met Asn Gly Leu Pro1 5 10 15Lys Leu Lys Ser Leu Thr Ile Gly Asn Asn Ile Asn Ser Leu Pro Ser 20 25 30Phe Phe Leu Ser Gly Val Leu Asp Ser Leu Lys Glu 35 40322127DNAStreptococcus pneumoniae 32atgaaaaaaa ctacaatatt atcattaact acagctgcgg ttattttagc agcatatgtc 60cctaatgaac caatcctagc agcatatgtc cctaatgaac caatcctagc agatactcct 120agttcggaag taatcaaaga gactaaagtt ggaagtatta ttcaacaaaa taatatcaaa 180tataaggttc taactgtaga aggtaacata ggaactgttc aagtgggtaa tggagttact 240cctgtagagt ttgaagctgg tcaagatgga aaaccattca cgattcctac aaaaatcaca 300gtaggtgata aagtatttac cgttactgaa gtagctagtc aagcttttag ttattatcca 360gatgaaacag gtagaattgt ctactatcct agctctatta ctatcccatc aagcataaaa 420aaaatacaaa aaaaaggctt ccatggaagt aaagctaaaa ctattatttt tgacaaaggc 480agtcagctgg agaaaattga agatagagct tttgattttt ctgaattaga agagattgaa 540ttgcctgcat ctctagaata tattggaaca agtgcatttt cttttagtca aaaattgaaa 600aagctaacct tttcctcaag ttcaaaatta gaattaatat cacatgaggc ttttgctaat 660ttatcaaatt tagagaaact aacattacca aaatcggtta aaacattagg aagtaatcta 720tttagactca ctactagctt aaacatgttg atgttgagag gaatgatcgt tgcctcagtt 780gatggtgttt cgtttcaaag taaaactcaa ttaatttatt atccaagtca aaaaaatgac 840gaaagttata aaacgcctaa ggagacaaaa gaacttgcat catattcgtt taataaaaat 900tcttacttga aaaaactcga attgaatgaa ggtttacaaa aaatcggtac ttttgcattt 960gcggatgcga ccaaacttga agaaattagc ttaccaaata gtttagaaac tattgaacgt 1020ttagcctttt acggtaattt agaattaaaa gaacttatat taccagataa tgttaaaaat 1080tttggtaaac acgttatgaa cggtttacca aaatttttaa cattatctgg taataatatc 1140aactcattgc cgtccttctt cctaagtggc gtcttagatt cattaaagga aattcatatt 1200aagaataaaa gtacagagtt ttctgtgaaa aaagatacat ttgcaattcc tgaaactgtt 1260aagttctatg taacatcaga acatataaaa gatgttctta aatcaaattt atctactagt 1320aatgatatca ttgttgaaaa agtagataat ataaaacaag aaactgatgt agctaaacct 1380aaaaagaatt ctaatcaggg agtagttggt tgggttaaag acaaaggttt atggtattac 1440ttaaacgaat caggttcaat ggctactggt tgggttaaag acaaaggttt atggtattac 1500ttaaacgaat caggttcaat ggctactggt tgggttaaag acaaaggttt atggtattac 1560ttaaatgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 1620ttaaacgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 1680ttaaacgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 1740ttaaatgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 1800ttaaacgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 1860ttaaatgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 1920ttaaacgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 1980ttaaacgaat caggttcaat ggctactggt tgggttaaag acaaaggctt atggtattac 2040ttaaatgaat caggttcaat ggctactggt tggtttaaag tttctggtaa atggtactat 2100acctataatt caggagattt tatttag 2127332106DNAStreptococcus pneumoniae 33atgaaaaaaa ctacaatatt atcattaact acagctgcgg ttattttagc agatgtccct 60aatgaaccaa tcctagcaga tactcccagt tcggaagtaa tcaaagagac taaagttgga 120agtattattc aacaaaataa tatcaaatat aaggttctaa ctgtagaagg taacatagga 180actgttcaag tgggtaatgg agttactcct gtagagtttg aagctggtca agatggaaaa 240ccattcacga ttcctacaaa aatcacagta ggtgataaag tatttaccgt tactgaagta 300gctagtcaag cttttagtta ttatccagat gaaacaggta gaattgtcta ctatcctagc 360tctattacta tcccatcaag cataaaaaaa atacaaaaaa aaggcttcca tggaagtaaa 420gctaaaacta ttatttttga caaaggcagt cagctggaga aaattgaaga tagagctttt 480gatttttctg aattagaaga gattgaattg cctgcatctc tagaatatat tggaacaagt 540gcattttctt ttagtcaaaa attgaaaaag ctaacctttt cctcaagttc aaaattagaa 600ttaatatcac atgaggcttt tgctaattta tcaaatttag agaaactaac attaccaaaa 660tcggttaaaa cattaggaag taatctattt agactcacta ctagcttaaa acatgttgat 720gttgaagaag gaaatgaatc gtttgcctca gttgatggtg ttttgttttc aaaagataaa 780acccaattaa tttattatcc aagtcaaaaa aatgacgaaa gttataaaac gcctaaggag 840acaaaagaac ttgcatcata ttcgtttaat aaaaattctt acttgaaaaa actcgaattg 900aatgaaggtt tagaaaaaat cggtactttt gcatttgcag atgcgattaa acttgaagaa 960attagcttac caaatagttt agaaactatt gaacgtttag ccttttacgg taatttagaa 1020ttaaaagaac ttatattacc aaataatgtt aaaaattttg gtaaacacgt tatgaacggt 1080ttaccaaaat taaaaagttt aacaattggt aataatatca actcattgcc gtccttcttc 1140ctaagtggcg tcttagattc attaaaggaa attcatatta agaataaaag tacagagttt 1200tctgtgaaaa aagatacatt tgcaattcct gaaactgtta agttctatgt aacatcagaa 1260catataaaag atgttcttaa atcaaattta tctactagta atgatatcat tgttgaaaaa 1320gtagataata taaaacaaga aactgatgta gctaaaccta aaaagaattc taatcaggga 1380gtagttggtt gggttaaaga caaaggttta tggtattact taaacgaatc aggttcaatg 1440gctactggtt gggttaaaga caaaggttta tggtattact taaacgaatc aggttcaatg 1500gctactggtt gggttaaaga caaaggttta tggtattact taaatgaatc aggttcaatg 1560gctactggtt gggttaaaga caaaggctta tggtattact taaacgaatc aggttcaatg 1620gctactggtt gggttaaaga caaaggctta tggtattact taaacgaatc aggttcaatg 1680gctactggtt gggttaaaga caaaggctta tggtattact taaatgaatc aggttcaatg 1740gctactggtt gggttaaaga caaaggctta tggtattact taaacgaatc aggttcaatg 1800gctactggtt gggttaaaga caaaggctta tggtattact taaatgaatc aggttcaatg 1860gctactggtt gggttaaaga caaaggctta tggtattact taaacgaatc aggttcaatg 1920gctactggtt gggttaaaga caaaggctta tggtattact taaacgaatc aggttcaatg 1980gctactggtt gggttaaaga caaaggctta tggtattact taaatgaatc aggttcaatg 2040gctactggtt ggtttaaagt ttctggtaaa tggtactata cctataattc aggagatttt 2100atttag 210634393DNAStreptococcus pneumoniae 34ttagagaaac taacattacc aaaatcggtt
aaaacattag gaagtaatct atttagactc 60actactagct taaacatgtt gatgttgaga ggaatgatcg ttgcctcagt tgatggtgtt 120tcgtttcaaa gtaaaactca attaatttat tatccaagtc aaaaaaatga cgaaagttat 180aaaacgccta aggagacaaa agaacttgca tcatattcgt ttaataaaaa ttcttacttg 240aaaaaactcg aattgaatga aggtttacaa aaaatcggta cttttgcatt tgcggatgcg 300accaaacttg aagaaattag cttaccaaat agtttagaaa ctattgaacg tttagccttt 360tacggtaatt tagaattaaa agaacttata tta 39335544DNAStreptococcus pneumoniae 35ttagagaaac taacattacc aaaatcggtt aaaacattag gaagtaatct atttagactc 60actactagct taaaacatgt tgatgttgaa gaaggaaatg aatcgtttgc ctcagttgat 120ggtgttttgt tttcaaaaga taaaacccaa ttaatttatt atccaagtca aaaaaatgac 180gaaagttata aaacgcctaa ggagacaaaa gaacttgcat catattcgtt taataaaaat 240tcttacttga aaaaactcga attgaatgaa ggtttagaaa aaatcggtac ttttgcattt 300gcagatgcga ttaaacttga agaaattagc ttaccaaata gtttagaaac tattgaacgt 360ttagcctttt acggtaattt agaattaaaa gaacttatat taccaaataa tgttaaaaat 420tttggtaaac acgttatgaa cggtttacca aaattaaaaa gtttaacaat tggtaataat 480atcaactcat tgccgtcctt cttcctaagt ggcgtcttag attcattaaa ggaaattcat 540atta 544361137DNAStreptococcus pneumoniae 36tatgtcccta atgaaccaat cctagcagca tatgtcccta atgaaccaat cctagcagat 60actcctagtt cggaagtaat caaagagact aaagttggaa gtattattca acaaaataat 120atcaaatata aggttctaac tgtagaaggt aacataggaa ctgttcaagt gggtaatgga 180gttactcctg tagagtttga agctggtcaa gatggaaaac cattcacgat tcctacaaaa 240atcacagtag gtgataaagt atttaccgtt actgaagtag ctagtcaagc ttttagttat 300tatccagatg aaacaggtag aattgtctac tatcctagct ctattactat cccatcaagc 360ataaaaaaaa tacaaaaaaa aggcttccat ggaagtaaag ctaaaactat tatttttgac 420aaaggcagtc agctggagaa aattgaagat agagcttttg atttttctga attagaagag 480attgaattgc ctgcatctct agaatatatt ggaacaagtg cattttcttt tagtcaaaaa 540ttgaaaaagc taaccttttc ctcaagttca aaattagaat taatatcaca tgaggctttt 600gctaatttat caaatttaga gaaactaaca ttaccaaaat cggttaaaac attaggaagt 660aatctattta gactcactac tagcttaaac atgttgatgt tgagaggaat gatcgttgcc 720tcagttgatg gtgtttcgtt tcaaagtaaa actcaattaa tttattatcc aagtcaaaaa 780aatgacgaaa gttataaaac gcctaaggag acaaaagaac ttgcatcata ttcgtttaat 840aaaaattctt acttgaaaaa actcgaattg aatgaaggtt tacaaaaaat cggtactttt 900gcatttgcgg atgcgaccaa acttgaagaa attagcttac caaatagttt agaaactatt 960gaacgtttag ccttttacgg taatttagaa ttaaaagaac ttatattacc agataatgtt 1020aaaaattttg gtaaacacgt tatgaacggt ttaccaaaat ttttaacatt atctggtaat 1080aatatcaact cattgccgtc cttcttccta agtggcgtct tagattcatt aaaggaa 1137371119DNAStreptococcus pneumoniae 37gatgtcccta atgaaccaat cctagcagat actcccagtt cggaagtaat caaagagact 60aaagttggaa gtattattca acaaaataat atcaaatata aggttctaac tgtagaaggt 120aacataggaa ctgttcaagt gggtaatgga gttactcctg tagagtttga agctggtcaa 180gatggaaaac cattcacgat tcctacaaaa atcacagtag gtgataaagt atttaccgtt 240actgaagtag ctagtcaagc ttttagttat tatccagatg aaacaggtag aattgtctac 300tatcctagct ctattactat cccatcaagc ataaaaaaaa tacaaaaaaa aggcttccat 360ggaagtaaag ctaaaactat tatttttgac aaaggcagtc agctggagaa aattgaagat 420agagcttttg atttttctga attagaagag attgaattgc ctgcatctct agaatatatt 480ggaacaagtg cattttcttt tagtcaaaaa ttgaaaaagc taaccttttc ctcaagttca 540aaattagaat taatatcaca tgaggctttt gctaatttat caaatttaga gaaactaaca 600ttaccaaaat cggttaaaac attaggaagt aatctattta gactcactac tagcttaaaa 660catgttgatg ttgaagaagg aaatgaatcg tttgcctcag ttgatggtgt tttgttttca 720aaagataaaa cccaattaat ttattatcca agtcaaaaaa atgacgaaag ttataaaacg 780cctaaggaga caaaagaact tgcatcatat tcgtttaata aaaattctta cttgaaaaaa 840ctcgaattga atgaaggttt agaaaaaatc ggtacttttg catttgcaga tgcgattaaa 900cttgaagaaa ttagcttacc aaatagttta gaaactattg aacgtttagc cttttacggt 960aatttagaat taaaagaact tatattacca aataatgtta aaaattttgg taaacacgtt 1020atgaacggtt taccaaaatt aaaaagttta acaattggta ataatatcaa ctcattgccg 1080tccttcttcc taagtggcgt cttagattca ttaaaggaa 111938366DNAStreptococcus pneumoniae 38agttcggaag taatcaaaga gactaaagtt ggaagtatta ttcaacaaaa taatatcaaa 60tataaggttc taactgtaga aggtaacata ggaactgttc aagtgggtaa tggagttact 120cctgtagagt ttgaagctgg tcaagatgga aaaccattca cgattcctac aaaaatcaca 180gtaggtgata aagtatttac cgttactgaa gtagctagtc aagcttttag ttattatcca 240gatgaaacag gtagaattgt ctactatcct agctctatta ctatcccatc aagcataaaa 300aaaatacaaa aaaaaggctt ccatggaagt aaagctaaaa ctattatttt tgacaaaggc 360agtcag 3663927DNAArtificial SequenceSynthetically generated oligonucleotide 39cgcggatcca tatgtcccta atgaacc 274049DNAArtificial SequenceSynthetically generated oligonucleotide 40gcgctcgagt tcctttaatg aatctaagac gccacttagg aagaaggac 4941390PRTArtificial SequenceSynthetic construct 41Met Asp Ile Gly Ile Asn Ser Asp Pro Tyr Val Pro Asn Glu Pro Ile1 5 10 15Leu Ala Asp Thr Pro Ser Ser Glu Val Ile Lys Glu Thr Lys Val Gly 20 25 30Ser Ile Ile Gln Gln Asn Asn Ile Lys Tyr Lys Val Leu Thr Val Glu 35 40 45Gly Asn Ile Gly Thr Val Gln Val Gly Asn Gly Val Thr Pro Val Glu 50 55 60Phe Glu Ala Gly Gln Asp Gly Lys Pro Phe Thr Ile Pro Thr Lys Ile65 70 75 80Thr Val Gly Asp Lys Val Phe Thr Val Thr Glu Val Ala Ser Gln Ala 85 90 95Phe Ser Tyr Tyr Pro Asp Glu Thr Gly Arg Ile Val Tyr Tyr Pro Ser 100 105 110Ser Ile Thr Ile Pro Ser Ser Ile Lys Lys Ile Gln Lys Lys Gly Phe 115 120 125His Gly Ser Lys Ala Lys Thr Ile Ile Phe Asp Lys Gly Ser Gln Leu 130 135 140Glu Lys Ile Glu Asp Arg Ala Phe Asp Phe Ser Glu Leu Glu Glu Ile145 150 155 160Glu Leu Pro Ala Ser Leu Glu Tyr Ile Gly Thr Ser Ala Phe Ser Phe 165 170 175Ser Gln Lys Leu Lys Lys Leu Thr Phe Ser Ser Ser Ser Lys Leu Glu 180 185 190Leu Ile Ser His Glu Ala Phe Ala Asn Leu Ser Asn Leu Glu Lys Leu 195 200 205Thr Leu Pro Lys Ser Val Lys Thr Leu Gly Ser Asn Leu Phe Arg Leu 210 215 220Thr Thr Ser Leu Lys His Val Asp Val Glu Glu Gly Asn Glu Ser Phe225 230 235 240Ala Ser Val Asp Gly Val Leu Phe Ser Lys Asp Lys Thr Gln Leu Ile 245 250 255Tyr Tyr Pro Ser Gln Lys Asn Asp Glu Ser Tyr Lys Thr Pro Lys Glu 260 265 270Thr Lys Glu Leu Ala Ser Tyr Ser Phe Asn Lys Asn Ser Tyr Leu Lys 275 280 285Lys Leu Glu Leu Asn Glu Gly Leu Glu Lys Ile Gly Thr Phe Ala Phe 290 295 300Ala Asp Ala Ile Lys Leu Glu Glu Ile Ser Leu Pro Asn Ser Leu Glu305 310 315 320Thr Ile Glu Arg Leu Ala Phe Tyr Gly Asn Leu Glu Leu Lys Glu Leu 325 330 335Ile Leu Pro Asn Asn Val Lys Asn Phe Gly Lys His Val Met Asn Gly 340 345 350Leu Pro Lys Leu Lys Ser Leu Thr Ile Gly Asn Asn Ile Asn Ser Leu 355 360 365Pro Ser Phe Phe Leu Ser Gly Val Leu Asp Ser Leu Lys Glu Leu Glu 370 375 380His His His His His His385 390421162DNAStreptococcus pneumoniae 42ggatccatat gtccctaatg aaccaatcct agcagcatat gtccctaatg aaccaatcct 60agcagatact cccagttcgg aagtaatcaa agagactaaa gttggaagta ttattcaaca 120aaataatatc aaatataagg ttctaactgt agaaggtaac ataggaactg ttcaagtggg 180taatggagtt actcctgtag agtttgaagc tggtcaagat ggaaaaccat tcacgattcc 240tacaaaaatc acagtaggtg ataaagtatt taccgttact gaagtagcta gtcaagcttt 300tagttattat ccagatgaaa caggtagaat tgtctactat cctagctcta ttactatccc 360atcaagcata aaaaaaatac aaaaaaaagg cttccatgga agtaaagcta aaactattat 420ttttgacaaa ggcagtcagc tggagaaaat tgaagataga gcttttgatt tttctgaatt 480agaagagatt gaattgcctg catctctaga atatattgga acaagtgcat tttcttttag 540tcaaaaattg aaaaagctaa ccttttcctc aagttcaaaa ttagaattaa tatcacatga 600ggcttttgct aatttatcaa atttagagaa actaacatta ccaaaatcgg ttaaaacatt 660aggaagtaat ctatttagac tcactactag cttaaaacat gttgatgttg aagaaggaaa 720tgaatcgttt gcctcagttg atggtgtttt gttttcaaaa gataaaaccc aattaattta 780ttatccaagt caaaaaaatg acgaaagtta taaaacgcct aaggagacaa aagaacttgc 840atcatattcg tttaataaaa attcttactt gaaaaaactc gaattgaatg aaggtttaga 900aaaaatcggt acttttgcat ttgcagatgc gattaaactt gaagaaatta gcttaccaaa 960tagtttagaa actattgaac gtttagcctt ttacggtaat ttagaattaa aagaacttat 1020attaccaaat aatgttaaaa attttggtaa acacgttatg aacggtttac caaaattaaa 1080aagtttaaca attggtaata atatcaactc attgccgtcc ttcttcctaa gtggcgtctt 1140agattcatta aaggaactcg ag 11624351DNAArtificial SequencePrimer 43tagcctcgag ttaacctttg tctttaaccc aaccaactac tccctgatta g 514444DNAArtificial SequencePrimer 44ctaatgaacc acatatggca gatactccta gttcggaagt aatc 4445445PRTStreptococcus pneumoniae serotype 6 45Met Ala Asp Thr Pro Ser Ser Glu Val Ile Lys Glu Thr Lys Val Gly1 5 10 15Ser Ile Ile Gln Gln Asn Asn Ile Lys Tyr Lys Val Leu Thr Val Glu 20 25 30Gly Asn Ile Gly Thr Val Gln Val Gly Asn Gly Val Thr Pro Val Glu 35 40 45Phe Glu Ala Gly Gln Asp Gly Lys Pro Phe Thr Ile Pro Thr Lys Ile 50 55 60Thr Val Gly Asp Lys Val Phe Thr Val Thr Glu Val Ala Ser Gln Ala65 70 75 80Phe Ser Tyr Tyr Pro Asp Glu Thr Gly Arg Ile Val Tyr Tyr Pro Ser85 90 95Ser Ile Thr Ile Pro Ser Ser Ile Lys Lys Ile Gln Lys Lys Gly Phe 100 105 110His Gly Ser Lys Ala Lys Thr Ile Ile Phe Asp Lys Gly Ser Gln Leu 115 120 125Glu Lys Ile Glu Asp Arg Ala Phe Asp Phe Ser Glu Leu Glu Glu Ile 130 135 140Glu Leu Pro Ala Ser Leu Glu Tyr Ile Gly Thr Ser Ala Phe Ser Phe145 150 155 160Ser Gln Lys Leu Lys Lys Leu Thr Phe Ser Ser Ser Ser Lys Leu Glu 165 170 175Leu Ile Ser His Glu Ala Phe Ala Asn Leu Ser Asn Leu Glu Lys Leu 180 185 190Thr Leu Pro Lys Ser Val Lys Thr Leu Gly Ser Asn Leu Phe Arg Leu 195 200 205Thr Thr Ser Leu Lys His Val Asp Val Glu Glu Gly Asn Glu Ser Phe 210 215 220Ala Ser Val Asp Gly Val Leu Phe Ser Lys Asp Lys Thr Gln Leu Ile225 230 235 240Tyr Tyr Pro Ser Gln Lys Asn Asp Glu Ser Tyr Lys Thr Pro Lys Glu 245 250 255Thr Lys Glu Leu Ala Ser Tyr Ser Phe Asn Lys Asn Ser Tyr Leu Lys 260 265 270Lys Leu Glu Leu Asn Glu Gly Leu Glu Lys Ile Gly Thr Phe Ala Phe 275 280 285Ala Asp Ala Ile Lys Leu Glu Glu Ile Ser Leu Pro Asn Ser Leu Glu 290 295 300Thr Ile Glu Arg Leu Ala Phe Tyr Gly Asn Leu Glu Leu Lys Glu Leu305 310 315 320Ile Leu Pro Asp Asn Val Lys Asn Phe Gly Lys His Val Met Asn Gly 325 330 335Leu Pro Lys Leu Lys Ser Leu Thr Ile Gly Asn Asn Ile Asn Ser Leu 340 345 350Pro Ser Phe Phe Leu Ser Gly Val Leu Asp Ser Leu Lys Glu Ile His 355 360 365Ile Lys Asn Lys Ser Thr Glu Phe Ser Val Lys Lys Asp Thr Phe Ala 370 375 380Ile Pro Glu Thr Val Lys Phe Tyr Val Thr Ser Glu His Ile Lys Asp385 390 395 400Val Leu Lys Ser Asn Leu Ser Thr Ser Asn Asp Ile Ile Val Glu Lys 405 410 415Val Asp Asn Ile Lys Gln Glu Thr Asp Val Ala Lys Pro Lys Lys Asn 420 425 430Ser Asn Gln Gly Val Val Gly Trp Val Lys Asp Lys Gly 435 440 445461338DNAStreptococcus pneumoniae serotype 6 46atggcagata ctcctagttc ggaagtaatc aaagagacta aagttggaag tattattcaa 60caaaataata tcaaatataa ggttctaact gtagaaggta acataggaac tgttcaagtg 120ggtaatggag ttactcctgt agagtttgaa gctggtcaag atggaaaacc attcacgatt 180cctacaaaaa tcacagtagg tgataaagta tttaccgtta ctgaagtagc tagtcaagct 240tttagttatt atccagatga aacaggtaga attgtctact atcctagctc tattactatc 300ccatcaagca taaaaaaaat acaaaaaaaa ggcttccatg gaagtaaagc taaaactatt 360atttttgaca aaggcagtca gctggagaaa attgaagata gagcttttga tttttctgaa 420ttagaagaga ttgaattgcc tgcatctcta gaatatattg gaacaagtgc attttctttt 480agtcaaaaat tgaaaaagct aaccttttcc tcaagttcaa aattagaatt aatatcacat 540gaggcttttg ctaatttatc aaatttagag aaactaacat taccaaaatc ggttaaaaca 600ttaggaagta atctatttag actcactact agcttaaaac atgttgatgt tgaagaagga 660aatgaatcgt ttgcctcagt tgatggtgtt ttgttttcaa aagataaaac tcaattaatt 720tattatccaa gtcaaaaaaa tgacgaaagt tataaaacgc ctaaggagac aaaagaactt 780gcatcatatt cgtttaataa aaattcttac ttgaaaaaac tcgaattgaa tgaaggttta 840gaaaaaatcg gtacttttgc atttgcggat gcgattaaac ttgaagaaat tagcttacca 900aatagtttag aaactattga acgtttagcc ttttacggta atttagaatt aaaagaactt 960atattaccag ataatgttaa aaattttggt aaacacgtta tgaacggttt accaaaatta 1020aaaagtttaa caattggtaa taatatcaac tcattgccgt ccttcttcct aagtggcgtc 1080ttagattcat taaaggaaat tcatattaag aataaaagta cagagttttc tgtgaaaaaa 1140gatacatttg caattcctga aactgttaag ttctatgtaa catcagaaca tataaaagat 1200gttcttaaat caaatttatc tactagtaat gatatcattg ttgaaaaagt agataatata 1260aaacaagaaa ctgatgtagc taaacctaaa aagaattcta atcagggagt agttggttgg 1320gttaaagaca aaggttaa 1338471926DNAStreptococcus pneumoniae 47atgaaaaaaa ctacaatatt atcattaact acagctgcgg ttattttagc agcatatgtc 60cctaatgaac caatcctagc agatactcct agttcggaag taatcaaaga gactaaagtt 120ggaagtatta ttcaacaaaa taatatcaaa tataaggttc taactgtaga aggtaacata 180ggaactgttc aagtgggtaa tggagttact cctgtagagt ttgaagctgg tcaagatgga 240aaaccattca cgattcctac aaaaatcaca gtaggtgata aagtatttac cgttactgaa 300gtagctagtc aagcttttag ttattatcca gatgaaacag gtagaattgt ctactatcct 360agctctatta ctatcccatc aagcataaaa aaaatacaaa aaaaaggctt ccatggaagt 420aaagctaaaa ctattatttt tgacaaaggc agtcagctgg agaaaattga agatagagct 480tttgattttt ctgaattaga agagattgaa ttgcctgcat ctctagaata tattggaaca 540agtgcatttt cttttagtca aaaattgaaa aagctaacct tttcctcaag ttcaaaatta 600gaattaatat cacatgaggc ttttgctaat ttatcaaatt tagagaaact aacattacca 660aaatcggtta aaacattagg aagtaatcta tttagactca ctactagctt aaaacatgtt 720gatgttgaag aaggaaatga atcgtttgcc tcagttgatg gtgttttgtt ttcaaaagat 780aaaactcaat taatttatta tccaagtcaa aaaaatgacg aaagttataa aacgcctaag 840gagacaaaag aacttgcatc atattcgttt aataaaaatt cttacttgaa aaaactcgaa 900ttgaatgaag gtttagaaaa aatcggtact tttgcatttg cggatgcgat taaacttgaa 960gaaattagct taccaaatag tttagaaact attgaacgtt tagcctttta cggtaattta 1020gaattaaaag aacttatatt accagataat gttaaaaatt ttggtaaaca cgttatgaac 1080ggtttaccaa aattaaaaag tttaacaatt ggtaataata tcaactcatt gccgtccttc 1140ttcctaagtg gcgtcttaga ttcattaaag gaaattcata ttaagaataa aagtacagag 1200ttttctgtga aaaaagatac atttgcaatt cctgaaactg ttaagttcta tgtaacatca 1260gaacatataa aagatgttct taaatcaaat ttatctacta gtaatgatat cattgttgaa 1320aaagtagata atataaaaca agaaactgat gtagctaaac ctaaaaagaa ttctaatcag 1380ggagtagttg gttgggttaa agacaaaggt ttatggtatt acttaaacga atcaggttca 1440atggctactg gttgggttaa agacaaaggt ttatggtatt acttaaacga atcaggttca 1500atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaacga atcaggttca 1560atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaatga atcaggttca 1620atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaacga atcaggttca 1680atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaacga atcaggttca 1740atggctactg gttgggttaa agacaaaggc ttatggtatt acttaaatga atcaggttca 1800atggctactg gttggtttac agtttctggt aaatggtact atacctataa ttcaggagat 1860ttattagtaa acacgactac acccgatggc tatcgagtca atgctaacgg tgagtgggta 1920ggataa 19264821DNAArtificial SequencePrimer 48aactgttcaa gtgggtaatg g 214922DNAArtificial SequencePrimer 49tgaacttgag gaaaaggtta gc 225038DNAArtificial SequencePrimer 50atgaaaaaac tacaatatta tcattaacta cagctgcg 385133DNAArtificial SequencePrimer 51ccataaacct ttgtctttaa cccaaccaac tac 3352311PRTStreptococcus pneumoniae 52Ile His Ile Lys Asn Lys Ser Thr Glu Phe Ser Val Lys Lys Asp Thr1 5 10 15Phe Ala Ile Pro Glu Thr Val Lys Phe Tyr Val Thr Ser Glu His Ile 20 25 30Lys Asp Val Leu Lys Ser Asn Leu Ser Thr Ser Asn Asp Ile Ile Val 35 40 45Glu Lys Val Asp Asn Ile Lys Gln Glu Thr Asp Val Ala Lys Pro Lys 50 55 60Lys Asn Ser Asn Gln Gly Val Val Gly Trp Val Lys Asp Lys Gly Leu65 70 75 80Trp Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys 85 90 95Asp Lys Gly Leu Trp Tyr
Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr 100 105 110Gly Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly 115 120 125Ser Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu 130 135 140Asn Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu145 150 155 160Trp Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys 165 170 175Asp Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr 180 185 190Gly Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly 195 200 205Ser Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu 210 215 220Asn Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys Asp Lys Gly Leu225 230 235 240Trp Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr Gly Trp Val Lys 245 250 255Asp Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly Ser Met Ala Thr 260 265 270Gly Trp Val Lys Asp Lys Gly Leu Trp Tyr Tyr Leu Asn Glu Ser Gly 275 280 285Ser Met Ala Thr Gly Trp Phe Lys Val Ser Gly Lys Trp Tyr Tyr Thr 290 295 300Tyr Asn Ser Gly Asp Phe Ile305 310
Patent applications by David E. Briles, Birmingham, AL US
Patent applications by Jeremy Yethon, Toronto CA
Patent applications by Joe Wang, Toronto CA
Patent applications by Susan K. Hollingshead, Birmingham, AL US
Patent applications by Sanofi Pasteur, Ltd.
Patent applications by THE UAB RESEARCH FOUNDATION
Patent applications in class Streptococcus (e.g., Group B Streptococcus, pneumococcus or Streptococcus pneumoniae, etc.)
Patent applications in all subclasses Streptococcus (e.g., Group B Streptococcus, pneumococcus or Streptococcus pneumoniae, etc.)