Patent Application
20110097360
This invention relates to polypeptides, including pili proteins, from Streptococcus pneumoniae (S. pneumoniae), including fragments and variants thereof, and methods of their use in the treatment of and immunization against S. pneumoniae infections.
The Gram-positive bacterium Streptococcus pneumoniae (also known as Spn or pneumococcus) is a major cause of morbidity and mortality world-wide and represents one of the four major infectious disease killers, together with HIV, malaria, and tuberculosis (Bruyn, G. A. W. & van Furth, R. (1991) Eur. J. Clin. Microbiol. Infect. Dis. 10, 897-910; Ryan, M. W. & Antonelli, P. J. (2000) Laryngoscope 110, 961-964; Cutts, F. T., Zaman, S. M., Enwere, G., Jaffar, S., Levine, O, S., Okoko, C. Oluwalana, A., Vaughan, S., Obaro, A., Leach, A., et al. (2005) Lancet 365, 1139-1146; Swiatlo, E., Champlin, F. R., Holman, S. C., Wilson, W. W.&Watt, J. M. (2002) Infect. Immun. 70, 412-415; Sandgren, A., Albiger, B., Orihuela, C., Tuomanen, E., Normark, S. & Henriques-Normark, B. (2005) J. Infect. Dis. 192, 791-800). It is a main cause of respiratory tract infections such as otitis media, sinusitis, and community acquired pneumonia, but also an important pathogen in invasive diseases such as septicemia and meningitis. Even though pneumococcus is a devastating pathogen, it also harmlessly colonizes healthy children attending day-care centers to a high extent (Henriques Normark, B., Christensson, B., Sandgren, A., Noreen, B., Sylvan, S., Burman, L. G. & Olsson-Liljequist, B. (2003) Microb. Drug Resist. 9, 337-344; Nunes, S., Sá-Leão, R., Carriço, J., Alves, C. R., Mato, R., Avô, A. B., Saldanha, J., Almeida, J. S., Sanches, I. S. & de Lencastre, H. (2005) J. Clin. Microbiol. 43, 1285-1293). A major virulence factor in pneumococcal disease is the polysaccharide capsule, by which pneumococci are grouped into at least ninety different serotypes (Henrichsen, J. (1995) J. Clin. Microbiol. 33, 2759-2762). Other genetic factors, such as CbpA (choline-binding protein A) and pneumolysin, have been described to be of importance for virulence (Lau, G. W., Haataja, S., Lonetto, M., Kensit, S. E., Marra, A., Bryant, A. P., McDevitt, D., Morrison, D. A. & Holden, D. W. (2001) Mol. Microbiol. 40, 555-571; Rosenow, C., Ryan, P., Weiser, J. N., Johnson, S., Fontan, P., Ortqvist, A. & Masure, H. R. (1997) Mol. Microbiol. 25, 819-829; Tuomanen, E. (1999) Current Opin. Biol. 2, 35-39).
Infection by S. pneumoniae leads to invasive disease triggered by initial colonization of the nasopharynx, but the mechanisms of adhesion are not well understood. In vitro adhesion of encapsulated pneumococci is much lower than for nonencapsulated nonvirulent derivatives (Swiatlo, E., Champlin, F. R., Holman, S. C., Wilson, W. W.&Watt, J. M. (2002) Infect. Immun. 70, 412-415), even though capsule expression is essential for successful colonization of the upper airways. These observations suggest that in vivo, pneumococci are adhesive despite the production of a thick capsule (Sandgren, A., Albiger, B., Orihuela, C., Tuomanen, E., Normark, S. & Henriques-Normark, B. (2005) J. Infect. Dis. 192, 791-800).
In other Gram-positive bacteria, such as Corynebacterium diphtheriae (Ton-That, H., Marraffini, L. A. & Schneewind, O. (2004) Mol. Microbiol. 53, 251-261; Ton-That, H. & Schneewind, O. (2003) Mol. Microbiol. 50, 1429-1438), Actinomyces spp. (Kelstrup, J., Theilade, J. & Fejerskov, O. (1979) Scand. J. Dent. Res. 87, 415-423), and recently group A streptococci (GAS) and group B streptococci (GBS) (Mora, M., Bensi, G., Capo, S., Falugi, F., Zingaretti, C., Manetti, A. G. O., Maggi, T., Taddei, A. R., Grandi, G. & Telford, J. L. (2005) Proc. Natl. Acad. Sci. USA 102, 15641-15646; Lauer, P., Rinaudo, C. D., Soriani, M., Margarit, I., Mainone, D., Rosini, R., Taddei, A. R., Mora, M., Rappuoli, R., Grandi, G. & Telford, J. L. (2005) Science 309, 105), pili-like surface structures have been identified by electron microscopy and characterized genetically as well as biochemically (Ton-That, H., Marraffini, L. A. & Schneewind, O. (2004) Mol. Microbiol. 53, 251-261; Ton-That, H. & Schneewind, O. (2003) Mol. Microbiol. 50, 1429-1438; Mora, M., Bensi, G., Capo, S., Falugi, F., Zingaretti, C., Manetti, A. G. O., Maggi, T., Taddei, A. R., Grandi, G. & Telford, J. L. (2005) Proc. Natl. Acad. Sci. USA 102, 15641-15646; Lauer, P., Rinaudo, C. D., Soriani, M., Margarit, I., Mainone, D., Rosini, R., Taddei, A. R., Mora, M., Rappuoli, R., Grandi, G. & Telford, J. L. (2005) Science 309, 105). In Actinomyces spp. type 1 fimbrial genes mediate adhesion to dental and mucosal surfaces (Li, T., Khah, M. K., Slavnic, S., Johansson, I. & Stromberg, N. (2001) Infect Immun. 69, 7224-7233). However, there is a need for functional data on the physiological role and function in infectious disease of pili and other antigens in pathogenic Streptococcus spp.
Gram-positive pili are extended polymers formed by a transpeptidase reaction involving covalent cross-linking of subunit proteins containing specific amino acid motifs, which are assembled by specific sortases. Sortases are also responsible for covalent attachment of the pilus to the peptidoglycan cell wall.
The present disclosure describes polypeptides from Streptococcus pneumoniae. In some aspects the polypeptides described herein include pili peptides from S. pneumoniae. In other aspects, other polypeptides from S. pneumoniae are described. The Streptococcus pneumoniae polypeptides described herein are useful in methods of treatment for and immunization against S. pneumoniae infections.
In some aspects, the disclosure features pili polypeptides from a second pili island identified in Streptococcus pneumoniae INV104B (pilus II island (INV104B)). In other aspects, the disclosure features pili polypeptides identified in S. pneumoniae 23F, INV200, and OXC141. The pili are thought to play a role in the pathogenesis of S. pneumoniae.
In some aspects, the disclosure features isolated pilus encoded by the Streptococcus pneumoniae pilus II island (INV104B). In some embodiments, the pilus includes a sortase. In some embodiments, the pilus includes an LPXTG cell wall anchored protein, e.g., a polypeptide having the amino acid sequence of SEQ ID NO:2, 4 and/or 6, or a processed form thereof.
In some embodiments, the pili are separated from cells by enzymatic digestion (e.g., with one or more enzymes such as peptidoglycan hydrolases (e.g., mutanolysin, lysostaphin, and lysozyme)). In some embodiments, the pili are separated from cells by mechanical shearing (e.g., by ultrasonication). In some embodiments, the pili are substantially free of bacterial cells. In some embodiments, the disclosure features methods of producing the pilus (e.g., S. pneumoniae pili), wherein the methods include subjecting a bacterial cell that produces the pilus to enzymatic digestion or mechanical shearing and isolating the pilus from the cell.
In other aspects, the disclosure features immunogenic compositions including more or more of the isolated pili (e.g., S. pneumoniae pili).
In other aspects, the disclosure features an isolated Streptococcus pneumoniae sortase, wherein the sortase is one of SEQ ID NO:282, SEQ ID NO:1386, SEQ ID NO:676, or SEQ ID NO:1123.
In other aspects, the disclosure features an isolated Streptococcus pneumoniae LPXTG cell wall anchored protein, wherein the LPXTG cell wall anchored protein is one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.
In further aspects, the disclosure features methods of isolating pili encoded by the Streptococcus pneumoniae pilus II island (INV104B), wherein the methods include subjecting bacterial cells that produce pili encoded by the Streptococcus pneumoniae pilus II island (INV104B) to enzymatic digestion (e.g., mutanolysin) or mechanical shearing (e.g., ultrasonication) and isolating the pili from the cells. In some embodiments, isolating includes a density gradient centrifugation. In some embodiments, isolating includes reduction of polydispersity, such as separating components by size, e.g., using gel filtration chromatography.
In other aspects, the disclosure features antibodies that bind specifically to a pilus encoded by the Streptococcus pneumoniae pilus II island (INV104B). In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single-chain antibody, or a Fab fragment.
In other aspects, the disclosure features an immunogenic composition comprising a purified Streptococcus pneumoniae pilus II island (INV104B) polypeptide in oligomeric form. In some embodiments, the polypeptide is a hyperoligomer. In other embodiments, the polypeptide is a fragment of a LPXTG cell wall anchored protein encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In further aspects, the disclosure features methods of inducing an immune response against Streptococcus pneumoniae. In some embodiments, the methods comprise administering an effective amount of pili encoded by the Streptococcus pneumoniae pilus II island (INV104B) to a subject.
In other aspects, the disclosure features methods of detecting a Streptococcus pneumoniae infection in a subject. In some embodiments, the methods comprise assaying a sample from the subject for the presence of an antibody to pili encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In other aspects, the disclosure features methods of detecting a Streptococcus pneumoniae infection in a subject. In some embodiments, the methods comprise contacting a sample with an antibody and detecting binding of the antibody to a component of the sample. In some embodiments, the antibody binds to a pili component. In other embodiments, the antibody binds to a pili complex.
In other aspects, the disclosure features methods of treating a subject having a Streptococcus pneumoniae infection. In some embodiments, the methods comprise administering to the subject an effective amount of an agent that binds specifically to pili encoded by the Streptococcus pneumoniae pilus II island (INV104B). In some embodiments, the agent is an antibody. In some embodiments, the antibody blocks attachment of Streptococcus pneumoniae to cells. In some embodiments, the antibody specifically binds to one or more LPXTG cell wall anchored proteins encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In other aspects, the disclosure features methods of determining the course of treatment for a subject having a Streptococcus pneumoniae infection. In some embodiments, the method comprises assaying a sample from the subject for the presence of an antibody to pili encoded by the Streptococcus pneumoniae pilus II island (INV104B), and administering to the subject an anti-inflammatory agent if the presence of the antibody is detected. In other embodiments, the method comprises assaying a sample from the subject for the presence of an antibody to pili encoded by the Streptococcus pneumoniae pilus II island (INV104B), and administering to the subject an antibiotic agent if the presence of the antibody is not detected.
In other aspects, the disclosure features isolated pilus or pilus-like multimers that comprise an amino acid sequence of a pilus protein encoded by the Streptococcus pneumoniae pilus II island (INV104B) that has up to 30 amino acid substitutions, insertions, or deletions. In some embodiments, the amino acid sequence has up to 20 amino acid substitutions, insertions, or deletions. In other embodiments, the amino acid sequence has up to 10 amino acid substitutions, insertions, or deletions. In still other embodiments, amino acid sequence has up to 5 amino acid substitutions, insertions, or deletions.
In further aspects, the disclosure features polypeptides that have the amino acid sequence of one or more LPXTG cell wall anchored proteins encoded by the Streptococcus pneumoniae pilus II island (INV104B). In other aspects, the disclosure features immunogenic fragments of one or more LPXTG cell wall anchored proteins encoded by the Streptococcus pneumoniae pilus II island (INV104B). In other aspects, the disclosure features polynucleotides that encode polypeptides that have the amino acid sequence of one or more LPXTG cell wall anchored proteins encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In other aspects, the disclosure features purified polypeptides with the amino acid sequence of SEQ ID NO:2, 4, 6, 7, 8, and 9. In other aspects, the disclosure features purified polypeptides having ten consecutive residues of SEQ ID NO:2, 4, 6, 7, 8, and 9. In still other aspects, the disclosure features purified polypeptides with amino acid sequences at least 85% identical to SEQ ID NO:2, 4, 6, 7, 8, and 9.
In additional aspects, the disclosure features purified polypeptides with at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID NO:29 through SEQ ID NO:1742, or immunogenic fragments thereof. In other aspects, the disclosure features purified polypeptides with an amino acid sequence selected from the group consisting of SEQ ID NO:29 through SEQ ID NO:1742, or immunogenic fragments thereof.
In some embodiments, the disclosure features purified OCX141 polypeptides with at least 85% sequence identity to a sequence selected from the group SEQ ID NO: 53, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 99, SEQ ID NO: 104, SEQ ID NO: 117, SEQ ID NO: 135, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 198, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 242, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 433, SEQ ID NO: 439, SEQ ID NO: 444, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545, SEQ ID NO: 581, or SEQ ID NO: 593, and immunogenic fragments thereof.
In other embodiments, the disclosure features purified INV200 polypeptides with at least 85% sequence identity to a sequence selected from the group SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 639, SEQ ID NO: 645, SEQ ID NO: 747, SEQ ID NO: 751, SEQ ID NO: 752, SEQ ID NO: 783, SEQ ID NO: 786, SEQ ID NO: 787, SEQ ID NO: 810, SEQ ID NO: 812, SEQ ID NO: 813, SEQ ID NO: 824, SEQ ID NO: 831, SEQ ID NO: 842, SEQ ID NO: 847, SEQ ID NO: 875, SEQ ID NO: 876, SEQ ID NO: 879, SEQ ID NO: 880, SEQ ID NO: 882, SEQ ID NO: 913, SEQ ID NO: 914, SEQ ID NO: 925, SEQ ID NO: 926, SEQ ID NO: 947, SEQ ID NO: 948, SEQ ID NO: 968, SEQ ID NO: 987, SEQ ID NO: 988, SEQ ID NO: 990, SEQ ID NO: 992, SEQ ID NO: 1003, SEQ ID NO: 1007, SEQ ID NO: 1008, SEQ ID NO: 1036, SEQ ID NO: 1082, SEQ ID NO: 1120, or SEQ ID NO: 1123, and immunogenic fragments thereof.
In further embodiments, the disclosure features purified 23F polypeptides with at least 85% sequence identity to a sequence selected from the group SEQ ID NO: 1297, SEQ ID NO: 1309, SEQ ID NO: 1311, SEQ ID NO: 1343, SEQ ID NO: 1362, SEQ ID NO: 1364, SEQ ID NO: 1434, SEQ ID NO: 1451, SEQ ID NO: 1455, SEQ ID NO: 1466, SEQ ID NO: 14678, SEQ ID NO: 1470, SEQ ID NO: 1474, SEQ ID NO: 1484, SEQ ID NO: 1485, SEQ ID NO: 1486, SEQ ID NO: 1487, or SEQ ID NO: 1491, and immunogenic fragments thereof.
In other aspects, the disclosure features immunogenic fragments of an LPXTG cell wall anchored protein encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In further aspects, the disclosure features isolated nucleic acids with the polynucleotide sequence of SEQ ID NO:1, 3, and 5. In other aspects, the disclosure features isolated nucleic acids that hybridize under stringent conditions to a hybridization probe, wherein the probe has the polynucleotide sequence of SEQ ID NO:1, 3, and 5 or the complement of SEQ ID NO:1, 3, and 5. In still other aspects, the disclosure features an isolated nucleic acid having a sequence that encodes an amino acid sequence that is at least 85% identical to SEQ ID NO:2, 4, 6, 7, 8, and 9.
In other aspects, the disclosure features isolated nucleic acids having a sequence that encodes an amino acid sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NO: 29 though SEQ ID NO:1742. In other aspects, the disclosure features isolated nucleic acids having a sequence that encodes an amino acid sequence that is selected from the group consisting of SEQ ID NO: 29 though SEQ ID NO:1742.
In other aspects, the disclosure features methods of inducing an immune response against Streptococcus pneumoniae. In some embodiments, the methods include administering an effective amount of an immunogenic fragment of a LPXTG cell wall anchored protein encoded by the Streptococcus pneumoniae pilus II island (INV104B) to a subject. In some embodiments, the subject is a human.
In other aspects, the disclosure features an antibody to a pilus protein encoded by the Streptococcus pneumoniae pilus II island (INV104B) in a cell. In some embodiments, the methods include expressing a nucleic acid encoding the antibody to the pilus protein encoded by the Streptococcus pneumoniae pilus II island (INV104B) in the cell. In some embodiments, the pilus protein is a LPXTG cell wall anchored protein.
In still other aspects, the disclosure features methods of purifying Streptococcus pneumoniae from a sample comprising Streptococcus pneumoniae. These methods include: providing an affinity matrix comprising an antibody bound to a solid support; contacting the sample with the affinity matrix to form an affinity matrix-Streptococcus pneumoniae complex; separating the affinity matrix-Streptococcus pneumoniae complex from the remainder of the sample; and releasing Streptococcus pneumoniae from the affinity matrix.
In further aspects, the disclosure features methods of delivering a cytotoxic agent or a diagnostic agent to Streptococcus pneumoniae. These methods include: providing the cytotoxic agent or the diagnostic agent conjugated to an antibody or fragment thereof; and exposing the Streptococcus pneumoniae to the antibody-agent or fragment-agent conjugate.
In other aspects, the disclosure features methods of identifying a binding modulator for pili encoded by the Streptococcus pneumoniae pilus II island (INV104B). These methods include contacting an animal cell susceptible to Streptococcus pneumoniae pili binding with a candidate compound and a bacterial cell having pili encoded by the Streptococcus pneumoniae pilus II island (INV104B), and determining whether binding of the bacterial cell to the animal cell is inhibited. In some embodiments, inhibition of the binding activity is indicative of an inhibitor of binding by pili encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In other aspects, the disclosure features methods of identifying binding modulators to the activities of pili encoded by the Streptococcus pneumoniae pilus II island (INV104B). These methods include contacting an cell susceptible to Streptococcus pneumoniae pili binding with a candidate compound and a pili encoded by the Streptococcus pneumoniae pilus II island (INV104B), and determining whether binding of the pili to the cell is inhibited. In some embodiments, inhibition of binding activity is indicative of an inhibitor of binding by pili encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In further aspects, the disclosure features methods of identifying a binding modulator for pili encoded by the Streptococcus pneumoniae pilus II island (INV104B). These methods include contacting an cell susceptible to Streptococcus pneumoniae pili binding with a candidate compound and a pilus protein or cell binding fragment thereof encoded by the Streptococcus pneumoniae pilus II island (INV104B), and determining whether binding of the pilus protein or cell binding fragment thereof to the cell is inhibited. In some embodiments, inhibition of binding activity is indicative of an inhibitor of binding by pili encoded by the Streptococcus pneumoniae pilus II island (INV104B).
In still other aspects, the disclosure features methods of isolating pili encoded by the Streptococcus pneumoniae pilus II island (INV104B). These methods include subjecting Streptococcus pneumoniae cells that produce pili encoded by the Streptococcus pneumoniae pilus II island (INV104B) to ultrasonication or digestion with a lytic enzyme; separating non-cellular components by density gradient centrifugation; and isolating pili encoded by the Streptococcus pneumoniae pilus II island (INV104B). In some embodiments, the lytic enzyme is mutanolysin. In other embodiments, the Streptococcus pneumoniae cells that produce pili encoded by the Streptococcus pneumoniae pilus II island (INV104B) are Streptococcus pneumoniae TIGR4 cells
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying figures and the description below. Other features, objects, and advantages of the invention will be apparent from the description and figures, and from the additional embodiments below.
The Applicants have identified new polypeptide sequences from Streptococcus pneumoniae (also known as pneumococcus), including new pili polypeptides and other polypeptides useful as antigens. New pili polypeptide sequences were identified in the unfinished genome sequence for S. pneumoniae isolate INV104, as sequenced by The Institute for Genomic Research (see worldwide web site “tigr.org”). These pili polypeptide sequences are encoded by a pathogenicity island, referred to herein as the pilus II island (INV104B), which is present in some, but not all, clinical pneumococcal isolates. Pili are important for pneumococcal adherence to lung epithelial cells as well as for colonization. Additionally, the Sanger partial genomes for S. pneumoniae strains 23F, INV200, and OXC141 were analyzed (see worldwide web site “sanger.ac.uk/Projects/Microbes/”) to identify genes encoding polypeptides absent from INV104, the results of which included additional pili polypeptides as well as a large number of other non-INV104 polypeptides. Accordingly, this disclosure features, inter alia, S. pneumoniae pili, pilus, and other polypeptide compositions and the use of the same in methods of treatment for, diagnosis of, and immunization against S. pneumoniae infections. As used herein the term S. pneumoniae Polypeptide(s) is meant to include S. pneumoniae pilus II island (INV104B) pili polypeptides, pili polypeptides from 23F, INV200, and OXC141, and other polypeptides from S. pneumoniae. Also as used herein the term S. pneumoniae Pilus Polypeptide(s) is meant to include S. pneumoniae pilus II island (INV104B) pili polypeptides and pili polypeptides from 23F, INV200, and OXC141.
S. pneumoniae Pili of the Pilus II Island of Inv104B
Pneumococcal pili encoded by a 6.5 kb insert between genes corresponding to sp1008 and sp1009 of S. pneumoniae isolate INV104B (ST227, serotype 1) of strain TIGR4 (as illustrated by
An exemplary nucleic acid sequence for LPXTG-1 (orf01290-long) is hereby provided:
An exemplary amino acid sequence for LPXTG-1 is hereby provided:
LPXTG-1 contains a sortase substrate motif, VPXTG (SEQ ID NO:16), shown in underscore in SEQ ID NO:2, above.
The orf01290-long sequence (SEQ ID NO:1) has an intermediary stop codon that is bolded and underscored in SEQ ID NO:1. An exemplary nucleic acid sequence halted at this stop codon (orf01290-short) would have the following transcribed gene sequence (LPXTG-1A):
An exemplary amino acid sequence for LPXTG-1A (from orf01290-short) is hereby provided:
LPXTG-1A contains a sortase substrate motif, VPXTG (SEQ ID NO:16), shown in underscore in SEQ ID NO:4, above.
An exemplary nucleic acid sequence for LPXTG-2 (orf01287) is hereby provided:
An exemplary amino acid sequence for LPXTG-2 is hereby provided:
LPXTG-2 contains a sortase substrate motif, VTXTG (SEQ ID NO:21), shown in underscore in SEQ ID NO:6, above.
Polypeptide Sequences Identified in 23F, INV200, and OXC141 S. pneumoniae Strains
The Sanger partial genomes for S. pneumoniae strains 23F, INV200, and OXC141 do not contain the S. pneumoniae pilus II island (INV104B) region. However, 23F, INV200, and OXC141 encode sortases and LPXTG cell wall anchored proteins, which are disclosed herein. For example, 23F and OXC141 each encode at least one sortase (sort-23F (orf01917; SEQ ID NO:1386) and sort-OXC141 (orf01672; SEQ ID NO:282)) and INV200 encodes at least three cell wall anchored proteins (Anchor-1, Anchor-2, and Anchor-3) that are not encoded by S. pneumoniae strain INV104B.
An exemplary amino acid sequence for Anchor-1 (INV200-orf00426) is hereby provided:
Anchor-1 contains a sortase substrate motif, LPNTG (SEQ ID NO:10), shown in underscore in SEQ ID NO:7, above.
An exemplary amino acid sequence for Anchor-2 (INV200-orf00441) is hereby provided:
Anchor-2 contains a sortase substrate motif, LPATG (SEQ ID NO:10), shown in underscore in SEQ ID NO:8, above.
An exemplary amino acid sequence for Anchor-3 (INV200-orf03448) is hereby provided:
Anchor-3 contains a sortase substrate motif, LPNTG (SEQ ID NO:10), shown in underscore in SEQ ID NO:9, abOve.
23F, INV200, and OXC141 also encode additional polypeptide sequences that are not encoded by S. pneumoniae strain INV104B. These additional polypeptide sequences are disclosed herein in Example 2 as specific examples of sequences that can be used in the methods described herein and as antigens in immunogenic compositions for the production of antibodies and/or the stimulation of an immune response in a subject.
Other Bacterial Pili Polypeptides and Polypeptides from S. pneumoniae
The methods and compositions described herein can be used with pili polypeptides or other polypeptides from any Gram-positive bacterium including, for example, S. pneumoniae. Known and putative pili proteins have been identified in GAS (e.g., Streptococcus pyogenes) (Mora et al., 2005, Proc. Natl. Acad. Sci. USA, 102:15641-6), GBS (e.g., Streptococcus agalactiae) (Lauer et al., 2005, Science, 309:105; WO 2006/078318), Actinomycetes naeslundii (Yeung et al., 1998, Infect. Immun., 66:1482-91), Corynebacterium diphtheriae (Ton-That et al., 2003, Mol. Microbiol., 50:1429-38; Ton-That and Schneewind, 2004, Trends. Microbiol., 12:228-34), Clostridium perfringens, and Enterococcus faecalis. Examples of Gram-positive bacteria include, without limitation, firmicutes such as those of genera Streptococcus (e.g., S. pneumoniae, S. agalactiae, S. pyogenes, S. suis, S. zooepidemicus, S. viridans, S. mutans, S. gordonii, S. equi), Bacillus (e.g., B. anthracis, B. cereus, B. subtilis), Listeria (e.g., L. innocua, L. monocytogenes), Staphylococcus (e.g., S. aureus, S. epidermidis, S. caprae, S. saprophyticus, S. lugdunensis, S. schleiferi), Enterococcus (e.g., E. faecalis, E. faecium), Lactobacillus, Lactococcus (e.g., L. lactis), Leuconostoc (e.g., L. mesenteroi des), Pectinatus, Pediococcus, Acetobacterium, Clostridium (e.g., C. botulinum, C. difficile, C. perfringens, C. tetani), Ruminococcus (e.g., R. albus), Heliobacterium, Heliospirillum, and Sporomusa; and actinobacteria such as those of genera Actinomycetes (e.g., A. naeslundii), Corynebacterium (e.g., C. diphtheriae, C. efficiens), Arthrobacter, Bifidobacterium (e.g., B. longum), Frankia, Micrococcus, Micromonospora, Mycobacterium (e.g., M. tuberculosis, M. leprae, M. Bovis, M. africanum, M. microti), Nocardia (e.g., N. asteroides), Propionibacteriun, and Streptomyces (e.g., S. somaliensis, S. avermitilis, S. coelicolor).
Isolated Pili Polypeptides and Other Polypeptides from S. pneumoniae
Isolated S. pneumoniae Polypeptides, can be used in the methods described herein and as antigens in immunogenic compositions for the production of antibodies and/or the stimulation of an immune response in a subject. Examples of useful S. pneumoniae LPXTG cell wall anchor pili polypeptides include LPXTG-1, LPXTG-1A, LPXTG-2, Anchor-1, Anchor-2, and Anchor-3 (i.e., SEQ ID NOs:2, 4, 6, 7, 8, and 9). Examples of useful S. pneumoniae sortase polypeptides include sort-1, sort-2, sort-23F, and sort-OXC141 (i.e., SEQ ID NOs:676, 1123, 1386, and 282). Variants of S. pneumoniae Polypeptides can also be used in the methods described herein and as antigens in immunogenic compositions for the production of antibodies and/or the stimulation of an immune response in a subject. For example, S. pneumoniae Polypeptides containing at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, with a S. pneumoniae Polypeptide sequence are also useful in the new methods. Furthermore, a S. pneumoniae Polypeptide sequence with up to 50, e.g., 1, 3, 5, 10, 15, 20, 25, 30, or 40 amino acid insertions, deletions, or substitutions, e.g., conservative amino acid substitutions will be useful in the compositions and methods described herein.
The determination of percent identity between two amino acid sequences can be accomplished using the BLAST 2.0 program, which is available to the public at ncbi.nlm.nih.gov/BLAST. Sequence comparison is performed using an ungapped alignment and using the default parameters (BLOSUM 62 matrix, gap existence cost of 11, per residue gap cost of 1, and a lambda ratio of 0.85). The mathematical algorithm used in BLAST programs is described in Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402.
As used herein, “conservative amino acid substitution” means a substitution of an amino acid in a polypeptide within an amino acid family. Families of amino acids are recognized in the art and are based on physical and chemical properties of the amino acid side chains. Families include the following: amino acids with basic side chains (e.g. lysine, arginine, and histidine); amino acids with acidic side chains (e.g., aspartic acid and glutamic acid); amino acids with uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine); amino acids with nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan); amino acids with branched side chains (e.g., threonine, valine, and isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). An amino acid can belong to more than one family.
Fragments of S. pneumoniae Polypeptides, e.g., immunogenic fragments, are also useful in the methods and compositions described herein. Typically, the fragments are at least 8, 10, 15, 20, 50, 100, 200, or 500 contiguous amino acid residues of a S. pneumoniae Polypeptide. Examples of S. pneumoniae LPXTG polypeptides with useful fragments include LPXTG-1, LPXTG-1A, LPXTG-2, Anchor-1, Anchor-2, or Anchor-3 (e.g., SEQ ID NO:2, 4, 6, 7, 8, or 9). Non-limiting examples of S. pneumoniae sortase polypeptides with useful fragments include sort-1, sort-2, sort-23F, and sort-OXC141 (i.e., SEQ ID NOs:676, 1123, 1386, and 282). In some embodiments the fragments retain at least one biological activity of the full-length protein, such as covalent attachment to a peptidoglycan cell wall or the ability to cross-link to another fragment or protein through an LPXTG motif.
In some embodiments the immunogenic compositions described herein comprise one or more S. pneumoniae Pilus Polypeptides that may be formulated or purified in an oligomeric (pilus) form. In some embodiments, the oligomeric form is a hyperoligomer. In some embodiments the immunogenic compositions described herein comprise one or more S. pneumoniae Pilus Polypeptides that have been isolated in an oligomeric (pilus) form. The oligomer or hyperoligomer pilus structures comprising S. pneumoniae Pilus Polypeptides may be purified or otherwise formulated for use in immunogenic compositions.
One or more of the S. pneumoniae Polypeptides from S. pneumoniae open reading frame polynucleotide sequences may be replaced by a polynucleotide sequence coding for a fragment of the replaced ORF. In some embodiments, one or more of the S. pneumoniae Polypeptides from S. pneumoniae open reading frames may be replaced by a sequence having sequence homology to the replaced ORF.
One or more of the S. pneumoniae Pilus Polypeptide sequences typically include an LPXTG motif (such as LPXTG (SEQ ID NO:10)) or other sortase substrate motif. The LPXTG sortase substrate motif of a S. pneumoniae pilus protein may be generally represented by the formula X1X2X3X4G (SEQ ID NO:1746), wherein X1 is an L, a V, an E, a Y, an I, or a Q; wherein X2 is a P if Xi is an L; wherein X2 is a V if X1 is a E or a Q; wherein X2 is a V or a P if X1 is a V; wherein X3 is any amino acid residue; wherein X4 is a T if X1 is a V, E, or Q; and wherein X4 is a T, S, or A if X1 is an L. Non-limiting examples of LPXTG motifs include YPXTG (SEQ ID NO:11), IPXTG (SEQ ID NO:12), LPXSG (SEQ ID NO:13), VVXTG (SEQ ID NO:14), EVXTG (SEQ ID NO:15), VPXTG (SEQ ID NO:16), QVXTG (SEQ ID NO:17), LPXAG (SEQ ID NO:18), QVPTG (SEQ ID NO:19), FPXTG (SEQ ID NO:20) and VTXTG (SEQ ID NO:21).
The S. pneumoniae Pilus Polypeptides described herein can effect the ability of the S. pneumoniae bacteria to adhere to and invade epithelial cells. These pilus polypeptides may also affect the ability of S. pneumoniae to translocate through an epithelial cell layer. In some embodiments, one or more S. pneumoniae Pilus Polypeptides from S. pneumoniae are capable of binding to or otherwise associating with an epithelial cell surface or synthetic model thereof. In some embodiments one or more S. pneumoniae Pilus Polypeptides bind to or associate with one or more of fibrinogen, fibronectin, or collagen.
The S. pneumoniae sortase proteins are predicted to be involved in the secretion and anchoring of the LPXTG containing surface proteins. The S. pneumoniae pilus II island (INV104B) sortase proteins disclosed herein are encoded by genes (sort-1 and sort-2) found in the same 6.5 kb insert between genes corresponding to sp1008 and sp1009 of TIGR4 as the LPXTG-1 and LPXTG-2 genes discussed above. 23F and OXC141 also encode sortases: sort-23F (SEQ ID NO:1386) and sort-OXC141 (SEQ ID NO:282). Sortase proteins and variants of sortase proteins useful in the methods described herein can be obtained from Gram-positive bacteria.
The S. pneumoniae Pilus Polypeptides can be covalently attached to the bacterial cell wall by membrane-associated transpeptidases, such as a sortase. The sortase may function to cleave the surface protein, preferably between the threonine and glycine residues of an LPXTG motif. The sortase may then assist in the formation of an amide link between the threonine carboxyl group and a cell wall precursor such as lipid II. The precursor can then be incorporated into the peptidoglycan via the transglycoslylation and transpeptidation reactions of bacterial wall synthesis. See Comfort et al., Infection & Immunity (2004) 72(5): 2710-2722.
In some embodiments, the compositions described herein comprise oligomeric, pilus-like structures comprising S. pneumoniae Pilus Polypeptides such as LPXTG-1, LPXTG-1A, LPXTG-2, Anchor-1, Anchor-2, or Anchor-3 (e.g., SEQ ID NO:2, 4, 6, 7, 8, or 9). The oligomeric, pilus-like structure may comprise numerous units of pilus poplypeptides. In some embodiments, the oligomeric, pilus-like structures comprise two or more pilus proteins. In some embodiments, the oligomeric, pilus-like structure comprises a hyper-oligomeric pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each subunit comprises a pilus protein or a fragment thereof. The oligomeric subunits may be covalently associated via a conserved lysine within a pilin motif. The oligomeric subunits may be covalently associated via an LPXTG motif, in some embodiments, via a threonine or serine amino acid residue.
S. pneumoniae Pilus Polypeptides or fragments thereof can be incorporated into the oligomeric, pilus-like structures described herein and will, in some embodiments, include a pilin motif. Oligomeric, pilus-like structures may be used alone or in combination. In some embodiments, the compositions described herein comprise a S. pneumoniae pilus II island (INV104B) pilus in oligomeric form. Further, in some embodiments the S. pneumoniae pilus II island (INV104B) pilus can be organized in a hyperoligomeric form.
Pili encoded by Streptococcus pneumoniae can be purified from cells, such as bacterial cells, that express Streptococcus pneumoniae pili or pili-like structures by separating the pili from the cells, e.g., by mechanical shearing or enzymatic digestion, and isolating the separated pili.
Suitable bacterial cells for purification of pili include piliated Gram-positive bacterial strains that express Streptococcus pneumoniae Pili Polypeptides, non-piliated Gram-positive bacteria that have been transformed with one or more Gram-positive pilus proteins, such as S. pneumoniae LPXTG-1, LPXTG-1A, LPXTG-2, Anchor-1, Anchor-2, and Anchor-3 (e.g., SEQ ID NOs:2, 4, 6, 7, 8, and 9), and Gram-negative or other cells transformed with one or more Gram-positive pilus proteins, such as S. pneumoniae LPXTG-1, LPXTG-1A, LPXTG-2, Anchor-1, Anchor-2, and Anchor-3 (e.g., SEQ ID NOs:2, 4, 6, 7, S, and 9). Typically, a cell used for purification of pili will produce only the type or types of pili desired, e.g., endogenous or heterologous pili. For the production of heterologous pili, the cell can be altered, e.g., by mutation or recombinant DNA methods, to not produce endogenous pili. Typically, a pili-producing Gram-positive bacterial cell useful for purification will express one or more compatible sortases such that the pili are expressed on the cell surface. Examples of S. pneumoniae pilus II island (INV104B), 23F, INV200, and OXC141 LPXTG cell wall anchor polypeptides that could be purified include LPXTG-1, LPXTG-1A, LPXTG-2, Anchor-1, Anchor-2, and Anchor-3 (e.g., SEQ ID NOs:2, 4, 6, 7, 8, and 9). Examples of S. pneumoniae pilus II island (INV104B), 23F, INV200, and OXC141 sortases that could be purified include sort-1, sort-2, sort-23F, and sort-OXC141 (e.g., SEQ ID NOs:676, 1123, 1386, and 282).
Separation of pili from Gram-positive bacterial cells is typically accomplished by mechanical shearing, enzymatic digestion, decreasing or inhibiting sortase activity, or treatment with a compound that interferes with cell wall integrity. Mechanical shearing can physically remove the pili from the cells, whereas other methods can eliminate the point of attachment of the pili (e.g., by degradation of cell wall or pilus components). Following separation of the pili from the cells, the pili and cells can be separated, e.g., by centrifugation.
Non-limiting examples of mechanical shearing methods include ultrasonication, glass bead shearing, and mixing. Methods of sonication are discussed, for example, in Yamaguchi et al., 2004, Current Microbiol., 49:59-65. Methods of glass bead shearing are discussed, for example, in Levesque et al., 2001, J. Bacteriol., 183:2724-32. General methods of mechanical shearing are discussed, for example, in Wolfgang et al., 1998, Mol. Microbiol., 29:321-30; Trachtenberg et al., 2005, J. Mol. Biol., 346:665-676; Parge et al., 1990, J. Biol. Chem., 265:2278-85; Isaacson et al., 1981, J. Bacteriol., 146:784-9; Korhonen et al., 1980, Infect. Immun., 27:569-75; Hahn et al., 2002, J. Mol. Biol., 323:845-57; St. Geme et al., 1996, Proc. Natl. Acad. Sci. USA, 93:11913-18; Weber et al., 2005, J. Bacteriol., 187:2458-68; and Mu et al., 2002, J. Bacteriol., 184:4868-74.
Non-limiting examples of enzymes suitable for enzymatic digestion include cell-wall degrading enzymes such as mutanolysin, lysostaphin, and lysozymes. Methods of enzymatic digestion are discussed, for example, in Bender et al., 2003, J. Bacteriol., 185:6057-66; Ton-That et al., 2004, Mol. Microbiol., 53:251-61; and Ton-That et al., 2003, Mol. Microbiol., 50:1429-38. For downstream administration to subjects, one can use multiple enzymes to remove cell-wall components that may cause an undesired host reaction.
Non-limiting examples of methods of inhibiting or decreasing sortase activity include decreasing, for example, SrtA activity by introduction of a loss-of-function allele of SrtA, deleting the endogenous SrtA gene, expression of a nucleic acid that decreases SrtA expression (e.g., an antisense or miRNA), and treating the cells with a compound that inhibits SrtA activity (see, e.g., Marrafini et al., Microbiol. Mol. Biol. Rev., 70:192-221, 2006).
Exemplary sortase A inhibitors include methane-thiosulfonates (e.g., MTSET and (2-sulfonatoethyl) methane-thiosulfonate) (Ton-That and Schneewind, J. Biol. Chem., 274:24316-24320, 1999), p-hydroxymercuribenzoic acid, glucosylstero β-sitosterol-3-O-glucopyranol (Kim et al., Biosci. Biotechnol. Biochem., 67:2477-79, 2003), berberine chloride (Kim et al., Biosci. Biotechnol. Biochem., 68:421-24, 2004), peptidyl-diazomethane (LPAT-CHN2) (Scott et al., Biochem. J., 366:953-58, 2002), peptidyl-chloromethane (LPAT-CH2Cl), peptidyl-vinyl sulfone [LPAT-SO2(Ph)] (Conolly et al., J. Biol. Chem., 278:34061-65, 2003), vinyl sulfones (e.g., di-, ethyl-, methyl-, and phenyl vinyl sulfones) (Frankel et al., J. Am. Chem. Soc., 126:3404-3405, 2004), LPXTG motif peptides with the threonine residue replaced by a phosphinate group (e.g., LPEΨ{PO2H—CH2}G) (Kruger et al., Bioorg. Med. Chem., 12:3723-29, 2004), substituted (Z)-diaryl-acrylonitriles (Oh et al., J. Med. Chem., 47:2418-21, 2004), and extracts of various medicinal plants (Kim et al., Biosci. Biotechnol. Biochem., 66:2751-54, 2002).
Non-limiting examples of compounds that interfere with cell wall integrity include glycine and antibiotics such as penicillins (e.g., methicillin, amoxicillin, ampicillin), cephalosporins (e.g., cefalexin, cefproxil, cefepime), glycopeptides (e.g., vancomycin, teicoplanin, ramoplanin), and cycloserine.
Separated pili can be separated from other components by density, for example by using density gradient centrifugation. For example, the pili can be separated by centrifugation on a sucrose gradient.
Typically, a sample containing Gram-positive pili will contain pili oligomers of different molecular weights due to differing numbers of pilus protein subunits present in the pili. To reduce polydispersity, a sample containing Gram-positive pili can be separated by size. For example, a gel filtration or size exclusion column can be used. An ultrafiltration membrane can also be used to reduce polydispersity.
Gram-positive pili can also be isolated using affinity methods such as affinity chromatography. For example, a protein that binds specifically to a Gram-positive pilus, e.g., an antibody that binds specifically to a pilus component or an antibody that binds preferentially to pili, can be immobilized on a solid substrate (e.g., a chromatography substrate), then a sample containing Gram-positive pili can be exposed to the immobilized binding protein. Such affinity isolation methods can also be used to isolate, purify, or enrich preparations of cells that express Gram-positive pili.
Gram-positive pili can also be isolated using any other protein purification method known in the art, e.g., precipitations, column chromatography methods, and sample concentrations. Additional methods are described, e.g., in Ruffolo et al., 1997, Infect. Immun., 65:339-43. Methods of protein purification are described in detail in, e.g., Scopes, R. K., Protein Purification: Principles and Practice, 3rd. ed., 1994, Springer, N.Y.
The presence of Gram-positive pili in fractions during purification can be followed by electrophoresis (e.g., polyacrylamide electrophoresis), measuring binding of an agent that specifically binds to the gram positive pili (e.g., an antibody against a pilus protein or an antibody that binds preferentially to pili), and/or measuring an activity of the pili such as protein or cell binding.
The S. pneumoniae Polypeptides described herein may also be used to prepare antibodies specific to those S. pneumoniae Polypeptides. In some embodiments the antibodies are specific to an oligomeric or hyper-oligomeric form of a S. pneumoniae Pilus Polypeptide. The compositions described herein also include combinations of antibodies specific to S. pneumoniae pilus polypeptides and other polypeptides from S. pneumoniae selected to provide protection against an increased range of serotypes and strain isolates. For example, such a combination may comprise a first and second antibody, wherein the first antibody is specific to a first S. pneumoniae Polypeptide and the second antibody is specific to a second S. pneumoniae Polypeptide or non-S. pneumoniae Polypeptide.
The specific S. pneumoniae Polypeptide antibodies described herein include one or more biological moieties that, through chemical or physical means, can bind to or associate with an epitope of a S. pneumoniae Polypeptide. The antibodies described herein include antibodies which specifically bind to a S. pneumoniae Polypeptide. The compositions described herein include antibodies obtained from both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (see, e.g., Winter et al. (1991) Nature 349: 293-299; and U.S. Pat. No. 4,816,567; F(ab′)2 and F(ab) fragments; Fv molecules (non-covalent heterodimers, see, e.g., Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5897-5883); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties of the parent antibody molecule. The compositions described herein further include antibodies obtained through non-conventional processes, such as phage display.
The antibodies described herein can be polyclonal, monoclonal, recombinant, e.g., chimeric or humanized, fully human, non-human, e.g., murine, or single chain antibodies. Methods of making such antibodies are known. In some cases, the antibodies have effector function and can fix complement. The antibodies can also be coupled to toxins, reporter groups, or imaging agents.
In some embodiments, the specific S. pneumoniae antibodies described herein are monoclonal antibodies. These monoclonal antibodies include an antibody composition having a homogeneous antibody population. The monoclonal antibodies described herein may be obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas. See, e.g., Cote, et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p 77.
Chimeric, humanized, e.g., completely human, antibodies are desirable for applications that include repeated administration, e.g., therapeutic treatment (and some diagnostic applications) of a human subject.
The antibodies described herein can also be used in the prophylactic or therapeutic treatment of S. pneumoniae infection. The antibodies may block the attachment or some other activity of S. pneumoniae on host cells. Additionally, the antibodies can be used to deliver a toxin or therapeutic agent such as an antibiotic to S. pneumoniae cells.
The antibodies described herein may be used in diagnostic applications, for example, to detect the presence or absence of S. pneumoniae Polypeptides in a biological sample. Anti-pili, pilus polypeptide or other polypeptide from S. pneumoniae antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, contrast agents, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerytlirin; examples of contrast agents include electron dense materials useful for electron microscopy, such as gold particles, or magnetically active materials useful for magnetic resonance imaging, such as supermagnetic iron particles; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H. Such diagnostic antibodies can be used in methods to detect the presence of piliated S. pneumoniae containing Streptococcus pneumoniae pilus II island (INV104B) pili in an infected patient, e.g., by testing a sample from the patient. The course of treatment can then be selected based on the presence or absence of Streptococcus pneumoniae Polypeptides. For example, a patient infected with non-piliated S. pneumoniae could be treated with an antibiotic, whereas a patient infected with piliated S. pneumoniae containing S. pneumoniae Pili Polypeptides could also be treated with a pili-binding compound, such as an antibody, and/or an anti-inflammatory agent (e.g., IL-6 or an anti-TNF agent such as an anti-TNF antibody).
In some aspects, the methods described herein (also referred to herein as “screening assays”) can be used to identify modulators, i.e., candidate compounds or agents identified from one or more test compounds (e.g., antibodies, proteins, peptides, peptidomimetics, peptoids, small inorganic molecules, small non-nucleic acid organic molecules, nucleic acids (e.g., antisense nucleic acids, siRNA, oligonucleotides, or synthetic oligonucleotides), or other drugs) that inhibit an activity, e.g., a binding activity of S. pneumoniae Pili Polypeptides. Compounds thus identified can be used to modulate the binding activity of S. pneumoniae containing S. pneumoniae Pili Polypeptides or the attachment of such S. pneumoniae in a therapeutic protocol, to elaborate the biological function S. pneumoniae.
In some embodiments, assays are provided for screening test compounds to identify those that can bind to S. pneumoniae Pili Polypeptides or a portion thereof. Compounds that bind to S. pneumoniae Pili Polypeptides can be tested for their ability to modulate an activity associated with S. pneumoniae pili such as attachment, infection, or an inflammatory response.
The test compounds used in the methods described herein can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation, but which, nevertheless, remain bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem., 37:2678-2685); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des., 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993, Proc. Natl. Acad. Sci. USA, 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA, 91:11422; Zuckermann et al., 1994, J. Med. Chem., 37:2678; Cho et al., 1993, Science, 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl., 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl., 33:2061; and in Gallop et al., 1994, J. Med. Chem., 37:1233).
Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques, 13:412-421), or on beads (Lam, 1991, Nature, 354:82-84), chips (Fodor, 1993, Nature, 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA, 89:1865-1869), or on phage (Scott and Smith, 1990, Science, 249:386-390; Devlin, 1990, Science, 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA, 87:6378-6382; Felici, 1991, J. Mol. Biol., 222:301-310; and Ladner supra).
In some embodiments, the assay is a cell-based assay in which a cell, e.g., a bacterial cell, that expresses a S. pneumoniae Polypeptides or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate S. pneumoniae activity is determined, for example, by monitoring cell binding. The cell, for example, can be of mammalian origin, e.g., murine, rat, or human origin. The cell can be an epithelial cell, e.g., a A549 lung epithelial cell.
The ability of the test compound to modulate S. pneumoniae Pili Polypeptides from binding to a ligand or substrate, e.g., a cell or a protein such as fibrinogen, fibronectin, or collagen can be evaluated, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to S. pneumoniae pilus II island (INV104B) pili or pilus protein can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, S. pneumoniae Polypeptides can be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate S. pneumoniae Polypeptide binding to a substrate in a complex. For example, compounds (e.g., S. pneumoniae Polypeptide binding partners) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
The ability of a compound to interact with a S. pneumoniae Polypeptide with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with a S. pneumoniae Polypeptide without labeling either the compound or the S. pneumoniae Polypeptide (McConnell et al., 1992, Science 257:1906-1912). As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and a S. pneumoniae Polypeptide.
In some embodiments, a cell-free assay is provided in which a S. pneumoniae Polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the S. pneumoniae Polypeptide or biologically active portion thereof is evaluated. In general, biologically active portions of a S. pneumoniae Polypeptide to be used in the new assays include fragments that participate in interactions with other S. pneumoniae Polypeptide molecules.
Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
An interaction between two molecules can be detected using fluorescence energy transfer (FET) (see, e.g., Lakowicz et al., U.S. Pat. No. 5,631,169 and Stavrianopoulos et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor.’ Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
In some embodiments, determining the ability of a S. pneumoniae Polypeptide to bind to a target molecule (e.g., a fibrinogen, fibronectin, or collagen polypeptide or fragment thereof) can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (e.g., Sjolander et al., 1991, Anal. Chem., 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol., 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.
In some embodiments, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. The target gene product can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly, with a detectable labelsdiscussed herein.
Multiple target gene products can be anchored onto a solid phase using protein microarray technology, which is also known by other names including: protein chip technology and solid-phase protein array technology. Protein microarray technology is well known to those of ordinary skill in the art and is based on, but not limited to, obtaining an array of identified peptides or proteins on a fixed substrate, binding target molecules or biological constituents to the peptides, and evaluating such binding. See, e.g., G. MacBeath and S. L. Schreiber, “Printing Proteins as Microarrays for High-Throughput Function Determination,” Science 289(5485):1760-1763, 2000. Microarray substrates include but are not limited to glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, or nylon. The microarray substrates may be coated with a compound to enhance synthesis of a probe (e.g., a peptide) on the substrate. Coupling agents or groups on the substrate can be used to covalently link the first amino acid to the substrate. A variety of coupling agents or groups are known to those of skill in the art. Peptide probes can be synthesized directly on the substrate in a predetermined grid. Alternatively, peptide probes can be spotted on the substrate, and in such cases the substrate may be coated with a compound to enhance binding of the probe to the substrate. In these embodiments, presynthesized probes are applied to the substrate in a precise, predetermined volume and grid pattern, preferably utilizing a computer-controlled robot to apply probe to the substrate in a contact-printing manner or in a non-contact manner such as ink jet or piezo-electric delivery. Probes may be covalently linked to the substrate. In some embodiments, one or more control peptide or protein molecules are attached to the substrate. Control peptide or protein molecules allow determination of factors such as peptide or protein quality and binding characteristics, reagent quality and effectiveness, hybridization success, and analysis thresholds and success.
In some embodiments it is desirable to immobilize a S. pneumoniae Polypeptide, an anti-pilus or pilus protein antibody, or a S. pneumoniae Polypeptide binding protein to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a S. pneumoniae Polypeptide, or interaction of a S. pneumoniae Polypeptide with a target molecule in the presence or absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/pilus II island (INV104B) pilus protein fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose™ beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or S. pneumoniae pilus II island (INV104B) pili or pilus protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of S. pneumoniae Polypeptide binding or activity determined using standard techniques.
Other techniques for immobilizing either a S. pneumoniae Polypeptide or a binding target on matrices include using conjugation of biotin and streptavidin. Biotinylated S. pneumoniae Polypeptides or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kits from Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
To conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).
In some embodiments, the assay is performed utilizing antibodies that bind specifically to S. pneumoniae Polypeptides or binding targets, but do not interfere with binding of the S. pneumoniae Polypeptide to its target. Such antibodies can be derivatized to the wells of the plate, and unbound target or S. pneumoniae Polypeptide trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the S. pneumoniae Polypeptide or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the S. pneumoniae Polypeptide or target molecule.
In some embodiments, cell-free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (for example, Rivas et al., 1993, Trends Biochem. Sci., 18:284-287); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (e.g., Ausubel et al., eds., 1999, Current Protocols in Molecular Biology, J. Wiley: New York.); and immunoprecipitation (for example, Ausubel et al., eds., 1999, Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to those skilled in the art (e.g., Heegaard, 1998, J. Mol. Recognit., 11:141-148 and Hage et al., 1997, J. Chromatogr. B. Biomed. Sci. Appl., 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.
In some embodiments, the assay includes contacting a S. pneumoniae Pili Polypeptide, or biologically active portion thereof with a known cell or compound (e.g., a protein) that binds to S. pneumoniae Pili Polypeptide, to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to affect binding of the S. pneumoniae Pili Polypeptide to the cell or compound.
A general assay for binding of bacterial cells that express S. pneumoniae pilus II island (INV104B) pili involves incubating bacterial cells that express S. pneumoniae pilus II island (INV104B) pili with A549 lung epithelial cells, washing to remove nonadherent bacterial cells, and detecting adherent bacterial cells. Bacterial adherence can be measured by any means in the art, e.g., detecting binding of an antibody to the adherent bacterial cells or lysing the epithelial cells and counting the number of associated bacterial cells. HEP2 cells, CHO cells, or HeLa cells can also be used in assays of binding of bacterial cells that express S. pneumoniae pilus II island (INV104B) pili.
Immunogenic compositions described herein that include S. pneumoniae Polypeptides may further comprise one or more antigenic agents. Exemplary antigens include those listed below. Additionally, the compositions described herein may be used to treat or prevent infections caused by any of the below-listed microbes. Antigens for use in the immunogenic compositions include, but are not limited to, one or more of the following set forth below, or antigens derived from one or more of the following set forth below:
N. meningitidis: a protein antigen from N. meningitides serogroup A, C, W135, Y, and/or B (Bruyn, G. A. W. & van Furth, R. (1991) Eur. J. Clin. Microbiol. Infect. Dis. 10, 897-910; Ryan, M. W. & Antonelli, P. J. (2000) Laryngoscope 110, 961-964; Cutts, F. T., Zaman, S. M., Enwere, G., Jaffar, S., Levine, O, S., Okoko, C. Oluwalana, A., Vaughan, S., Obaro, A., Leach, A., et al. (2005) Lancet 365, 1139-1146; Swiatlo, E., Champlin, F. R., Holman, S. C., Wilson, W. W.&Watt, J. M. (2002) Infect. Immun. 70, 412-415; Sandgren, A., Albiger, B., Orihuela, C., Tuomanen, E., Normark, S. & Henriques-Normark, B. (2005) J. Infect. Dis. 192, 791-800; Henriques Normark, B., Christensson, B., Sandgren, A., Noreen, B., Sylvan, S., Burman, L. G. & Olsson-Liljequist, B. (2003) Microb. Drug Resist. 9, 337-344; Nunes, S., Sá-, Leão, R., Carriço, J., Alves, C. R., Mato, R., Avô, A. B., Saldanha, J., Almeida, J. S., Sanches, I. S. & de Lencastre, H. (2005) J. Clin. Microbiol. 43, 1285-1293); an outer-membrane vesicle (OMV) preparation from N. meningitides serogroup B. (Henrichsen, J. (1995) J. Clin. Microbiol. 33, 2759-2762; Lau, G. W., Haataja, S., Lonetto, M., Kensit, S. E., Marra, A., Bryant, A. P., McDevitt, D., Morrison, D. A. & Holden, D. W. (2001) Mol. Microbiol. 40, 555-571; Rosenow, C., Ryan, P., Weiser, J. N., Johnson, S., Fontan, P., Ortqvist, A. & Masure, H. R. (1997) Mol. Microbiol. 25, 819-829; Tuomanen, E. (1999) Current Opin. Biol. 2, 35-39); a saccharide antigen, including LPS, from N. meningitides serogroup A, B, C W135 and/or Y, such as the oligosaccharide from serogroup C (see PCT/US99/09346; PCT IB98/01665; and PCT IB99/00103);
Streptococcus pneumoniae: a saccharide or protein antigen, particularly a saccharide from Streptococcus pneumoniae or a protein or antigenic peptide of PhtD (BVH-11-2, SP1003, spr0907) (Adamou et al., Infect. Immun., 69:949-53, 2001; Hamel et al., Infect. Immun., 72:2659-70, 2004); PhtE (BVH-3, SP1004, spr0908) (Adamou et al., Infect. Immun., 69:949-53, 2001; Hamel et al., Infect. Immun., 72:2659-70, 2004); PhtB (PhpA, BVH-11, SP1174, Spr1060) (Adamou et al., Infect. Immun., 69:949-53, 2001; Zhang et al., Infect. Immun., 69:3827-36, 2001; Hamel et al., Infect. Immun., 72:2659-70, 2004); PhtA (BVH-11-3, SP1175, sprl 061) (Adamou et al., Infect. Immun., 69:949-53, 2001; Wizemann et al., Infect. Immun., 69:1593-98, 2001; Zhang et al., Infect. Immun., 69:3827-36, 2001; Hamel et al., Infect. Immun., 72:2659-70, 2004); NanA (SP1693, spr1536) (Tong et al., Infect. Immun., 73:7775-78, 2005); SP1872 (spr1687) (Brown et al., Infect. Immun., 69:6702-06, 2001); PspC (CbpA, SP2190, spr1995) (Ogunniyi et al., Infect. Immun., 69:5997-6003, 2001); PspA (SP0177, spr0121, spr1274) (Briles et al., Vaccine, 19:S87-S95, 2001); SP0498 (spr0440); LytB (SP0965, spr0867) (Wizemann et al., Infect. Immun., 69:1593-98, 2001); AliB (SP1527, spr1382); PpmA (SP0981, spr0884) (Overweg et al., Infect. Immun., 68:4180-4188, 2000); LytC(SP1573, spr1431) (Wizemann et al., Infect. Immun., 69:1593-98, 2001); PsaA (Briles et al., Vaccine, 19:S87-S95, 2001); PdB (Ogunniyi et al., Infect. Immun., 69:5997-6003, 2001); RPhp (Zhang et al., Infect. Immun., 69:3827-36, 2001); PiuA (Jomaa et al., Vaccine, 24:5133-39, 2006); PiaA (Jomaa et al., Vaccine, 24:5133-39, 2006); 6PGD (Daniely et al., Clin. Exp. Immunol., 144:254-263, 2006); or PppA (Green et al., Infect. Immun., 73:981-89, 2005);
Streptococcus agalactiae: such as, Group B streptococcus antigens;
Streptococcus pyogenes: such as, Group A streptococcus antigens;
Enterococcus faecalis or Enterococcus faecium: such as, a trisaccharide repeat or other Enterococcus derived antigens provided in U.S. Pat. No. 6,756,361;
Helicobacter pylori: including: Cog, Vac, Nap, HopX, HopY and/or urease antigen;
Bordetella pertussis: such as pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) from B. pertussis, optionally also combination with pertactin and/or agglutinogens 2 and 3 antigen;
Staphylococcus aureus: including S. aureus type 5 and 8 capsular polysaccharides optionally conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAX™, or antigens derived from surface proteins, invasins (leukocidin, kinases, hyaluronidase), surface factors that inhibit phagocytic engulfment (capsule, Protein A), carotenoids, catalase production, Protein A, coagulase, clotting factor, and/or membrane-damaging toxins (optionally detoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin, leukocidin);
Staphylococcus epidermis: particularly, S. epidermidis slime-associated antigen (SAA);
Staphylococcus saprophyticus: (causing urinary tract infections) particularly the 160 kDa hemagglutinin of S. saprophyticus antigen;
Pseudomonas aeruginosa: particularly, endotoxin A, Wzz protein, P. aeruginosa LPS, more particularly LPS isolated from PAO1 (O5 serotype), and/or Outer Membrane Proteins, including Outer Membrane Proteins F (OprF) (Infect Immun. 2001 May; 69(5): 3510-3515);
Bacillus anthracis (anthrax): such as B. anthracis antigens (optionally detoxified) from A-components (lethal factor (LF) and edema factor (EF)), both of which can share a common B-component known as protective antigen (PA);
Moraxella catarrhalis: (respiratory) including outer membrane protein antigens (HMW-OMP), C-antigen, and/or LPS;
Yersinia pestis (plague): such as F1 capsular antigen (Infect Immun. 2003 January; 71(1)): 374-383, LPS (Infect Immun. 1999 October; 67(10): 5395), Yersinia pestis V antigen (Infect Immun. 1997 November; 65(11): 4476-4482);
Yersinia enterocolitica (gastrointestinal pathogen): particularly LPS (Infect Immun. 2002 August; 70(8): 4414);
Yersinia pseudotuberculosis: gastrointestinal pathogen antigens;
Mycobacterium tuberculosis: such as lipoproteins, LPS, BCG antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6 optionally formulated in cationic lipid vesicles (Infect Immun. 2004 October; 72(10): 6148), Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase associated antigens (Proc. Natl. Acad Sci U.S.A. 2004 Aug. 24; 101(34): 12652), and/or MPT51 antigens (Infect Immun. 2004 July; 72(7): 3829);
Legionella pneumophila (Legionnaires' Disease): L. pneumophila antigens—optionally derived from cell lines with disrupted asd genes (Infect Immun. 1998 May; 66(5): 1898);
Rickettsia: including outer membrane proteins, including the outer membrane protein A and/or B (OmpB) (Biochem Biophys Acta. 2004 Nov. 1; 1702(2):145), LPS, and surface protein antigen (SPA) (J Autoimmun. 1989 June; 2 Suppl:81);
E. coli: including antigens from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), and/or enterohemorrhagic E. coli (EHEC);
Vibrio cholerae: including proteinase antigens, LPS, particularly lipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specific polysaccharides, V. cholera O139, antigens of IEM108 vaccine (Infect Immun. 2003 October; 71(10):5498-504), and/or Zonula occludens toxin (Zot);
Salmonella typhi (typhoid fever): including capsular polysaccharides preferably conjugates (Vi, i.e. vax-TyVi);
Salmonella typhimurium (gastroenteritis): antigens derived therefrom are contemplated for microbial and cancer therapies, including angiogenesis inhibition and modulation of flk;
Listeria monocytogenes (systemic infections in immunocompromised or elderly people, infections of fetus): antigens derived from L. monocytogenes are preferably used as carriers/vectors for intracytoplasmic delivery of the conjugates/associated compositions described herein;
Porphyromonas gingivalis: such as, P. gingivalis outer membrane protein (OMP);
Tetanus: such as tetanus toxoid (TT) antigens, e.g., used as a carrier protein in conjunction/conjugated with the compositions described herein;
Diphtheria: such as a diphtheria toxoid or a diphtheria toxoid mutant, e.g., CRM197, additionally antigens capable of modulating, inhibiting or associated with ADP ribosylation are contemplated for combination/co-administration/conjugation with the compositions described herein, the diphtheria toxoids can be used as carrier proteins;
Borrelia burgdorferi (Lyme disease): such as antigens associated with P39 and P13 (an integral membrane protein, Infect Immun. 2001 May; 69(5): 3323-3334), VlsE Antigenic Variation Protein (J Clin Microbiol. 1999 December; 37(12): 3997);
Haemophilus influenzae B: such as a saccharide antigen therefrom;
Klebsiella: such as an OMP, including OMP A, or a polysaccharide optionally conjugated to tetanus toxoid;
Neiserria gonorrhoeae: including, a Por (or porin) protein, such as PorB (see Zhu et al., Vaccine (2004) 22:660-669), a transferring binding protein, such as TbpA and TbpB (see Price et al., Infection and Immunity (2004) 71(1):277-283), a opacity protein (such as Opa), a reduction-modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations (see Plante et al., J Infectious Disease (2000) 182:848-855), also see, e.g., WO99/24578, WO99/36544, WO99/57280, WO02/079243);
Chlamydia pneumoniae: particularly C. pneumoniae protein antigens;
Chlamydia trachomatis: including antigens derived from serotypes A, B, Ba and C are (agents of trachoma, a cause of blindness), serotypes L1, L2 & L3 (associated with Lymphogranuloma venereum), and serotypes, D-K;
Treponema pallidum (Syphilis): particularly a TmpA antigen; and
Haemophilus ducreyi (causing chancroid): including outer membrane protein (DsrA).
Where not specifically referenced, further bacterial antigens as described herein may be capsular antigens, polysaccharide antigens or protein antigens of any of the above. Further bacterial antigens may also include an outer membrane vesicle (OMV) preparation. Additionally, antigens include live, attenuated, split (for enveloped viruses), and/or purified versions of any of the aforementioned bacteria. The bacterial or microbial derived antigens described herein may be gram-negative or gram-positive and aerobic or anaerobic.
Additionally, any of the above bacterial-derived saccharides (polysaccharides, LPS, LOS or oligosaccharides) can be conjugated to another agent or antigen, such as a carrier protein (for example CRM197). Such conjugation may be direct conjugation effected by reductive amination of carbonyl moieties on the saccharide to amino groups on the protein, as provided in U.S. Pat. No. 5,360,897 and Can J Biochem Cell Biol. 1984 May; 62(5):270-5. Alternatively, the saccharides can be conjugated through a linker, such as, with succinamide or other linkages provided in Bioconjugate Techniques, 1996 and CRC, Chemistry of Protein Conjugation and Cross-Linking, 1993.
Influenza: including whole viral particles (attenuated), split, or subunit comprising hemagglutinin (HA) and/or neuraminidase (NA) surface proteins, the influenza antigens may be derived from chicken embryos or propagated on cell culture, and/or the influenza antigens are derived from influenza type A, B, and/or C, among others;
Respiratory syncytial virus (RSV): including the F protein of the A2 strain of RSV (J Gen Virol. 2004 November; 85(Pt 11):3229) and/or G glycoprotein;
Parainfluenza virus (PIV): including PIV type 1, 2, and 3, preferably containing hemagglutinin, neuraminidase and/or fusion glycoproteins;
Poliovirus: including antigens from a family of picornaviridae, preferably poliovirus antigens such as OPV or, preferably IPV;
Measles: including split measles virus (MV) antigen optionally combined with the Protollin and or antigens present in MMR vaccine;
Mumps: including antigens present in MMR vaccine;
Rubella: including antigens present in MMR vaccine as well as other antigens from Togaviridae, including dengue virus;
Rabies: such as lyophilized inactivated virus (RabAvert™);
Flaviviridae viruses: such as (and antigens derived therefrom) yellow fever virus, Japanese encephalitis virus, dengue virus (types 1, 2, 3, or 4), tick borne encephalitis virus, and West Nile virus;
Caliciviridae; antigens therefrom;
HIV: including HIV-1 or HIV-2 strain antigens, such as gag (p24gag and p55gag), env (gp160 and gp41), pol, tat, nef, rev vpu, miniproteins, (preferably p55 gag and gp140v delete) and antigens from the isolates HIVIIIb, HIVSF2, HIVLAV, HIVLAI, HIVMN, HIV-1CM235, HIV-1US4, HIV-2; simian immunodeficiency virus (SW) among others;
Rotavirus: including VP4, VP5, VP6, VP7, VP8 proteins (Protein Expr Purif. 2004 December; 38(2):205) and/or NSP4;
Pestivirus: such as antigens from classical porcine fever virus, bovine viral diarrhea virus, and/or border disease virus;
Parvovirus: such as parvovirus B19;
Coronavirus: including SARS virus antigens, particularly spike protein or proteases therefrom, as well as antigens included in WO 04/92360;
Hepatitis A virus: such as inactivated virus;
Hepatitis B virus: such as the surface and/or core antigens (sAg), as well as the presurface sequences, pre-S1 and pre-S2 (formerly called pre-S), as well as combinations of the above, such as sAg/pre-S1, sAg/pre-S2, sAg/pre-S1/pre-S2, and pre-S1/pre-S2, (see, e.g., AHBV Vaccines—Human Vaccines and Vaccination, pp. 159-176; and U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum et al., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991) 65:5457-5464);
Hepatitis C virus: such as E1, E2, E1/E2 (see, Houghton et al., Hepatology (1991) 14:381), NS345 polyprotein, NS 345-core polyprotein, core, and/or peptides from the nonstructural regions (International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436);
Delta hepatitis virus (HDV): antigens derived therefrom, particularly 8-antigen from HDV (see, e.g., U.S. Pat. No. 5,378,814);
Hepatitis E virus (HE V); antigens derived therefrom;
Hepatitis G virus (HGV); antigens derived therefrom;
Varcicella zoster virus: antigens derived from varicella zoster virus (VZV) (J. Gen. Virol. (1986) 67:1759);
Epstein-Barr virus: antigens derived from EBV (Baer et al., Nature (1984) 310:207);
Cytomegalovirus: CMV antigens, including gB and gH (Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp. 125-169);
Herpes simplex virus: including antigens from HSV-1 or HSV-2 strains and glycoproteins gB, gD and gH (McGeoch et al., J. Gen. Virol. (1988) 69:1531 and U.S. Pat. No. 5,171,568);
Human Herpes Virus: antigens derived from other human herpesviruses such as HHV6 and HHV7; and
HPV: including antigens associated with or derived from human papillomavirus (HPV), for example, one or more of E1-E7, L1, L2, and fusions thereof, particularly the compositions described herein may include a virus-like particle (VLP) comprising the L1 major capsid protein, more particular still, the HPV antigens are protective against one or more of HPV serotypes 6, 11, 16 and/or 18.
Further provided are antigens, compositions, methods, and microbes included in Vaccines, 4th Edition (Plotkin and Orenstein ed. 2004); Medical Microbiology 4th Edition (Murray et al. ed. 2002); Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991), which are contemplated in conjunction with the compositions described herein.
Additionally, antigens include live, attenuated, split, and/or purified versions of any of the aforementioned viruses.
Fungal antigens for use herein that are associated with vaccines include those described in: U.S. Pat. Nos. 4,229,434 and 4,368,191 for prophylaxis and treatment of trichopytosis caused by Trichophyton mentagrophytes; U.S. Pat. Nos. 5,277,904 and 5,284,652 for a broad spectrum dermatophyte vaccine for the prophylaxis of dermatophyte infection in animals, such as guinea pigs, cats, rabbits, horses and lambs, these antigens comprises a suspension of killed T. equinum, T. mentagrophytes (var. granulare), M. canis and/or M. gypseum in an effective amount optionally combined with an adjuvant; U.S. Pat. Nos. 5,453,273 and 6,132,733 for a ringworm vaccine comprising an effective amount of a homogenized, formaldehyde-killed fungi, i.e. Microsporum canis culture in a carrier; and U.S. Pat. No. 5,948,413 involving extracellular and intracellular proteins for pythiosis. Additional antigens identified within antifungal vaccines include Ringvac bovis LTF-130 and Bioveta.
Further, fungal antigens for use herein may be derived from Dermatophytres, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme.
Fungal pathogens for use as antigens or in derivation of antigens in conjunction with the compositions described herein comprise Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candicla glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitiS, Blastomyces dermaticlis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulation, Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, and Saksenaea spp.
Other fungi from which antigens are derived include Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp.
Processes for producing fungal antigens are well known in the art (see U.S. Pat. No. 6,333,164). In some methods a solubilized fraction extracted and separated from an insoluble fraction obtainable from fungal cells of which cell wall has been substantially removed or at least partially removed, characterized in that the process comprises: obtaining living fungal cells; obtaining fungal cells of which cell wall has been substantially removed or at least partially removed; bursting the fungal cells of which cell wall has been substantially removed or at least partially removed; obtaining an insoluble fraction; and extracting and separating a solubilized fraction from the insoluble fraction.
In some embodiments, microbes (bacteria, viruses and/or fungi) against which the present compositions and methods can be implement include those that cause sexually transmitted diseases (STDs) and/or those that display on their surface an antigen that can be the target or antigen composition as described herein. In some embodiment, compositions are combined with antigens derived from a viral or bacterial STD. Antigens derived from bacteria or viruses can be administered in conjunction with the compositions described herein to provide protection against at least one of the following STDs, among others: chlamydia, genital herpes, hepatitis (particularly HCV), genital warts, gonorrhea, syphilis and/or chancroid (see, WO00/15255).
In some embodiments, the compositions described herein are co-administered with one or more antigens for the prevention or treatment of an STD.
Antigens derived from the following viruses associated with STDs, which are described in greater detail above, are preferred for co-administration with the compositions described herein: hepatitis (particularly HCV), HPV, HIV, or HSV.
Additionally, antigens derived from the following bacteria associated with STDs, which are described in greater detail above, are preferred for co-administration with the compositions described herein: Neiserria gonorrhoeae, Chlamydia pneumoniae, Chlamydia trachomatis, Treponema pallidum, or Haemophilus ducreyi.
The S. pneumoniae antigen may be a respiratory antigen and could further be used in an immunogenic composition for methods of preventing and/or treating infection by a respiratory pathogen, including a virus, bacteria, or fungi such as respiratory syncytial virus (RSV), PIV, SARS virus, influenza, Bacillus anthracis, particularly by reducing or preventing infection and/or one or more symptoms of respiratory virus infection. A composition comprising an antigen described herein, such as one derived from a respiratory virus, bacteria or fungus is administered in conjunction with the compositions described herein to an individual which is at risk of being exposed to that particular respiratory microbe, has been exposed to a respiratory microbe or is infected with a respiratory virus, bacteria or fungus. The composition(s) described herein is/are co-administered at the same time or in the same formulation with one or more antigens of the respiratory pathogen. Administration of the composition results in reduced incidence and/or severity of one or more symptoms of respiratory infection.
In some embodiments, the compositions described herein are used in conjunction with one or more antigens for treatment of a pediatric population, as in a pediatric antigen. In some embodiments, the age of the subjects in the pediatric population is less than about 3 years old, or less than about 2 years, or less than about 1 years old. In some embodiments the pediatric antigen (in conjunction with the composition described herein) is administered multiple times over at least 1, 2, or 3 years.
In some embodiments, the compositions described herein are used in conjunction with one or more antigens for treatment of a geriatric population, as in a geriatric antigen. In some embodiments, the age of the subjects in the geriatric population is greater than about 50 years old, greater than about 55 years old, greater than about 60 years old, greater than about 65 years old, greater than about 70 years old, greater than about 75 years old, greater than about 80 years old, or greater than about 85 years old. In some embodiments the geriatric antigen (in conjunction with the composition described herein) is administered multiple times over at least 1, 2, or 3 years.
Other antigens for use in conjunction with the compositions of the present include hospital acquired (nosocomial) associated antigens.
In some embodiments, parasitic antigens are contemplated in conjunction with the compositions described herein. Examples of parasitic antigens include those derived from organisms causing malaria and/or Lyme disease.
In some embodiments, the antigens in conjunction with the compositions described herein are associated with or effective against a mosquito born illness. In some embodiments, the antigens in conjunction with the compositions described herein are associated with or effective against encephalitis. In some embodiments the antigens in conjunction with the compositions described herein are associated with or effective against an infection of the nervous system.
In some embodiments, the antigens in conjunction with the compositions described herein are antigens transmissible through blood or body fluids.
In some aspects, methods of producing microparticles having adsorbed antigens are provided. The methods comprise: (a) providing an emulsion by dispersing a mixture comprising (i) water, (ii) a detergent, (iii) an organic solvent, and (iv) a biodegradable polymer selected from the group consisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and a polycyanoacrylate. The polymer is typically present in the mixture at a concentration of about 1% to about 30% relative to the organic solvent, while the detergent is typically present in the mixture at a weight-to-weight detergent-to-polymer ratio of from about 0.00001:1 to about 0.1:1 (more typically about 0.0001:1 to about 0.1:1, about 0.001:1 to about 0.1:1, or about 0.005:1 to about 0.1:1); (b) removing the organic solvent from the emulsion; and (c) adsorbing an antigen on the surface of the microparticles. In some embodiments, the biodegradable polymer is present at a concentration of about 3% to about 10% relative to the organic solvent.
In some embodiments, microparticles for use herein are formed from materials that are sterilizable, non-toxic and biodegradable. Such materials include, without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid, polycaprolactone, polyorthoester, polyanhydride, PACA, and polycyanoacrylate. In some embodiments, microparticles for use with the methods described herein are derived from a poly(α-hydroxy acid), in particular, from a poly(lactide) (“PLA”) or a copolymer of D,L-lactide and glycolide or glycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or “PLGA”), or a copolymer of D,L-lactide and caprolactone. The microparticles may be derived from any of various polymeric starting materials which have a variety of molecular weights and, in the case of the copolymers such as PLG, a variety of lactide:glycolide ratios, the selection of which will be largely a matter of choice, depending in part on the coadministered macromolecule. These parameters are discussed more fully below.
Further antigens may also include an outer membrane vesicle (OMV) preparation.
Antigens can also be adsorbed to peptidoglycans of various gram-positive bacteria to make gram-positive enhancer matrix (GEM) particles, as described in Bosma et al., Appl. Env. Microbiol., 72:880-889, 2006, the entire contents of which are incorporated herein by reference. This method relies on the non-covalent binding of the LysM motif (Buist et al., J. Bact., 177:1554-63, 1995; Bateman and Bycroft, J. Mol. Biol., 299:1113-19, 2000) to the cell wall peptidoglycan of acid-treated cells. Briefly, a polypeptide antigen linked to one or more LysM motifs (e.g., non-covalently or covalently (e.g., as a fusion protein or by conjugation) is added to acid-treated gram-positive bacteria. The antigen peptides bind with high affinity and can be used in immunogenic compositions. Exemplary acids used in these methods include trichloroacetic acid (e.g., at 0.1%-10%), acetic acid (e.g., at 5.6 M), HCl (e.g., at 0.01 M), lactic acid (e.g., at 0.72 M), and formic acid (e.g., at 0.56 M).
Additional formulation methods and antigens (especially tumor antigens) are provided in U.S. Pat. No. 6,884,435.
The following references, each of which is specifically incorporated by reference in its entirety, include antigens useful in conjunction with the compositions described herein:
The S. pneumoniae polypeptides used with the compositions described herein may be present in the compositions as individual separate polypeptides, but in some embodiments at least two (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) of the antigens can be expressed as a single polypeptide chain (a “hybrid” or “fusion” polypeptide). Such fusion polypeptides offer two principal advantages: first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable fusion partner that overcomes the problem; second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful.
The fusion polypeptide may comprise one or more polypeptide sequences encoded by the S. pneumoniae Polypeptides as identified herein. Accordingly, the compositions described herein may include a fusion peptide comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from a protein encoded by the S. pneumoniae pilus II island (INV104B) or a fragment thereof. In some embodiments, the first and second amino acid sequences in the fusion polypeptide comprise different epitopes.
In some embodiments, hybrids (or fusions) consisting of amino acid sequences from two, three, four, five, six, seven, eight, nine, or ten antigens encoded by the S. pneumoniae Polypeptide sequences discussed herein are preferred. In some embodiments, hybrids consisting of amino acid sequences from two, three, four, or five antigens encoded by the S. pneumoniae Polypeptide sequences discussed herein are preferred.
Different hybrid polypeptides may be mixed together in a single formulation. Within such combinations, a sequence encoded by the S. pneumoniae Polypeptide sequences discussed herein may be present in more than one hybrid polypeptide and/or as a non-hybrid polypeptide. In some embodiments, however, that an antigen is present either as a hybrid or as a non-hybrid, but not as both.
Hybrid polypeptides can be represented by the formula NH2-A-{-X-L-}n-B—COOH, wherein: X is an amino acid sequence of a S. pneumoniae Polypeptide as described herein or a fragment thereof; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
If a —X— moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid protein. In some embodiments, the leader peptides will be deleted except for that of the —X— moiety located at the N-terminus of the hybrid protein i.e. the leader peptide of X1 will be retained, but the leader peptides of X2 . . . Xn will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of X1 as moiety -A-.
For each n instances of {-X-L-}, linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH2—X1-L1-X2-L2-COOH; NH2—X1—X2—COOH; NH2—Xi-L1-X2—COOH; NH2—X1—X2-L2-COOH; etc. Linker amino acid sequence(s)-L-will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptide sequences that facilitate cloning, poly-glycine linkers (i.e. comprising Glyn where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG, with the Gly-Ser dipeptide being formed from a BamHI restriction site, thus aiding cloning and manipulation, and the (Gly)4 tetrapeptide being a typical poly-glycine linker.
In the hybrid polypeptide formula provided above, -A- is an optional N-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification. Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. If X1 lacks its own N-terminus methionine, -A- is preferably an oligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine.
In the hybrid polypeptide formula provided above, —B— is an optional C-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification, or sequences which enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.
In the hybrid polypeptide formula provided above, most preferably, n is 2 or 3.
This description provides nucleic acids encoding the S. pneumoniae Polypeptide sequences and/or the hybrid fusion polypeptides. This description also provides nucleic acids encoding S. pneumoniae Polypeptide antigen, and/or the hybrid fusion polypeptides described herein. Furthermore, the description provides nucleic acids which can hybridize to these nucleic acids, preferably under “high stringency” conditions (e.g. 65° C. in a 0.1×SSC, 0.5% SDS solution).
The polypeptides described herein can be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, non-glycosylated, lipidated, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other host cell proteins).
The nucleic acids described herein can be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself, etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other host cell nucleic acids).
The terms “nucleic acid” and “nucleic acids,” as used herein, include DNA and RNA, and also their analogs, such as those containing modified backbones (e.g., phosphorothioates, etc.), and also peptide nucleic acids (PNA), etc. The compositions described herein include nucleic acids comprising sequences complementary to those described above (e.g., for antisense or probing purposes).
This description also discloses a process for producing a polypeptide, comprising the step of culturing a host cell transformed with nucleic acid as described herein under conditions which induce polypeptide expression.
This description also discloses a process for producing a polypeptide, comprising the step of synthesizing at least part of the polypeptide by chemical means.
This description also discloses a process for producing nucleic acid, comprising the step of amplifying nucleic acid using a primer-based amplification method (e.g. PCR).
This description also discloses a process for producing nucleic acid, comprising the step of synthesizing at least part of the nucleic acid by chemical means.
The S. pneumoniae Polypeptide sequences described herein may be isolated from native S. pneumoniae, or they may be recombinantly produced, for instance in a heterologous host. For example, the S. pneumoniae pilus II island (INV104B) antigens described herein may be isolated from S. pneumoniae containing the S. pneumoniae pilus II island (INV104B), or they may be recombinantly produced, for instance, in a heterologous host.
The heterologous host may be prokaryotic (e.g., a bacterium) or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g. M. tuberculosis), S. gordonii, L. lactis, yeasts, etc.
Recombinant production of polypeptides may be facilitated by adding a tag protein to the S. pneumoniae Polypeptide sequences as described herein to be expressed as a fusion protein comprising the tag protein and the polypeptide. For example, recombinant production of polypeptides is facilitated by adding a tag protein to the S. pneumoniae pilus II island (INV104B) pilus antigen to be expressed as a fusion protein comprising the tag protein and the S. pneumoniae pilus II island (INV104B) pilus antigen. Such tag proteins can facilitate purification, detection and stability of the expressed protein. Tag proteins suitable for use with the compositions described herein include a polyarginine tag (Arg-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP-tag-chitin-binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination anti-termination factor (NusA), E. coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). Preferred tag proteins include GST. A full discussion on the use of tag proteins can be found at Terpe et al., “Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems,” Appl. Microbiol. Biotechnol. (2003) 60:523-533.
After purification, the tag proteins may optionally be removed from the expressed fusion protein, i.e., by specifically tailored enzymatic treatments known in the art. Commonly used proteases include enterokinase, tobacco etch virus (TEV), thrombin, and factor Xa.
The compositions described herein are preferably immunogenic compositions, and are more preferably vaccine compositions. In some embodiments, the pH of the composition is between 6 and 8, preferably about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen-free. The composition may be isotonic with respect to humans.
Vaccines as described herein may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Accordingly, methods are described for the therapeutic or prophylactic treatment of a S. pneumoniae infection in an animal susceptible to such S. pneumoniae infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic composition described herein. For example, methods are described for the therapeutic or prophylactic treatment of a S. pneumoniae infection in an animal susceptible to streptococcal infection comprising administering to said animal a therapeutic or prophylactic amount of the immunogenic compositions described herein.
The compositions described herein can also be used as a medicament. In some embodiments, the medicament is preferably able to raise an immune response in a mammal (i.e. it is an immunogenic composition) and is, in some embodiments, a vaccine. The compositions described herein can also be used in the manufacture of a medicament for raising an immune response in a mammal. In some embodiments, the medicament is a vaccine.
The compositions described herein can also be use in kits containing one or more containers of the compositions. Such compositions can be in liquid form or can be lyophilized, as can individual antigens. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such compositions may comprise a first component comprising one or more S. pneumoniae Pili Polypeptides. Preferably, the S. pneumoniae Pili Polypeptides are in an oligomeric or hyperoligomeric form.
The kit can further contain a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other buffers, diluents, filters, needles, and syringes. The kit can also contain a second or third container with another active agent, for example an antibiotic.
The kit can also contain a package insert containing written instructions for methods of inducing immunity against S. pneumoniae or for treating S. pneumoniae infections. The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
A delivery device pre-filled with the immunogenic compositions described herein can also be used.
A method for inducing an immune response in a mammal can include the step of administering an effective amount of a composition as described herein. In some embodiments, the immune response is protective and preferably involves antibodies and/or cell-mediated immunity. This immune response will, in some embodiments, induce long lasting (e.g., neutralizing) antibodies and a cell mediated immunity that can quickly respond upon exposure to one or more S. pneumoniae antigens. In some embodiments, the method raise a booster response.
Methods of neutralizing S. pneumoniae infection in a mammal can include administering to the mammal an effective amount of the immunogenic compositions as described herein, a vaccine as described herein, or antibodies which recognize an immunogenic composition as described herein.
In some embodiments, the mammal is a human. Where the vaccine is for prophylactic use, the human can be a male or a female (either of child bearing age or a teenager). Alternatively, the human may be elderly (e.g., over the age of 50, 55, 60, 65, 70, 75, 80, or 85) and may have an underlying disease such as diabetes or cancer. In some embodiments, where the vaccine is for therapeutic use, the human can be a pregnant female or an elderly adult.
In some embodiments, the uses and methods described herein are for the prevention and/or treatment of a disease caused by S. pneumoniae. The compositions may also be effective against other streptococcal bacteria. The compositions may also be effective against other Gram positive bacteria.
Some methods of checking efficacy of therapeutic treatment involves monitoring S. pneumoniae bacterial infection after administration of a composition as described herein. A non-limiting means of checking efficacy of prophylactic treatment involves monitoring immune responses against the S. pneumoniae antigens in the compositions described herein after administration of the composition.
A non-limiting means of assessing the immunogenicity of the component proteins of the immunogenic compositions described herien is to express the proteins recombinantly and to screen patient sera or mucosal secretions by immunoblot. A positive reaction between the protein and the patient serum indicates that the patient has previously mounted an immune response to the protein in question—that is, the protein is an immunogen. This method may also be used to identify immunodominant proteins and/or epitopes.
Another means of checking efficacy of therapeutic treatment involves monitoring S. pneumoniae infection after administration of the compositions described herein. One way of checking efficacy of prophylactic treatment involves monitoring immune responses both systemically (such as monitoring the level of IgG1 and IgG2a production) and mucosally (such as monitoring the level of IgA production) against the S. pneumoniae antigens in the compositions described herein after administration of the composition. Typically, S. pneumoniae serum specific antibody responses are determined post-immunization, but pre-challenge, whereas mucosal S. pneumoniae specific antibody body responses are determined post-immunization and post-challenge.
The vaccine compositions described herein can, in some embodiments, be evaluated in in vitro and in vivo animal models prior to host, e.g., human, administration.
The efficacy of immunogenic compositions described herein can also be determined in vivo by challenging animal models of S. pneumoniae infection, e.g., guinea pigs or mice, with the immunogenic compositions. The immunogenic compositions may or may not be derived from the same serotypes as the challenge serotypes. In some embodiments, the immunogenic compositions are derivable from the same serotypes as the challenge serotypes. In some embodiments, the immunogenic composition and/or the challenge serotypes are derivable from the group of S. pneumoniae serotypes.
In vivo efficacy models include but are not limited to: (i) a murine infection model using human S. pneumoniae serotypes; (ii) a murine disease model which is a murine model using a mouse-adapted S. pneumoniae strain, such as those strains which are particularly virulent in mice and (iii) a primate model using human S. pneumoniae isolates.
The immune response may be one or both of a TH1 immune response and a TH2 response. The immune response may be an improved or an enhanced or an altered immune response. The immune response may be one or both of a systemic and a mucosal immune response. In some embodiments the immune response is an enhanced system and/or mucosal response. An enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response. In some embodiments, the enhanced immune response includes an increase in the production of IgG1 and/or IgG2a and/or IgA. In some embodiments, the mucosal immune response is a TH2 immune response. In some embodiments, the mucosal immune response includes an increase in the production of IgA.
Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections. Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immune response may result in the production of IgG1, IgE, IgA and memory B cells for future protection.
A TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgG1, IgE, IgA and memory B cells. In some embodiments, the enhanced TH2 immune response will include an increase in IgG1 production.
A TH1 immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 immune response (such as IL-2, IFNγ, and TNFβ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. In some embodiments, the enhanced TH1 immune response will include an increase in IgG2a production.
The immunogenic compositions described herein, in particular, immunogenic compositions comprising one or more S. pneumoniae Polypeptide antigen, may be used either alone or in combination with other antigens optionally with an immunoregulatory agent capable of eliciting a Th1 and/or Th2 response.
The compositions described herein will generally be administered directly to a patient. Certain routes may be favored for certain compositions, as resulting in the generation of a more effective immune response, in some embodiments, a CMI response, or as being less likely to induce side effects, or as being easier for administration. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intradermally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (see, e.g., WO 99/27961) or transcutaneous (see, e.g., WO 02/074244 and WO 02/064162), intranasal (see, e.g., WO03/028760), ocular, aural, pulmonary or other mucosal administration.
The compositions described herein may be used to elicit systemic and/or mucosal immunity.
In some embodiments, the immunogenic composition comprises one or more S. pneumoniae Polypeptide antigen(s) which elicits a neutralizing antibody response and one or more S. pneumoniae Polypeptide antigen(s) which elicits a cell mediated immune response. In this way, the neutralizing antibody response prevents or inhibits an initial S. pneumoniae infection while the cell-mediated immune response capable of eliciting an enhanced Th1 cellular response prevents further spreading of the S. pneumoniae infection. The immunogenic composition may include one or more S. pneumoniae Polypeptide antigens; one or more S. pneumoniae pilus or other S. pneumoniae antigens; and one or more non-pilus S. pneumoniae antigens, e.g., cytoplasmic antigens. In some embodiments, the immunogenic composition comprises one or more S. pneumoniae surface antigens or the like and one or other antigens, such as a cytoplasmic antigen capable of eliciting a Th1 cellular response.
Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc.
The compositions described herein may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilized composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more antigens in liquid form and one or more lyophilized antigens.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, such as antibiotics, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention, or increases a measurable immune response or prevents or reduces a clinical symptom. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The compositions described herein can, in addition to the components mentioned above, comprise one or more ‘pharmaceutically acceptable carriers’, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. A thorough discussion of pharmaceutically acceptable excipients is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th ed., ISBN: 0683306472.
Vaccines as described herein may be administered in conjunction with other immunoregulatory agents. In some embodiments, compositions further comprise one or more adjuvants. Adjuvants for use with the vaccines described herein include, but are not limited to, one or more of the following set forth below:
Mineral containing compositions suitable for use as adjuvants with the compositions disclosed herein include mineral salts, such as aluminum salts and calcium salts. Including mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulfates, etc. (see, e.g., chapters 8 & 9 of Vaccine Design . . . (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.), or mixtures of different mineral compounds (e.g. a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption to the salt(s) being preferred. The mineral containing compositions may also be formulated as a particle of metal salt (WO 00/23105).
Aluminum salts may be included in vaccines described herein such that the dose of Al3+ is between 0.2 and 1.0 mg per dose.
Oil-emulsion compositions suitable for use as adjuvants with the compositions described herein include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). See WO90/14837. See also, Podda, “The adjuvanted influenza vaccines with novel adjuvants: experience with the MF59-adjuvanted vaccine”, Vaccine (2001) 19: 2673-2680; Frey et al., “Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non-elderly adults”, Vaccine (2003) 21:4234-4237. MF59 is used as the adjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.
In some embodiments adjuvants for use in the compositions are submicron oil-in-water emulsions. Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80™ (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span 85™ (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphosphoryloxy)-ethylamine (MTP-PE), for example, the submicron oil-in-water emulsion known as “MF59” (International Publication No. WO 90/14837; U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entireties; and Ott et al., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v Squalene (e.g. 4.3%), 0.25-0.5% w/v Tween 80™, and 0.5% w/v Span 85™ and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PE may be present in an amount of about 0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100 μg/dose. As used herein, the term “MF59-0” refers to the above submicron oil-in-water emulsion lacking MTP-PE, while the term MF59-MTP denotes a formulation that contains MTP-PE. For instance, “MF59-100” contains 100 μg MTP-PE per dose, and so on. MF69, another submicron oil-in-water emulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v Tween 80™, and 0.75% w/v Span 85™ and optionally MTP-PE. Yet another submicron oil-in-water emulsion is MF75, also known as SAF, containing 10% squalene, 0.4% Tween 80™, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidized into a submicron emulsion. MF75-MTP denotes an MF75 formulation that includes MTP, such as from 100-400 μg MTP-PE per dose.
Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO 90/14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entireties.
Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants with the compositions disclosed herein.
Saponin formulations, may also be used as adjuvants with the compositions disclosed herein. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-TLC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO96/33739).
Combinations of saponins and cholesterols can be used to form unique particles called Immunostimulating Complexs (ISCOMs). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of Quil A, QHA and QHC. ISCOMs are further described in EP0109942, WO 96/11711 and WO 96/33739. Optionally, the ISCOMS may be devoid of additional detergent. See WO 00/07621.
A review of the development of saponin based adjuvants can be found at Barr, et al., “ISCOMs and other saponin based adjuvants”, Advanced Drug Delivery Reviews (1998) 32:247-271. See also Sjolander, et al., “Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines”, Advanced Drug Delivery Reviews (1998) 32:321-338.
Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants with the compositions disclosed herein. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic and non-replicating, and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Q13-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p1). VLPs are discussed further in WO 03/024480, WO 03/024481, and Niikura et al., “Chimeric Recombinant Hepatitis E Virus-Like Particles as an Oral Vaccine Vehicle Presenting Foreign Epitopes”, Virology (2002) 293:273-280; Lenz et al., “Papillomarivurs-Like Particles Induce Acute Activation of Dendritic Cells”, Journal of Immunology (2001) 5246-5355; Pinto, et al., “Cellular Immune Responses to Human Papillomavirus (HPV)-16 L1 Healthy Volunteers Immunized with Recombinant HPV-16 L1 Virus-Like Particles”, Journal of Infectious Diseases (2003) 188:327-338; and Gerber et al., “Human Papillomavirus Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Enterotoxin Mutant R192G or CpG”, Journal of Virology (2001) 75(10):4752-4760. Virosomes are discussed further in, for example, Gluck et al., “New Technology Platforms in the Development of Vaccines for the Future”, Vaccine (2002) 20:B10-B16. Immunopotentiating reconstituted influenza virosomes (IRIV) are used as the subunit antigen delivery system in the intranasal trivalent INFLEIXAL™ product {Mischler & Metcalfe (2002) Vaccine 20 Suppl 5:B17-23} and the INFLUVAC PLUS™ product.
Adjuvants suitable for use with the compositions described herein include bacterial or microbial derivatives such as:
Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. See Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.
Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in Meraldi et al., “OM-174, a New Adjuvant with a Potential for Human Use, Induces a Protective Response with Administered with the Synthetic C-Terminal Fragment 242-310 from the circumsporozoite protein of Plasmodium berghei”, Vaccine (2003) 21:2485-2491; and Pajak, et al., “The Adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo”, Vaccine (2003) 21:836-842.
Immunostimulatory oligonucleotides suitable for use as adjuvants with the compositions described herein include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, the guanosine may be replaced with an analog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al., “Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles”, Nucleic Acids Research (2003) 31(9): 2393-2400; WO02/26757 and WO99/62923 for examples of possible analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg, “CpG motifs: the active ingredient in bacterial extracts?”, Nature Medicine (2003) 9(7): 831-835; McCluskie, et al., “Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA”, FEMS Immunology and Medical Microbiology (2002) 32:179-185; WO98/40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199.
The CpG sequence may be directed to TLR9, such as the motif GTCGTT (SEQ ID NO:22) or TTCGTT (SEQ ID NO:23). See Kandimalla, et al., “Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpG DNAs,” Biochemical Society Transactions (2003) 31 (part 3): 654-658. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, et al., “CpG-A-Induced Monocyte IFN-gamma-Inducible Protein-10 Production is Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha”, J. Immunol. (2003) 170(8):4061-4068; Krieg, “From A to Z on CpG”, TRENDS in Immunology (2002) 23(2): 64-65 and WO01/95935. Preferably, the CpG is a CpG-A ODN.
In some embodiments, the CpG oligonucleotide is constructed so that the 5′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3′ ends to form “immunomers”. See, e.g., Kandimalla, et al., “Secondary structures in CpG oligonucleotides affect immunostimulatory activity”, BBRC (2003) 306:948-953; Kandimalla, et al., “Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic GpG DNAs”, Biochemical Society Transactions (2003) 31(part 3):664-658; Bhagat et al., “CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents” BBRC (2003) 300:853-861 and WO 03/035836.
Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants with the compositions described herein. In some embodiments, the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin “LT), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO95/17211 and as parenteral adjuvants in WO98/42375. In some embodiments, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety: Beignon, et al., “The LTR72 Mutant of Heat-Labile Enterotoxin of Escherichia coli Enhances the Ability of Peptide Antigens to Elicit CD4+ T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin,” Infection and Immunity (2002) 70(6):3012-3019; Pizza, et al., “Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants,” Vaccine (2001) 19:2534-2541; Pizza, et al., “LTK63 and LTR72, two mucosal adjuvants ready for clinical trials,” Int. J. Med. Microbiol. (2000) 290(4-5):455-461; Scharton-Kersten et al., “Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits and Unrelated Adjuvants,” Infection and Immunity (2000) 68(9):5306-5313; Ryan et al., “Mutants of Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Th1 and Th2 Cells,” Infection and Immunity (1999) 67(12):6270-6280; Partidos et al., “Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides,” Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al., “Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines,” Vaccines (2003) 2(2):285-293; and Pine et al., (2002) “Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxin from Escherichia coli (LTK63),” J. Control Release (2002) 85(1-3):263-270. Numerical reference for amino acid substitutions is preferably based on the aligmnents of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al., Mol. Microbiol. (1995) 15(6):1165-1167, specifically incorporated herein by reference in its entirety.
Bioadhesives and mucoadhesives may also be used as adjuvants with the compositions described herein. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al. (2001) J. Cont. Rele. 70:267-276) or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants with the compositions described herein. See, e.g., WO 99/27960.
Microparticles may also be used as adjuvants with the compositions described herein. Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to 30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide co glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).
Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0 626 169.
Adjuvants suitable for use with the compositions described herein include polyoxyethylene ethers and polyoxyethylene esters. See WO99/52549. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152).
In some embodiments polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
PCPP formulations are described, for example, in Andrianov et al., “Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions”, Biomaterials (1998) 19(1-3):109-115 and Payne et al., “Protein Release from Polyphosphazene Matrices”, Adv. Drug. Delivery Review (1998) 31(3):185-196.
Examples of muramyl peptides suitable for use as adjuvants with the compositions described herein include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), and N-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
Examples of imidazoquinolone compounds suitable for use adjuvants with the compositions described herein include Imiquamod and its homologues, described further in Stanley, “Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential,” Clin Exp Dermatol (2002) 27(7):571-577 and Jones, “Resiquimod 3M,” Curr Opin Investig Drugs (2003) 4(2):214-218.
The compositions described herein may also include combinations of the adjuvants identified above. For example, the following adjuvant compositions may be used with the compositions described herein:
(1) a saponin and an oil-in-water emulsion (WO 99/11241);
(2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g. 3dMPL) (see WO 94/00153);
(3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol;
(4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) (WO 98/57659);
(5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231);
(6) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion;
(7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™);
(8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dPML).
(9) one or more mineral salts (such as an aluminum salt)+an immunostimulatory oligonucleotide (such as a nucleotide sequence including a CpG motif). Combination No. (9) is a preferred adjuvant combination.
Human immunomodulators suitable for use as adjuvants with the compositions described herein include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.
Aluminum salts and MF59 are preferred adjuvants for use with injectable influenza vaccines. Bacterial toxins and bioadhesives are preferred adjuvants for use with mucosally-delivered vaccines, such as nasal vaccines.
The immunogenic compositions described herein may be administered in combination with an antibiotic treatment regime. In some embodiments, the antibiotic is administered prior to administration of the antigen as described herein or the composition comprising the one or more of the antigens as described herein.
In some embodiments, the antibiotic is administered subsequent to the administration of the one or more antigens as described herein or the composition comprising the one or more antigens as described herein. Examples of antibiotics suitable for use in the treatment streptococcal infections include but are not limited to penicillin or a derivative thereof or clindamycin or the like.
Each of the patents, patent applications, accession numbers and publications described herein is hereby incorporated by reference in its entirety.
Various modifications of the invention, in addition to those described herein, will be apparent to those of skill in the art in view of the foregoing description. Such modifications are also intended to fall within the scope of the appended embodiments. The present invention is further demonstrated in the following examples that are for purposes of illustration and are not intended to limit the scope of the present invention.
S. pneumoniae Chromosomal DNA Extraction
The S. pneumoniae strains listed in Table 1 were grown in 200 mL liquid culture (THYE medium) until the OD600 reached between 0.25 to 0.5. Next, the samples were centrifuged at 6000 RPM for 15-20 minutes (some pellets were stored at this point at −20° C.). The pellets were then resuspended with 2.7 mL (for a 3 mL final volume) of 50 mM EDTA at pH 8.0 and transferred to a 15 mL Falcon™ tube (BD Biosciences; Bedford, Mass.). 0.55 mL of freshly prepared Lysozyme (12 mg/mL Sigma L-6876 in 50 mM EDTA pH 8.0 (Sigma-Aldrich Co.; St. Louis, Mo.)) and 50 μL of 5000 U/mL Mutanolysin (Sigma M-9901) in water were then sequentially added to the Falcon™ tube. These samples were incubated for one hour at 37° C.
After incubation, 3.6 mL of Nuclei Lysis Solution (Wizard® Genomic DNA Purification Kit (Promega Corp.; Madison, Wis.)) was added and the samples were mixed by inversion six-times. The samples were then incubated for 5 minutes at 80° C., then cooled to room temperature. Next 184 of RNase solution (Wizard® Genomic DNA Purification Kit) was added and the samples were mixed by inversion ten-times. The samples were then incubated for thirty minutes at 37° C.
Each sample was then divided into six 1.2 mL Eppendorf tubes. To each Eppendorf tube was added 0.2 mL of Protein Precipitation Solution (Wizard® Genomic DNA Purification Kit) and mixed by inversion ten-times. Immediately after the tenth inversion, the samples were centrifuged at room temperature in an Eppendorf centrifuge for three minutes at full speed. The supernatants from the Eppendorf tubes were then pooled into a 15 mL Falcon tube (about 8 mL was typically recovered). Next 0.6 vol. of isopropanol was added and the samples were mixed by inversion ten-times.
Precipitated DNA was recovered by inserting a Pasteur pipet that had been heat sealed and bent in a flame into the Falcon tube and hooking the pellet. The precipitated DNA pellet was then washed with Ethanol 70%. Finally, the precipitated DNA pellet was dissolved into 3 mL of TE (10 mM Tris HCl, 1 mM EDTA, pH 8.0).
The details of PCR are well known to those of skill in the art. (See Mark A. Valasek, M. A., & Repa, J. J. (2005) Advan. Physiol. Educ. 29, 151-159; Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K, Current Protocols in Molecular Biology, Hoboken, N.J.: Wiley, 2005.). The specific parameters used herein are as follows (50 μL final volume of amplification):
PCR Amplification of Genomic DNA
Reagents
5 μL Buffer (10× concentrated)
0.5 μL BSA (0.5 mg/ml)
1 μL dNTP (10 mM concentrated)
1.5 μL oligo @ 10 μmol/μL concentration (15 pmoles)
1.5 μL oligo @ 10 μmol/μL concentration (15 pmoles)
0.25 μL Taq polymerase (F. Hoffmann-La Roche Ltd; Basel, Switzerland)
50 ng genomic DNA
water to 50 μL
Cycle Parameters:
94° C. for 3 minutes (one cycle)
94° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 1 minute and 20 seconds (6 cycles)
94° C. for 30 seconds, 58° C. for 30 seconds, 72° C. for 1 minute and 20 seconds (30 cycles)
72° C. for 8 minutes (one cycle)
Reagents
5 μL Buffer (10× concentrated)
0.5 μL BSA (0.5 mg/ml)
1 μL dNTP (10 mM concentrated)
1.5 μL oligo @ 10 μmol/μL concentration (15 pmoles)
1.5 μL oligo @ 10 μmol/μL concentration (15 pmoles)
0.25 μL Taq polymerase (F. Hoffmann-La Roche Ltd; Basel, Switzerland)
40.25 μL water
bacteria
Cycle Parameters
94° C. for 8 minutes (one cycle)
94° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 1 minute and 20 seconds (6 cycles)
94° C. for 30 seconds, 58° C. for 30 seconds, 72° C. for 1 minute and 20 seconds (30 cycles)
72° C. for 8 minutes (one cycle)
DNA samples were initially prepared (for steps 1 and 2) in parallel with separate containers for Cy3 and Cy5 labeling.
One gig genomic DNA, 2.6 μL random nonamers (GE Healthcare; Buckinghamshire, UK (formerly Amersham Pharmacia)), and 1.5 μL spikes (control DNAs) were combined and water was added to a final volume of 29.5 μL. The mixture was heated to 70° C. for five minutes, then cooled to room temperature for five minutes. Finally, the mixture was centrifuged briefly.
4 mL NEB2 buffer, 2 mL Nucleotide Mix (2 mM dATP, dGTP, dTTP and 1 mM dCTP) (individually obtained from GE Healthcare; Buckinghamshire, UK), and 2 mL dCTP Cy3 (or Cy5) (GE Healthcare; Buckinghamshire, UK (formerly Amersham Pharmacia) were added to the mixture from part (1). The combination was mixed gently and 2.5 mL DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs, Inc.; Ipswich, Mass.) was added. The combination was gently mixed further then incubated at 37° C. for 2.5 hours. Finally, the mixture was centrifuged briefly.
The two labeling reaction solutions were then combined and unincorporated nucleotides were removed by running the solution through a Qiaquick PCR clean-up spin column (Qiagen, Inc.; Valencia, Calif.). The samples were eluted from the column with two 304 portions of EB buffer. Volume was reduced to 7.5 μL in a speed vacuum.
Aluminum slides were incubated for two hours at 42° C. in a 5×SSC, 50% formamide, 0.2% SDS solution. Then the slides were dipped in water three-times in one dish, and two more times in a separate dish. These prepared slides were blown dry with nitrogen.
The DNA samples from step 2 were heat denatured for two minutes at 95° C. Then the tube was spun quickly. A hybridization mix containing 1 vol. of version II buffer and 2 vol. 100% formamide (deionized) was prepared and 22.5 μL of this mix was added to each sample. Then the samples were mixed, spun briefly and incubated for one hour at 42° C. Finally, the slides prepared at step 3 were loaded with sample and incubated overnight at 42° C.
The slides prepared at step 4 were submerged in a 1×SSC/0.2% SDS solution at 55° C. (after cover slips were removed). The slides were then removed and placed in a cradle in a second tray containing the same solution. The slides were incubated in the second tray at room temperature for five minutes on an orbiting shaker. Next, the cradle and slides were transferred to a third tray containing a 0.1×SSC/0.2% SDS solution at 55° C. The cradle was dipped five times in the solution in the third tray and then incubated at room temperature for ten minutes. The slides were then transferred to a fourth tray containing the same solution as the third tray where the slides were again dipped five times and incubated at room temperature for five minutes. Next the cradle and slides were transferred to a fifth tray containing a 0.1×SSC solution at 55° C., where the cradle and slides were dipped five times. The cradle and slides were then transferred to a sixth tray containing the same solution as the fifth tray where the slides were again dipped five times. Finally, the cradle and slides were transferred to a seventh tray containing water, where the cradle and slides were dipped twice quickly and then dried in the cradle with nitrogen gas.
Microarrav Data Analysis Protocols for Comparative Genomic Hybridization of Genomic DNA of S. pneumoniae Strains
Two color images of hybridized slides were acquired with the microarray scanner ScanArray (PerkinElmer Life and Analytical Sciences; Shelton, Conn.) at 10 μm of resolution. Acquisition parameters (photomultiplier gain and laser power) were adjusted to maintain a comparable fluorescence level between the two fluorophores channels (measuring the intensity level of specific control spots and the mean intensity of all the spots).
Images were analyzed with the software Genepix 5.x (Molecular Devices Corp.; Sunnyvale, Calif.). Automatic and manually curated flagging procedures for no good quality spots were applied. From the acquired images, the Genepix software extracted the intensity levels and computed default statistical parameters for each feature spotted on the microarray slide.
Data management was performed by a local installation of BASE (BioArray Software Environment, rel 1.2.10, Lund University). The data analysis consisted of two phases:
(a) Background correction and normalization—In this procedure, the local background intensity was subtracted to each spot intensity. The intensities were then normalized to the mean or median intensity of all the spots. The background correction and the normalization procedure (plug-in rel. 1.4) were applied in BASE with the following parameters:
(b) Data merging—Intensity values of repeated spots of a single slide or of repeated slides were merged computing the mean intensities of the separated two color channels. Spots that were flagged for low quality level or for background correction were filtered out. The algorithm also evaluated the probability of having different intensity levels between the averages of the two colors intensities (applying a T-test, plug-in rel. 1.7). The resulting dataset was stored in the BASE application and could be exported for other analyses.
The ratio of the two color intensity levels was transformed in log 2 scale.
The computed ratios were organized in a table dataset containing a column for each slide or group of merged slides (experiments), and a row for each spot or group merged spots (genes). The table was uploaded in the software TMeV (rel, 3.1, TIGR) for the clustering analysis.
Three main clustering algorithms were applied:
(a) Hierarchical clustering—This algorithm was applied to discover groups of genes showing similar presence/absence patterns among different experiments. In particular the algorithm was applied with the following settings of parameters:
A similarity tree (based on the unweighted pair group method with arithmetic mean algorithm, UPGMA) and a similarity matrix (based on the distance metric selected) were built to represent graphically and numerically the distances between experiments and between genes.
(b) Pavlidis template matching—This algorithm was applied to discover genes showing presence/absence patterns similar to a template pattern. Pavlidis template matching was applied to discover genes showing patterns correlated or anti-correlated to a reference gene among different experiments. The algorithm was applied using the threshold parameters Absolute R (selects patterns that are either correlated or anti-correlated with the template), a threshold p-value of 0.05, Pearson default distance metrics, and hierarchical clustering of the elements in the resulting matched and unmatched groups.
(c) Significance analysis of microarrays—This algorithm was applied to discover significant genes showing presence/absence patterns that able to divide the experiments in separated groups (defined a priori). The algorithm was applied to discover genes that were specific of a given group of experiments. The algorithm was applied with the following parameters:
Statistical test: Two-class unpaired
Number of permutations: 100
S0: Tusher et al. method
Q-values: No
Imputation engine: K-nearest neighbour imputer
Hierarchical clustering of the elements in the resulting matched and unmatched groups
The determination of the presence of the pilus II island (INV104B) in the S. Pneumoniae strains listed in Table 1 was performed on purified genomic DNA or directly on bacteria (colony PCR). The four oligos used for amplification are listed in Table 2.
Three diagnostic PCRs were performed for each strain listed in Table 1 with the following PCR Oligo combinations: 1008for-1009rev; 1008for-intrev; and intfor-1009rev. Clones positive for the pilus II island (INV104B) were successfully amplified by 1008for-intrev and intfor-1009rev. Strains negative for the pilus II island (INV104B) were successfully amplified by 1008for-1009rev.
The determination of the presence of the pilus island, i.e., the rlrA islet (as opposed to the pilus II island (INV104B)), in the S. Pneumoniae strains listed in Table 1 was performed. Similarly to the pilus II island (INV104B), specific primers were designed and used to amplify defined regions of the rlrA islet. Examples of methods for determining the presence of the rlrA islet are set forth in U.S. application Ser. No. 11/707,433, entitled “Purification of Bacterial Antigen,” filed Feb. 16, 2007; and U.S. Provisional Application Ser. No. 60/774,450, entitled “Purification of Bacterial Antigen,” filed on Feb. 17, 2006, the contents of which are incorporated herein by reference in their entirety.
Table 1 indicates that a great number of S. pneumoniae strains contain one or both pilus and pilus II island (INV104B).
1. Sequence Download and Assembly
The preliminary sequences of four strains of S. pneumoniae were downloaded from the Sanger Web site (see worldwide web site “sanger.ac.uk/Projects/Microbes/”). The Sanger sequences are composed by a variable number of non-overlapping contigs. Details for the downloaded sequences are listed in Table 3:
To identify the likely ordering of the contigs, the sequences were aligned against the TIGR4 complete sequence using MUMmer3.19, to form a single pseudo-molecule. To separate two subsequent contigs the following sequence was inserted:
SEQ ID NO:28 was designed to (i) generate a stop codon in all six reading frames so that no gene is predicted across junctions and (ii) provide a start site in all frames, pointing toward contigs to predict incomplete genes at their extremities. Contigs that did not align against the TIGR4 genome were put at the end of the pseudochromosome.
2. Gene Prediction
The genes were predicted using the glimmer3.02 suite. The Hidden Markov Model was trained using the predicted genes of TIGR4 as a training set.
3. Protein Set Comparison
All the proteins of each genome were compared against all the proteins of the other genomes using Fasta. A protein was considered to be conserved if its aminoacidic sequence could be aligned with a percent identity of at least 90% on at least 50% of its length.
The amino acid sequences of OXC141, INV200, and 23F that did not meet this criteria are set forth below. These sequences may provide polypeptides and/or proteins that can be used in compositions or methods of treatment for, diagnosis of, and immunization against S. pneumoniae infections.
4. Start Codons
The N-terminal residues in the amino acid sequences listed herein are given as the amino acid encoded by the first codon in the corresponding nucleotide sequence. In those sequences in which the first codon is not ATG, it will be understood that the first codon will be translated as methionine when the codon is a start codon, but will be translated as the indicated non-Met amino acid when the sequence is at the C-terminus of a fusion partner. The listed sequences specifically disclose and encompass each of the amino acid sequences listed having a N-terminus methionine residue (e.g. a formyl methionine residue) in place of any indicated non-Met residue.
In some embodiments, preferred OXC141 antigens are selected from the polypeptides orf00045 (SEQ ID NO: 53), orf00068 (SEQ ID NO: 65), orf00074 (SEQ ID NO: 70), orf00223 (SEQ ID NO: 99), orf00229 (SEQ ID NO: 104), orf00360 (SEQ ID NO: 117), orf00506 (SEQ ID NO: 135), orf00781 (SEQ ID NO: 177), orf00785 (SEQ ID NO: 178), orf01068 (SEQ ID NO: 198), orf01446 (SEQ ID NO: 235), orf01447 (SEQ ID NO: 236), orf01449 (SEQ ID NO: 237), orf01455 (SEQ ID NO: 242), orf01460 (SEQ ID NO: 247), orf01461 (SEQ ID NO: 248), orf01463 (SEQ ID NO: 250), orf01464 (SEQ ID NO: 251), orf01466 (SEQ ID NO: 252), orf01467 (SEQ ID NO: 253), orf02661 (SEQ ID NO: 433), orf02690 (SEQ ID NO: 439), orf02698 (SEQ ID NO: 444), orf03318 (SEQ ID NO: 538), orf03320 (SEQ ID NO: 539), orf03322 (SEQ ID NO: 540), orf03323 (SEQ ID NO: 541), orf03324 (SEQ ID NO: 542), orf03325 (SEQ ID NO: 543), orf03326 (SEQ ID NO: 544), orf03327 (SEQ ID NO: 545), orf03562 (SEQ ID NO: 581), orf03660 (SEQ ID NO: 593), and immunogenic fragments thereof.
In some embodiments, preferred INV200 antigens are selected from the polypeptides orf00159 (SEQ ID NO: 626), orf00162 (SEQ ID NO: 628), orf00163 (SEQ ID NO: 629), orf00164 (SEQ ID NO: 630), orf00165 (SEQ ID NO: 631), orf00166 (SEQ ID NO: 632), orf00201 (SEQ ID NO: 639), orf00209 (SEQ ID NO: 645), orf01109 (SEQ ID NO: 747), orf01137 (SEQ ID NO: 751), orf01138 (SEQ ID NO: 752), orf01309 (SEQ ID NO: 783), orf01313 (SEQ ID NO: 786), orf011315 (SEQ ID NO: 787), orf01431 (SEQ ID NO: 810), orf01433 (SEQ ID NO: 812), orf01434 (SEQ ID NO: 813), orf01537 (SEQ ID NO: 824), orf01588 (SEQ ID NO: 831), orf011642 (SEQ ID NO: 842), orf01656 (SEQ ID NO: 847), orf01800 (SEQ ID NO: 875), orf01801 (SEQ ID NO: 876), orf01810 (SEQ ID NO: 879), orf01812 (SEQ ID NO: 880), orf01818 (SEQ ID NO: 882), orf01988 (SEQ ID NO: 913), orf01989 (SEQ ID NO: 914), orf02105 (SEQ ID NO: 925), orf02106 (SEQ ID NO: 926), orf02263 (SEQ ID NO: 947), orf02264 (SEQ ID NO: 948), orf02459 (SEQ ID NO: 968), orf02538 (SEQ ID NO: 987), orf02539 (SEQ ID NO: 988), orf02541 (SEQ ID NO: 990), orf02545 (SEQ ID NO: 992), orf02604 (SEQ ID NO: 1003), orf02608 (SEQ ID NO: 1007), orf02609 (SEQ ID NO: 1008), orf02850 (SEQ ID NO: 1036), orf03197 (SEQ ID NO: 1082), orf03439 (SEQ ID NO: 1120), orf03448 (SEQ ID NO: 1123), and immunogenic fragments thereof.
In some embodiments, preferred 23F antigens are selected from the polypeptides orf01158 (SEQ ID NO: 1297), orf01305 (SEQ ID NO: 1309), orf01307 (SEQ ID NO: 1311), orf01631 (SEQ ID NO: 1343), orf01804 (SEQ ID NO: 1362), orf01807 (SEQ ID NO: 1364), orf02164 (SEQ ID NO: 1434), orf02189 (SEQ ID NO: 1451), orf02194 (SEQ ID NO: 1455), orf02219 (SEQ ID NO: 1466), orf02221 (SEQ ID NO: 1467), orf02224 (SEQ ID NO: 1470), orf02228 (SEQ ID NO: 1474), orf02242 (SEQ ID NO: 1484), orf02244 (SEQ ID NO: 1485), orf02246 (SEQ ID NO: 1486), orf02247 (SEQ ID NO: 1487), orf02652 (SEQ ID NO: 1491), and immunogenic fragments thereof.
The pilus 2 mediates adhesion to alveolar epithelial cells A459. Dual-label staining shows that a pilus 2-positive strain adheres to the surface of the A549 cells and that the pilus (visualized using labeled anti-01287 antibody) is contacting the cells.
Furthermore, isogenic knockout mutants of the pilus are significantly impaired in host cell interaction.
Incubation of A459 cells grown on glass coverslips with 01287 purified protein shows low level binding by confocal microscopy inspection. This observation was confirmed by incubating the protein with cells in suspension and quantifying the level of adherence by FACS analysis (
Purified pilus was imaged by confocal microscopy. It was shown to adhere to A459 cells grown on glass coverslips. Furthermore, purified pilus seems to increase adherence to the respiratory cells when added to strains expressing the pilus type 2. This effect is probably due to the interaction of the purified pilus with both bacteria and A549 cells. Purified pilus does not increase adherence of isogenic knockout mutants of the pilus 2 to the respiratory cells (
An exemplary nucleic acid sequence for LepA Peptidase (orf 01289) is hereby provided:
An exemplary amino acid sequence for LepA Peptidase is hereby provided:
An exemplary nucleic acid sequence for sort-1 (orf01285) is hereby provided:
An exemplary amino acid sequence for sort-1 is hereby provided:
An exemplary nucleic acid sequence for sort-2 (orf01282) is hereby provided:
An exemplary amino acid sequence for sort-2 is hereby provided:
A number of embodiments of the inventive methods and compositions have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.
bT4;
cT4/c;
d6B;
e23F/c;
f23F
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/002108 | 5/23/2008 | WO | 00 | 5/16/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60940167 | May 2007 | US |
