WO1993010238A1 - Pneumococcal fimbrial protein a vaccines - Google Patents

Pneumococcal fimbrial protein a vaccines Download PDF

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Publication number
WO1993010238A1
WO1993010238A1 PCT/US1992/009522 US9209522W WO9310238A1 WO 1993010238 A1 WO1993010238 A1 WO 1993010238A1 US 9209522 W US9209522 W US 9209522W WO 9310238 A1 WO9310238 A1 WO 9310238A1
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Prior art keywords
pneumococcal
acid sequence
polypeptide
amino acid
seq
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PCT/US1992/009522
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French (fr)
Inventor
Harold Russell
Jean A. Tharpe
Jacquelyn Sampson
Steven P. O'connor
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The Government Of The United States Of America As Represented By The Department Of Health And Human Services
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Publication of WO1993010238A1 publication Critical patent/WO1993010238A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates, in general, to pneumococcal fimbrial protein A (PfpA) .
  • the present invention relates to a DNA segment encoding a pneumococcal fimbrial protein A gene (pfpA) ; polypeptides encoded by the DNA segment; recombinant DNA molecules conteining the DNA segment; cells containing the recombinant DNA molecule; a method of producing a pneumococcal fimbrial protein A polypeptide; antibodies specific to pneumococcal fimbrial protein A; and a method of measuring the amount of pneumococcal fimbrial protein A in a sample.
  • pfpA pneumococcal fimbrial protein A gene
  • Streptococcus pneumoniae is an important cause of morbidity and mortality in the United States and developing countries (Sorensen, J. et al. (1986) Scand. J. Infect. Dis. 18:329-335; Wall, R. A. et al. (1986) Bull. WHO 64-4:553-558; Walsh, J. A., and K. S. Warren (1979) N. Eng. J. Med. 301:967-974; Williams, W. W. et al. (1988) Ann. Intern. Med. 108:616-625; Yolken, R. H. et al. (1984) J. Clin. Microbiol. 20:802-805). Pneumococcal disease is very prevalent among the very young, the elderly, and immunocompromised
  • pfpA pneumococcal fimbrial protein A gene
  • pfpA pneumococcal fimbrial protein A gene
  • PfpA pneumococcal fimbrial protein A
  • the present invention relates to a DNA segment coding for a polypeptide comprising an a ino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
  • the present invention relates to a polypeptide free of proteins with which it is naturally associated and comprising an amino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
  • the present invention relates to a recombinant DNA molecule comprising a vector and a DNA segment that codes for a polypeptide comprising an amino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
  • the present invention relates to a cell that contains the above-described recombinant DNA molecule.
  • the present invention relates to a method of producing a polypeptide comprising an amino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
  • the present invention relates to an antibody having binding affinity to a polypeptide encoding a pneumococcal fimbrial protein A gene, or a unique portion thereof.
  • the present invention relates to a method of measuring the amount of pneumococcal fimbrial protein A in a sample, comprising contacting the sample with the above-described antibodies and measuring the amount of immunocomplexes formed between the antibodies and any pneumococcal fimbrial protein A in the sample.
  • FIG. 1 Immunoblot of S. pneumoniae whole-cell antigen preparations with pneumococcus MAbs. Protein standards (STD) (in kilodaltons) and different serotypes of S. pneumoniae are shown. Lanes: 1, serotype 3; 2, serotype 6B; 3, serotype 7F; 4, serotype 8; 5, serotype 9V; 6, serotype 10A; 7, serotype 11A; 8, serotype 12F; 9, serotype 15B; 10, serotype 19A; 11, serotype 19F; 12, serotype 22F. The MAbs revealed an antigen at 37 kDa (arrow) in all serotypes tested.
  • STD Protein standards
  • FIG. 1 Immunofluorescence assay staining of S. pneumoniae cells with pneumococcal MAbs.
  • FIG. 1 Transmission electron microscopy of S. pneumoniae R36A after embedding, cutting, reacting with Mabs, and staining with gold-labeled goat anti-mouse immunoglobulin.
  • the present invention relates to a DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to pneumococcal fimbrial protein A, or at least 5 contiguous amino acids thereof.
  • the DNA segment comprises the sequence shown in SEQ ID NO:l, allelic or species variation thereof, or at least 15 contiguous nucleotides thereof (preferably, at least 20, 30, 40, or 50 contiguous nucleotides thereof) .
  • the DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or atr least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
  • the present invention relates to a polypeptide free of proteins with which it is naturally associated or a polypeptide bound to a solid support and comprising an amino acid sequence corresponding to pneumococcal fimbrial protein A, or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or allelic or species variation thereof equivalent thereto (for example, immunologically or functionally, equivalent thereto) , or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
  • the present invention relates to a recombinant DNA molecule comprising a vector (for example plasmid or viral vector) and a DNA segment (as described above) coding for a polypeptide corresponding to pneumococcal fimbrial protein A, as described above.
  • a vector for example plasmid or viral vector
  • a DNA segment as described above
  • the encoding segment is present in the vector operably linked to a promoter.
  • the present invention relates to a cell containing the above described recombinant DNA molecule.
  • Suitable host cells include procaryotes (such as bacteria, including Ij . coli) and both lower eucaryotes (for example yeast) and higher eucaryotes (for example, mammalian cells) .
  • Introduction of the recombinant molecule into the cell can be effected using methods known in the art.
  • the present invention relates to a method of producing a polypeptide having an amino acid sequence corresponding to pneumococcal fimbrial protein A comprising culturing the above-described cell under conditions such that the DNA segment is expressed and the polypeptide thereby produced and isolating the polypeptide.
  • the present invention relates to an antibody having binding affinity for pneumococcal fimbrial protein A, or a unique portion thereof.
  • pneumococcal fimbrial protein A comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
  • Antibodies can be raised to pneumococcal fimbrial protein A, or unique portions thereof, in its naturally occurring form and in its recombinant form. Binding fragments of such antibodies are also within the scope of the invention.
  • Pneumococcal fimbrial protein A may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens.
  • Pneumococcal fimbrial protein A or its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See for example, Microbiology, Hoeber Medical Division (Harper and Row, 1969) , Landsteiner, Specificity of Serological Reactions (Dover Publications, New York, 1962) and Williams et al.. Methods in Immunology and Immunochemistry, Vol.
  • a typical method involves hyperim unization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin is isolated.
  • the present invention relates to a hybridoma which produces a monoclonal antibody or binding fragment thereof having binding affinity for pneumococcal fimbrial protein A.
  • the pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
  • the hybridoma comprises 1E7A3D7C2.
  • the present invention relates to a diagnostic kit comprising: i) at least one of the above-described monoclonal antibodies, and ii) a conjugate comprising a binding partner of said monoclonal antibody and a label.
  • the present invention relates to a diagnostic kit comprising a conjugate comprising: i) at least one of the above-described monoclonal antibodies, and ii) a label.
  • the present invention relates to a method of measuring the amount of pneumococcal fimbrial protein A in a sample, comprising contacting the sample with the above-described antibodies and measuring the amount of immunocomplexes formed between the antibodies and any pneumococcal fimbrial protein A in the sample. Methods of measuring the amount of immunocomplexes formed can be those well known in the art, such as RIA, ELISA, and direct and indirect immunoassays.
  • the present invention relates to vaccines comprising the pneumococcal fimbrial protein A and the above-identified polypeptides derived therefrom and to methods of immunizing mammals (e.g., humans) with the vaccines.
  • the presently used commercial vaccine, Pneumovax is a mixture of 23 capsular polysaccharides from S_____ pneumoniae.
  • the vaccine is efficacious in adults but not effective in children less than two years of age. Since the polypeptides of the present invention are proteins, they can be used to protect against pneumococcal disease in children and adults.
  • the pneumococcal fimbrial protein A and the polypeptides derived therefrom described above may be conjugated to components of existing commercial vaccines. Data indicate that some children less than two years of age produce antibodies to the pneumococcal fimbrial protein A (the 37-Kda protein) .
  • Anaerobic bacteria were obtained from V.R. Dowell, CDC. These included Bacteroides asaccharolyticus, B. fragili ⁇ , B. intermedius, B. thetaiotaomicron, Eubacterium lentum, Fusobacterium necrophorum, F. nucleatum, Pepto ⁇ treptococcus anaerobius, P. asaccharolyticus , Propionibacterium acnes, and Staphylococcu ⁇ ⁇ accharolyticus.
  • Branhamella catarrhalis and Bordetella parapertussis were obtained from R. Weaver, CDC. Mycobacterium tuberculosis was provided by R.C. Good, CDC. R.
  • mice Female BALB/c mice were immunized with whole cell suspensions of S. pneumoniae R36A, a rough derivative of the capsular type 2 strain D39 (Avery, O. T. et al. (1944) J. Exp. Med. 79:137-157). The mice were immunized by intravenous injection three times and intraperitoneal injection one time. The maximum number of cells injected at any time was 10 8 . Fusion was done on day 25 by using standard procedures (Clafin, L. , and K. Williams (1978) Curr. Top. Microbiol. Immunol. 81:107-109).
  • Spleen cells of 4 mice were fused with Sp2/0-Agl4 myeloma cells (Schulman, M. et al. (1978) Nature (London) 276:269-270). Culture fluids of the growing hybridomas were tested for antibodies to S. pneumoniae whole cells in an ELISA. A clone designated 1E7A3D7C2 was one of 10 selected for further study. Further references to Mabs in this article refer to hybridoma clone 1E7A3D7C2.
  • ELISA Screening of hybridoma culture supernatants was done by ELISA.
  • U-bottom microtitration plates (Costar, Cambridge, Mass.) were sensitized with 50 ⁇ l of S. pneumoniae whole cell suspension (10 9 CFU/ml) diluted 1:4,000 in 0.1M carbonate buffer, pH 9.6, and kept for 16 h at 4°C. The plates were washed 5 times with 0.9% NaCl containing 0.05% Tween 20 (NaCl-T) .
  • the reaction was stopped by adding 1 ml of 4 M H 2 S0 4 and the optical density was read on a Dynatech ELISA Reader (Dynatech Laboratories, Inc., Alexandria, Va.) at 450 nm. An optical density of >0.200 was considered positive.
  • SDS-PAGE SDS-PAGE and immunoblot analysis.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by the method of Tsang et al. (Tsang, V. C. W. et al. (1983) Methods Enzymol. 92:377-391), using an 8% acrylamide resolving gel.
  • Equal volumes of sample buffer (5% SDS-10% 2-mercaptoethanol-20% glycerol in 0.01 M Tris HCL, [Ph 8.0]) and cell suspension containing 2.4 ⁇ g protein per ⁇ l were mixed, heated at 100°C for 5 min, and a 5- ⁇ l portion was applied to 1 of 15 wells.
  • Protein concentrations were determined by the method of Markwell et al. (Markwell, M. A. et al. (1978) Anal. Biochem. 87:206-210), with BSA as the standard.
  • Proteins separated by SDS-PAGE were either silver stained by the method of Morrissey (Morrissey, J. H. (1981) Anal. Biochem. 117:307-310) or electroblotted onto nitrocellulose (Schleicher & Schnell, Inc., Keene, N.H.).
  • the immunoblot procedure was done according to the method of Tsang et al. (Tsang, V. C. W. et al. (1983) Methods Enzymol. 92:377-391) with slight modifications.
  • the blots were given three 5-min washes with PBS, pH 7.2, containing 0.3% Tween-20 and were gently agitated overnight (16 h) at 25°C.
  • the blots were blocked for 1 h with casein-thimerosal buffer (CTB) (Kenna, J. G. et al. (1985) J. Immunol. Meth. 85:409-419). After three rinses with CTB, the blots were exposed to goat anti-mouse immunoglobulin horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, Calif.) for 2 h at 25 ⁇ C. Conjugate dilutions (1:2,000) were made in CTB. The blots were again rinsed three times with CTB and exposed to
  • the cells were transferred to fresh Lowicryl K4M two times during the next 24-hour period.
  • the Lowicryl K4M-treated cells were imbedded in gelatin capsules, which were placed inside a box lined with aluminum foil.
  • the capsules were hardened by holding them,in the box, 35 cm from a short-wave UV light source for 72 h at -20°C.
  • the box was brought to room temperature, and the capsules were allowed to continue hardening for up to 14 days.
  • the grids were rinsed two times with PBS and incubated on droplets of goat anti-mouse IgG-labeled colloidal gold particles (20 ⁇ m) (E. Y. Laboratories) for 1 h at room temperature.
  • the grids were rinsed two times and poststained with osmium tetroxide, uranyl acetate, and lead citrate.
  • the grids were examined with a Philips 410 transmission electron microscope.
  • Hybridoma clone 1E7A3D7C2 produced MAbs that reacted with a 37-kilodalton (kDa) protein antigen (pneumococcal fimbrial protein A) found in S. pneumoniae.
  • the MAbs reacted with an antigen fractionated in SDS-PAGE, yielding a single immunoblot band. This indicates that the MAb reacted with epitopes found only on the 37-kDa antigen (pneumococcal fimbrial protein A) .
  • all pneumococcal strains tested (24 serotypes) were tested for antigens reacting with the MAbs.
  • the latter strains represented 19 genera and 36 species of bacteria. None of the strains tested reacted with the pneumococcal MAbs, thus yielding a specificity of 100%
  • FIG. 1 shows the bright immunofluorescence of whole pneumococcus cells stained by the MAbs and fluorescein-labeled anti-mouse immunoglobulin in the IFA. Results from both the ELISA and the IFA indicate that the antigen has exposed epitopes on the surface of the cell or that the immunoglobulin and other immunologic reagents are able to penetrate the pneumococcal cell walls.
  • FIG. 3 shows that the cells were typical of gram-positive cocci in the process of division.
  • the figure also shows the reaction of MAbs and colloidal gold-labeled anti-mouse immunoglobulin G with thin sections of whole pneumococcal cells.
  • a large portion of the antigen appears to be intracellular since there is no coating or layering of the labeled MAbs around the cell.
  • the large patch of colloidal gold staining indicates that the MAbs bound antigen located inside the cell wall. There was no colloidal gold binding to control pneumococci that were exposed to the MAbs against L. pneumophila .
  • EXAMPLE 2 Cloning of the Pneumococcal Fimbrial Protein A Gene Streptococcus pneumoniae DNA digested with restriction enzyme Sau3Al was ligated to BamHI digested pUC13 and transformed into E. coli TB1. Recombinant clones were identified by colony immunoblot using the 37-kDa monoclonal antibody.
  • the plas id pSTR3-l is an example of the pneumococcal imbrial protein A gene cloned into pUC13.
  • EXAMPLE 3 Preparation of Purified 37 kDa Protein Antigen Two methods for preparing the 37 kDa protein are used. (1) Streptococcus pneumoniae is conventionally cultured and the cells harvested. Purified 37 kDa protein antigen (pneumococcal fimbrial protein A) is isolated from the Streptococcus pneumoniae cell mass by extraction with a non-ionic detergent and further purified by ammonium sulfate fractionation and isoelectric focusing. (2) E. coli TB1 strains containing plasmid pSTR3-l is cultured conventionally and the cells harvested. For improved yields, E.
  • Purified 37 kDa protein antigen pneumococcal fimbrial protein A
  • E. coli TB1 strains containing plasmid pSTR3-l is cultured conventionally and the cells harvested. For improved yields, E.
  • coli strains transformed with an expression vector that carries a strong, regulated prokaryatic promoter and which contains the gene coding for the 37 kDa protein, is used.
  • Suitable expression vectors are those that contain a bacteriophage ⁇ P L promoter (e.g., pKK1773-3) , a hybrid trp-lac promoter (e.g., pET-3a) or a bacteriophage T7 promoter.
  • the 37 kDa protein (PfpA) is then extracted from the separated cell mass.
  • mice carrying the xid (x-linked immunodeficiency) mutation were used in this protection study. They were tested for protection against challenge with a virulent type 3 Streptococcus pneumoniae strain, WU2. Mice were anesthetized with Ketamine/Ro pun and bled infraorbitally to obtain pre-immunization sera. 37 kDa protein (pneumococcal fimbrial protein A) was emulsified in complete Freund's adjuvant (CFA) to a protein concentration of 54 ⁇ g per ml.
  • CFA complete Freund's adjuvant
  • mice were injected subcutaneously into 2 axillary and 2 inguinal sites at 0.1 ml per site, delivering approximately 22 ⁇ g protein/mouse.
  • Ten control mice were treated identically with CFA and buffer substituting for protein.
  • IP intraperitoneally
  • controls were injected IP with buffer.
  • All mice were bled infraorbitally to obtain post-immunization sera, and challenged intravenously (IV) with 60 CFU of a log phase culture of S. pneumoniae strain WU2, a virulent capsular type 3 strain. Mice were observed for 21 days, and deaths were recorded.
  • Sera were collected prior to immunizations to establish baseline exposures,and also following the full immunization protocol (but before challenge) in order to correlate circulating antibody to the 37 kDa protein with protection.
  • mice carrying the xid mutation were injected according to the following protocol:
  • mice All mice were bled prior to immunization to establish baseline immunity.
  • Ten test mice were immunized subcutaneously in four sites with a total of 21 ⁇ g of 37 kDa protein antigen (pneumococcal fimbrial protein A) emulsified in Complete Freund's adjuvant (CFA) .
  • CFA Complete Freund's adjuvant
  • Ten control mice were immunized identically with CFA and buffer substituting for the antigen.
  • mice were boosted intraperitoneally (I.P.) with 100 ⁇ g of the 37 kDa protein antigen (test mice) or with buffer (controls) . No adjuvant was used with this booster immunization.
  • mice Eight days later, all mice were bled via the infraorbital sinus and the sera were collected and pooled into the two groups (immunized and controls) . At the same time, blood was collected from individual mice to assay for antibody responses.
  • mice were injected I.O. with 0.1 ml of pooled immune sera to attempt to passively transfer immunity.
  • Three additional mice were injected I.P. with 0.1 ml of pooled control mouse sera. (Only five mice were injected at this step because of the small amount of sera obtained from the immunized mice.)
  • mice were challenged intravenously (I.V.) with 140 colony-forming units (CFU) of a mid-log phase pneumococcal type 3 strain, WU2.
  • I.P. intravenously
  • CFU colony-forming units
  • mice were challenged I.V.with the same culture of WU2.
  • ELISA enzyme-linked immunosorbent assay
  • PREPARATION OF THE 37 kDa PROTEIN OR POLYPEPTIDE CONJUGATE Conjugates can be prepared by use of a carrier protein bound to the 37 kDa protein or polypeptides derived from 37 the kDa protein via a linker, to elicit a T cell dependent response.
  • carrier proteins could be any immunogenic protein, for example, keyhole limpet hemocyanin, bovine serum albumin, tetanous toxoid, diphtheria toxoid, and bacterial outer membrane proteins.
  • bacterial outer membrane proteins, useful as conjugates include outer membrane proteins of Neis ⁇ eria meningitidis and Haemophilu ⁇ influenzae .
  • Nei ⁇ eria meningitidis can be an organism selected from Nei ⁇ eria meningitidis , group A, B, or C.
  • the 37 kDa protein or polypeptides thereof can be used in a conjugate where the 37 kDa protein or polypeptides thereof are the T-cell dependent immunogenic carrier for polysaccharide antigens that are B-cell stimulators.
  • polysaccharide antigens are B-cell stimulators and that protective immunity is usually generated by a combination of B-cell and T-cell stimulation.
  • Protein antigens exhibit T-cell dependent properties; i.e., booster and carrier priming. T-cell dependent stimulation is important because children less than two years of age do not respond to T-cell independent antigens.
  • the attachment or conjugation of antigens can be accomplished by conventional processes, such as those described in U.S. Patent No. 4,808,700, involving the addition of chemicals that enable the formation of covalent chemical bonds between the carrier immunogen and the immunogen.
  • the 37 kDa protein antigen of this invention can be administered to mammals; e.g., human, in a variety of ways.
  • exemplary methods include parenteral (subcutaneous) administration given with a nontoxic adjuvant, such as an alum precipitate or peroral administration given after reduction or ablation of gastric activity; or in a pharmaceutical form that protects the antigen against inactivation by gastric juice (e.g., a protective capsule or microsphere) .
  • the dose and dosage regimen will depend mainly upon whether the antigen is being administered for therapeutic or prophylactic purposes, the patient, and the patient's history.
  • the total pharmaceutically effective amount of antigen administered per dose will typically be in the range of about 2 ⁇ g to 50 ⁇ g per patient.
  • the antigen will generally be formulated in a unit dosage in ectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • ectable form solution, suspension, emulsion
  • parenteral vehicle Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles include water, saline. Ringer's solution, dextrose solution, and 5% human serum albumin. Non aqueous vehicles, such as fixed oils and ethyl oleate, may also be used. Liposomes may be used as vehicles.
  • the vehicle may contain minor amounts of additives, such as substances which enhance isotonicity and chemical stability; e.g., buffers and preservatives.
  • ADDRESSEE BIRCH, STEWART, KOLASCH &
  • ATC GAG CTC CAC AGT 419 lie Ala Gly Asp Lys lie Glu Leu His Ser 50 55

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Abstract

The present invention relates, in general, to pneumococcal fimbrial protein A. In particluar, the present invention relates to a DNA segment encoding a pneumococcal fimbrial protein A gene; polypeptides encoded by said DNA segment; recombinant DNA molecules containing the DNA segment; cells containing the recombinant DNA molecule; a method of producing a pneumococcal fimbrial protein A polypeptide; antibodies specific to pneumococcal fimbrial protein A; a method of measuring the amount of pneumococcal fimbrial protein A in a sample, and, vaccines containing pneumococcal fimbrial protein A or a polypeptide derived therefrom.

Description

PNEUMOCOCCAL FIMBRIAL PROTEIN A VACCINES CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-in-Part of Serial No. 07/791,377 filed September 17, 1991. The entire contents of the above application is hereby incorporated by reference.
BACKGROUND OP THE INVENTION Field of the Invention
The present invention relates, in general, to pneumococcal fimbrial protein A (PfpA) . In particular, the present invention relates to a DNA segment encoding a pneumococcal fimbrial protein A gene (pfpA) ; polypeptides encoded by the DNA segment; recombinant DNA molecules conteining the DNA segment; cells containing the recombinant DNA molecule; a method of producing a pneumococcal fimbrial protein A polypeptide; antibodies specific to pneumococcal fimbrial protein A; and a method of measuring the amount of pneumococcal fimbrial protein A in a sample.
Background Information
Disease caused by Streptococcus pneumoniae (pneumococcus) is an important cause of morbidity and mortality in the United States and developing countries (Sorensen, J. et al. (1986) Scand. J. Infect. Dis. 18:329-335; Wall, R. A. et al. (1986) Bull. WHO 64-4:553-558; Walsh, J. A., and K. S. Warren (1979) N. Eng. J. Med. 301:967-974; Williams, W. W. et al. (1988) Ann. Intern. Med. 108:616-625; Yolken, R. H. et al. (1984) J. Clin. Microbiol. 20:802-805). Pneumococcal disease is very prevalent among the very young, the elderly, and immunocompromised
1
SUBSTITUTE SHEET persons. Despite its prevalence, diagnosis of the disease continues to be a problem.
Several tests have been developed to detect pneumococσus antigens and/or antibodies as a means of diagnosing pneumococcus infections (Coonrod, J. D., and M. W. Rytel (1973)
J. Lab Clin. Med. 81:778-786; Holmberg, H. et al. (1985) J.
Clin. Microbiol. 22:111-115; Ingram, D. L. et al. (1983) J.
Clin. Microbiol. 18:1119-1121; Jalonen, E. et al. (1989) J.
Infect. 19:127-134; Kanclerski, K. et al. (1988) J. Clin.
Microbiol. 26-1:96-100; Makela, P. H. (1982) Scand.J. Infect.
Dis. Suppl. 36:111-113; Perlino, C. A. (1984) J. Infect. Dis.
150:139-144; Sippel, J. E. et al. (1984) J. Clin. Microbiol.
20:884-886; Whitby, M. et al. (1985) J. Clin. Pathol.
38:341-344; Yolken, . H. et al. (1984) J. Clin. Microbiol.
20:802-805). The sensitivity of existing antigen detection tests utilizing body fluids such as serum and urine, remains very low (Ajello, G. W. et al. (1987) J. Clin. Microbiol. 25:1388-1391; Anhalt, J. P., and P. K. W. Yu (1975) J. Clin. Microbiol. 2:510-515; Bartram, C. E. Jr. et al. (1974) J. Lab. Clin. Med. 83:591-598; Congeni, B. L. et al. (1984) Ped. Infect. Dis. 3:417-419; Coonrod, J. D. (1983) Proceedings of the American Journal of Medicine Symposium, July 28, 1983, Am. J. Med. 75:85-92; Coovadia, Y. B. and K. K. Naidu (1985) J. Clin. Pathol. 38:561-564; Dilworth, J. A. (1975) J. Clin. Microbiol. 2:453-455; Doskeland, S. O. , and B. P. Berdal (1980) J. Clin. Microbiol. 11:380-384; Martin, S. J. et al. (1987) J. Clin. Microbiol. 25:248-250), except for antigen detection in cerebrospinal fluids (Henrichsen, J. et al. (1980) J. Clin. Microbiol. 11:589-592; Ingram, D. L. et al. (1983) J. Clin. Microbiol. 18:1119-1121; Lenthe-Eboa, S. et al. (1987) Eur. J. Clin. Microbiol. 6:28-34; Tilton, R. C. et al. (1984) J. Clin. Microbiol. 20:231-234; Yolken, R. H. et al. (1984) J. Clin. Microbiol. 20:802-805). The measurement of antibody response to pneumolysin by enzyme immunoassay (ELISA) appears to be promising for presumptive etiologic diagnosis (Jalonen, E. et al. (1989) J. Infect. 19:127-134; Kalin, M. et al. (1987) J. Clin. Microbiol. 25:226-229; Kanclerski, K. et al. (1988) J. Clin. Microbiol. 26-1:96-100), but the sensitivity and specificity of the test need improvement.
Although a positive blood culture is diagnostic for pneumococcus disease, most patients with bacterial pneumonia do not have bacteremia (Austrian, R. (1974) Prev. Med. 3:443-445; Austrian, R., and I. Gold (1964) Ann. Intern. Med. 60:759-776; Kalin, M. and A. A. Lindberg (1983) Scand. J. Infect. Dis. 15:247-255). The value of sputum cultures has also been questioned because of contamination of the specimens with pharyngeal flora that can include pneumococci (Barrett-Cooner, E. (1971) Ann. Rev. Resp. Dis. 103:845-848). Thus, clinical laboratories are rarely successful in establishing a firm bacterial etiology for those patients with respiratory infections diagnosed presumptively as pneumococcus pneumonia. Researchers have been in constant search for immunodiagnostic markers or tests to aid in the early diagnosis of pneumococcus infections. SUMMARY OF THE INVENTION It is a general object of this invention to provide pneumococcal fimbrial protein A (PfpA) (a 37-kilodalton protein) .
It is a specific object of this invention to provide a DNA segment which encodes a pneumococcal fimbrial protein A gene (pfpA) .
It is a further object of the invention to provide a polypeptide corresponding to a pneumococcal fimbrial protein A gene (pfpA) .
It is another object of the invention to provide a recombinant DNA molecule comprising a vector and a DNA segment encoding a pneumococcal fimbrial protein A gene (pfpA) .
It is a further object of the invention to provide a cell that contains the above-described recombinant molecule.
It is another object of the invention to provide a method of producing a polypeptide encoding a pneumococcal fimbrial protein A gene (pfpA) .
It is a further object of the invention to provide antibodies having binding affinity to a pneumococcal fimbrial protein A gene (pfpA) , or a unique portion thereof.
It is a further object of the invention to provide a method of measuring the amount of pneumococcal fimbrial protein A in a sample.
It is an additional object of the invention to provide vaccines capable of providing protection against pneumococcal pneumonia containing a pharmaceutical effective amount of pneumococcal fimbrial protein A (PfpA) or polypeptides derived therefrom and methods of administrating those vaccines.
Further objects and advantages of the present invention will be clear from the description that follows.
In one embodiment, the present invention relates to a DNA segment coding for a polypeptide comprising an a ino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
In another embodiment, the present invention relates to a polypeptide free of proteins with which it is naturally associated and comprising an amino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
In a further embodiment, the present invention relates to a recombinant DNA molecule comprising a vector and a DNA segment that codes for a polypeptide comprising an amino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
In yet another embodiment, the present invention relates to a cell that contains the above-described recombinant DNA molecule.
In a further embodiment, the present invention relates to a method of producing a polypeptide comprising an amino acid sequence corresponding to a pneumococcal fimbrial protein A gene.
In yet another embodiment, the present invention relates to an antibody having binding affinity to a polypeptide encoding a pneumococcal fimbrial protein A gene, or a unique portion thereof.
In a further embodiment, the present invention relates to a method of measuring the amount of pneumococcal fimbrial protein A in a sample, comprising contacting the sample with the above-described antibodies and measuring the amount of immunocomplexes formed between the antibodies and any pneumococcal fimbrial protein A in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Immunoblot of S. pneumoniae whole-cell antigen preparations with pneumococcus MAbs. Protein standards (STD) (in kilodaltons) and different serotypes of S. pneumoniae are shown. Lanes: 1, serotype 3; 2, serotype 6B; 3, serotype 7F; 4, serotype 8; 5, serotype 9V; 6, serotype 10A; 7, serotype 11A; 8, serotype 12F; 9, serotype 15B; 10, serotype 19A; 11, serotype 19F; 12, serotype 22F. The MAbs revealed an antigen at 37 kDa (arrow) in all serotypes tested.
Figure 2. Immunofluorescence assay staining of S. pneumoniae cells with pneumococcal MAbs.
Figure 3. Transmission electron microscopy of S. pneumoniae R36A after embedding, cutting, reacting with Mabs, and staining with gold-labeled goat anti-mouse immunoglobulin.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the present invention relates to a DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to pneumococcal fimbrial protein A, or at least 5 contiguous amino acids thereof. In one preferred embodiment, the DNA segment comprises the sequence shown in SEQ ID NO:l, allelic or species variation thereof, or at least 15 contiguous nucleotides thereof (preferably, at least 20, 30, 40, or 50 contiguous nucleotides thereof) . In a further preferred embodiment, the DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or atr least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
In a further embodiment, the present invention relates to a polypeptide free of proteins with which it is naturally associated or a polypeptide bound to a solid support and comprising an amino acid sequence corresponding to pneumococcal fimbrial protein A, or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) . In one preferred embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or allelic or species variation thereof equivalent thereto (for example, immunologically or functionally, equivalent thereto) , or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
In another embodiment, the present invention relates to a recombinant DNA molecule comprising a vector (for example plasmid or viral vector) and a DNA segment (as described above) coding for a polypeptide corresponding to pneumococcal fimbrial protein A, as described above. In a preferred embodiment, the encoding segment is present in the vector operably linked to a promoter.
In a further embodiment, the present invention relates to a cell containing the above described recombinant DNA molecule. Suitable host cells include procaryotes (such as bacteria, including Ij . coli) and both lower eucaryotes (for example yeast) and higher eucaryotes (for example, mammalian cells) . Introduction of the recombinant molecule into the cell can be effected using methods known in the art.
In another embodiment, the present invention relates to a method of producing a polypeptide having an amino acid sequence corresponding to pneumococcal fimbrial protein A comprising culturing the above-described cell under conditions such that the DNA segment is expressed and the polypeptide thereby produced and isolating the polypeptide.
In yet another embodiment, the present invention relates to an antibody having binding affinity for pneumococcal fimbrial protein A, or a unique portion thereof. In one preferred embodiment, pneumococcal fimbrial protein A comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) .
Antibodies (monoclonal or polyclonal) can be raised to pneumococcal fimbrial protein A, or unique portions thereof, in its naturally occurring form and in its recombinant form. Binding fragments of such antibodies are also within the scope of the invention.
Pneumococcal fimbrial protein A may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens. Pneumococcal fimbrial protein A or its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See for example, Microbiology, Hoeber Medical Division (Harper and Row, 1969) , Landsteiner, Specificity of Serological Reactions (Dover Publications, New York, 1962) and Williams et al.. Methods in Immunology and Immunochemistry, Vol. 1 (Academic Press, New York, 1967) , for descriptions of methods of preparing polyclonal antisera. A typical method involves hyperim unization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin is isolated.
In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts. Description of techniques for preparing such monoclonal antibodies may be found in Stites et al., editors, Basic and Clinical Immunology, (Lange Medical Publications, Los Altos, CA, Fourth edition) and references cited therein, and in particular in Kohler and Milstein in Nature 256:495-497 (1975), which discusses one method of generating monoclonal antibodies. In another embodiment, the present invention relates to a hybridoma which produces a monoclonal antibody or binding fragment thereof having binding affinity for pneumococcal fimbrial protein A. In one preferred embodiment, the pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof) . In another preferred embodiment, the hybridoma comprises 1E7A3D7C2.
In yet another embodiment, the present invention relates to a diagnostic kit comprising: i) at least one of the above-described monoclonal antibodies, and ii) a conjugate comprising a binding partner of said monoclonal antibody and a label.
In a further embodiment, the present invention relates to a diagnostic kit comprising a conjugate comprising: i) at least one of the above-described monoclonal antibodies, and ii) a label. In a further embodiment, the present invention relates to a method of measuring the amount of pneumococcal fimbrial protein A in a sample, comprising contacting the sample with the above-described antibodies and measuring the amount of immunocomplexes formed between the antibodies and any pneumococcal fimbrial protein A in the sample. Methods of measuring the amount of immunocomplexes formed can be those well known in the art, such as RIA, ELISA, and direct and indirect immunoassays.
In another embodiment, the present invention relates to vaccines comprising the pneumococcal fimbrial protein A and the above-identified polypeptides derived therefrom and to methods of immunizing mammals (e.g., humans) with the vaccines. The presently used commercial vaccine, Pneumovax, is a mixture of 23 capsular polysaccharides from S_____ pneumoniae. The vaccine is efficacious in adults but not effective in children less than two years of age. Since the polypeptides of the present invention are proteins, they can be used to protect against pneumococcal disease in children and adults. In one preferred embodiment, the pneumococcal fimbrial protein A and the polypeptides derived therefrom described above, may be conjugated to components of existing commercial vaccines. Data indicate that some children less than two years of age produce antibodies to the pneumococcal fimbrial protein A (the 37-Kda protein) .
The present invention is described in further detail in the following non-limiting Examples.
EXAMPLES The following protocols and experimental details are referenced in the Examples that follow: Bacterial strains. The S. pneumoniae strain R36A was kindly provided by D.E. Briles (University of Alabama at Birmingham) . Twenty-four serotypes of S. pneumoniae were provided by R. Facklam, Centers for Disease Control (CDC) , Atlanta, Ga. These serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11F, 11A, 12F, 14, 15B, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Enterococcus avium, E. casseliflavus, and E. gallinarum were also provided by R. Facklam. Anaerobic bacteria were obtained from V.R. Dowell, CDC. These included Bacteroides asaccharolyticus, B. fragiliε, B. intermedius, B. thetaiotaomicron, Eubacterium lentum, Fusobacterium necrophorum, F. nucleatum, Peptoεtreptococcus anaerobius, P. asaccharolyticus , Propionibacterium acnes, and Staphylococcuε εaccharolyticus. Branhamella catarrhalis and Bordetella parapertussis were obtained from R. Weaver, CDC. Mycobacterium tuberculosis was provided by R.C. Good, CDC. R. Barnes, CDC, provided Chlamydia pneumoniae. The following remaining bacteria were from the stock collection of the Immunology Laboratory, CDC: Bordetella pertussis, Enterobacter aerogeneε, E. agglomerans, E. cloacae, E. gergoviae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae (types a-f) , Legionella micdadei, L. pneumophil , Mycoplasma pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, streptococcus agalactiae, s. eguisimilis, S. pyogenes, and group G streptococci. Production of Mabs. Female BALB/c mice were immunized with whole cell suspensions of S. pneumoniae R36A, a rough derivative of the capsular type 2 strain D39 (Avery, O. T. et al. (1944) J. Exp. Med. 79:137-157). The mice were immunized by intravenous injection three times and intraperitoneal injection one time. The maximum number of cells injected at any time was 108. Fusion was done on day 25 by using standard procedures (Clafin, L. , and K. Williams (1978) Curr. Top. Microbiol. Immunol. 81:107-109). Spleen cells of 4 mice were fused with Sp2/0-Agl4 myeloma cells (Schulman, M. et al. (1978) Nature (London) 276:269-270). Culture fluids of the growing hybridomas were tested for antibodies to S. pneumoniae whole cells in an ELISA. A clone designated 1E7A3D7C2 was one of 10 selected for further study. Further references to Mabs in this article refer to hybridoma clone 1E7A3D7C2.
ELISA. Screening of hybridoma culture supernatants was done by ELISA. U-bottom microtitration plates (Costar, Cambridge, Mass.) were sensitized with 50 μl of S. pneumoniae whole cell suspension (109 CFU/ml) diluted 1:4,000 in 0.1M carbonate buffer, pH 9.6, and kept for 16 h at 4°C. The plates were washed 5 times with 0.9% NaCl containing 0.05% Tween 20 (NaCl-T) . Culture supernatants (50 μl) from the fusion plates were added to 50 μl of a solution containing 2% bovine serum albumin (BSA) , 10% normal rabbit serum, 0.3% Tween-20, and 0.02% Merthiolate in phosphate buffered saline (PBS), Ph 7.2, (ELISA diluent) (Wells, D. E. et al. (1987) J. Clin. Microbiol. 25:516-521) in the plates and were incubated for 30 in at 37°C. The plates were washed 5 times with NaCl-T. Fifty microliters of goat anti-mouse immunoglobulin horseradish peroxidase conjugate, diluted in ELISA diluent was added to each well. The plates were incubated for 30 min at 37°C. The plates were washed, and 50 μl of 3,3',5,5'-tetramethylbenzidine (0.1 mg/ml in 0.1M sodium acetate, 0.1 M citric acid [Ph 5.7] with 0.005% hydrogen peroxide) was added to each well and incubated for 30 min at 37°C. The reaction was stopped by adding 1 ml of 4 M H2S04 and the optical density was read on a Dynatech ELISA Reader (Dynatech Laboratories, Inc., Alexandria, Va.) at 450 nm. An optical density of >0.200 was considered positive.
SDS-PAGE and immunoblot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Tsang et al. (Tsang, V. C. W. et al. (1983) Methods Enzymol. 92:377-391), using an 8% acrylamide resolving gel. Equal volumes of sample buffer (5% SDS-10% 2-mercaptoethanol-20% glycerol in 0.01 M Tris HCL, [Ph 8.0]) and cell suspension containing 2.4 μg protein per μl were mixed, heated at 100°C for 5 min, and a 5-μl portion was applied to 1 of 15 wells. If the final protein content of the portion of sample to be tested was <1.2μg/μl, a volume up to 10 μl of sample was applied to achieve a final concentration of 6 μg of protein per well. Protein concentrations were determined by the method of Markwell et al. (Markwell, M. A. et al. (1978) Anal. Biochem. 87:206-210), with BSA as the standard.
Proteins separated by SDS-PAGE were either silver stained by the method of Morrissey (Morrissey, J. H. (1981) Anal. Biochem. 117:307-310) or electroblotted onto nitrocellulose (Schleicher & Schnell, Inc., Keene, N.H.). The immunoblot procedure was done according to the method of Tsang et al. (Tsang, V. C. W. et al. (1983) Methods Enzymol. 92:377-391) with slight modifications. The blots were given three 5-min washes with PBS, pH 7.2, containing 0.3% Tween-20 and were gently agitated overnight (16 h) at 25°C. The blots were blocked for 1 h with casein-thimerosal buffer (CTB) (Kenna, J. G. et al. (1985) J. Immunol. Meth. 85:409-419). After three rinses with CTB, the blots were exposed to goat anti-mouse immunoglobulin horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, Calif.) for 2 h at 25βC. Conjugate dilutions (1:2,000) were made in CTB. The blots were again rinsed three times with CTB and exposed to
3-3'diaminobenzadine-4-hydrochloride in PBS, pH 7.2 (0.5mg/ml), with 0.003% H202 for 5 min at 25°C. Reactivity was expressed as a visible colored band on the nitrocellulose paper. Low- molecular-mass protein standards (Bio-Rad) were used in PAGE and immunoblotting. Rabbit antisera to the protein standards were used to develop the standards (Carlone, G. M. (1986) Anal. Biochem. 155:89-91). Molecular masses were calculated by the method of Neville and Glossman (Neville, D. M., and H. Glossman (1974) Methods Enzymol. 32:92-102) using appropriate molecular mass standards.
IFA. A bacterial suspension containing approximately 400-500 CFU per field (10 μl) was allowed to dry at room temperature on each well of acetone-resistant, 12-well (5 mm diameter) , glass slides (25 x 75 mm) (Cel-Line Associates, Newfield, N.J.). The slides were then immersed in acetone for 10 min and air dried at room temperature. Mabs were added to the slides, which were incubated for 30 min at 37°C. After incubation, the slides were gently rinsed with PBS and soaked twice at 5-min intervals, blotted on filter paper, and air dried at room temperature. Fluorescein-labeled rabbit anti-mouse immunoglobulin (courtesy of W. F. Bibb, CDC) was then added, and the slides were incubated for 30 min at 37°C. They were then washed twice with PBS and gently blotted on filter paper. Slides were covered with carbonate-buffered mounting fluid, Ph 9.0, and cover slips and were then read with a Leitz Dialux 20 fluorescence microscope equipped with a HBO-100 mercury incident light source, an I cube filter system, a 4Ox dry objective lens, and 6.3 binoculars (E. Leitz, Inc., Rockleigh, N.J.).
Immunoelectron microscopy. Pneumococcal cells were washed two times with PBS and fixed in a mixture of 1% paraformaldehyde-0.1% glutaraldehyde (freshly made) for 20 min at 4°C. The cells were dehydrated in a graded alcohol series and then in a 1:1 mixture of absolute ethanol and Lowicryl K4M (Ladd Research Industries, Inc. ,Burlington, Vt.) for 1 h at 4°C. The cells were pelleted and suspended in a 1:2 mixture of absolute ethanol and Lowicryl K4M for 1 h at 4°C. They were again pelleted and suspended in Lowicryl K4M (undiluted) for 16 h at 4βC.
The cells were transferred to fresh Lowicryl K4M two times during the next 24-hour period. The Lowicryl K4M-treated cells were imbedded in gelatin capsules, which were placed inside a box lined with aluminum foil. The capsules were hardened by holding them,in the box, 35 cm from a short-wave UV light source for 72 h at -20°C. The box was brought to room temperature, and the capsules were allowed to continue hardening for up to 14 days.
Samples of the capsule were cut into 100-μm thin sections and picked up on nickel grids. Grids containing the sample were placed on a droplet of ovalbumin solution in PBS containing sodium azide (E. Y. Laboratories, Inc., San Mateo, Calif.) for 5 min. The grids (wet) were transferred to a solution of primary Mabs diluted in a solution of BSA reagent (1% BSA in PBS containing 0.1% Triton X-100, Tween 20, and sodium azide) (E. Y. Laboratories) and incubated for 1 h at room temperature or 18 to 48 h at 4°C in a moist chamber. For antibody binding controls, other grids were wetted with Mabs against Legionella pneumophila . The grids were rinsed two times with PBS and incubated on droplets of goat anti-mouse IgG-labeled colloidal gold particles (20 μm) (E. Y. Laboratories) for 1 h at room temperature. The grids were rinsed two times and poststained with osmium tetroxide, uranyl acetate, and lead citrate. The grids were examined with a Philips 410 transmission electron microscope.
CBA/CaHN/J Mice. X-linked immune deficiency (xid) of CBA/N mice as prepared by Wicker, L. S. and I. Seher, Curr. Top. Microbiol. Immunol. 124:86-101 were used to study the protection afforded by the 37 kDa protein.
EXAMPLE 1 Monoclonal Antibodies Hybridoma clone 1E7A3D7C2 produced MAbs that reacted with a 37-kilodalton (kDa) protein antigen (pneumococcal fimbrial protein A) found in S. pneumoniae. The MAbs reacted with an antigen fractionated in SDS-PAGE, yielding a single immunoblot band. This indicates that the MAb reacted with epitopes found only on the 37-kDa antigen (pneumococcal fimbrial protein A) . The MAbs produced by the immunization of mice with pneumococcal cells reacted with all pneumococcal strains tested (24 serotypes) to yield a sensitivity of 100%. For specificity, 55 different nonpneumococcal strains of bacteria that can also cause respiratory infections (Donowitz, G. R., and G. L. Mandell (1985) In: Principles and practices in infectious diseases, 2nd ed. (G.L. Mandell, R.G. Douglas, and J.E. Bennett, ed.) John Wiley & Sons, Inc., New York, pp.394-404) were tested for antigens reacting with the MAbs. The latter strains represented 19 genera and 36 species of bacteria. None of the strains tested reacted with the pneumococcal MAbs, thus yielding a specificity of 100%
Of 44 patients known to have pneumococcus disease, 34 (77%) had antibodies that reacted with the 37-kDa antigen (pneumococcal fimbrial protein A) by Western immunoblot
(Fig 1) .
The MAbs reacted with whole pneumococcal cells to yield a positive test result in both the ELISA and IFA. Figure 2 shows the bright immunofluorescence of whole pneumococcus cells stained by the MAbs and fluorescein-labeled anti-mouse immunoglobulin in the IFA. Results from both the ELISA and the IFA indicate that the antigen has exposed epitopes on the surface of the cell or that the immunoglobulin and other immunologic reagents are able to penetrate the pneumococcal cell walls.
Several strains of group A streptococci were tested for immunofluorescence after reacting with the pneumococcus MAbs. None of the heterologous bacterial cells fluoresced in this test, indicating that the IFA reaction was specific for pneumococcus cells.
To further determine the location on the cell of the 37-kDa antigen (pneumococcal fimbrial protein A) epitopes reacting with the MAbs, immunolabeling experiments were performed. Figure 3 shows that the cells were typical of gram-positive cocci in the process of division. The figure also shows the reaction of MAbs and colloidal gold-labeled anti-mouse immunoglobulin G with thin sections of whole pneumococcal cells. A large portion of the antigen appears to be intracellular since there is no coating or layering of the labeled MAbs around the cell. The large patch of colloidal gold staining indicates that the MAbs bound antigen located inside the cell wall. There was no colloidal gold binding to control pneumococci that were exposed to the MAbs against L. pneumophila .
EXAMPLE 2 Cloning of the Pneumococcal Fimbrial Protein A Gene Streptococcus pneumoniae DNA digested with restriction enzyme Sau3Al was ligated to BamHI digested pUC13 and transformed into E. coli TB1. Recombinant clones were identified by colony immunoblot using the 37-kDa monoclonal antibody. The plas id pSTR3-l is an example of the pneumococcal imbrial protein A gene cloned into pUC13.
EXAMPLE 3 Preparation of Purified 37 kDa Protein Antigen Two methods for preparing the 37 kDa protein are used. (1) Streptococcus pneumoniae is conventionally cultured and the cells harvested. Purified 37 kDa protein antigen (pneumococcal fimbrial protein A) is isolated from the Streptococcus pneumoniae cell mass by extraction with a non-ionic detergent and further purified by ammonium sulfate fractionation and isoelectric focusing. (2) E. coli TB1 strains containing plasmid pSTR3-l is cultured conventionally and the cells harvested. For improved yields, E. coli strains, transformed with an expression vector that carries a strong, regulated prokaryatic promoter and which contains the gene coding for the 37 kDa protein, is used. Suitable expression vectors are those that contain a bacteriophage λPL promoter (e.g., pKK1773-3) , a hybrid trp-lac promoter (e.g., pET-3a) or a bacteriophage T7 promoter. The 37 kDa protein (PfpA) is then extracted from the separated cell mass.
PROTECTION EXPERIMENTS WITH 37 kDa PROTEIN Experiment No. 1
Twenty CBA/CaHN/J mice carrying the xid (x-linked immunodeficiency) mutation were used in this protection study. They were tested for protection against challenge with a virulent type 3 Streptococcus pneumoniae strain, WU2. Mice were anesthetized with Ketamine/Ro pun and bled infraorbitally to obtain pre-immunization sera. 37 kDa protein (pneumococcal fimbrial protein A) was emulsified in complete Freund's adjuvant (CFA) to a protein concentration of 54 μg per ml. Ten mice were injected subcutaneously into 2 axillary and 2 inguinal sites at 0.1 ml per site, delivering approximately 22 μg protein/mouse. Ten control mice were treated identically with CFA and buffer substituting for protein. Fourteen days later, the ten test mice were injected intraperitoneally (IP) with 100 μg of the 37 kDa protein; controls were injected IP with buffer. Eight days following the IP immunizations, all mice were bled infraorbitally to obtain post-immunization sera, and challenged intravenously (IV) with 60 CFU of a log phase culture of S. pneumoniae strain WU2, a virulent capsular type 3 strain. Mice were observed for 21 days, and deaths were recorded.
Sera were collected prior to immunizations to establish baseline exposures,and also following the full immunization protocol (but before challenge) in order to correlate circulating antibody to the 37 kDa protein with protection.
Days post challenge: 1—no deaths
2—3 control mice dead
3—2 control mice dead
4—2 control mice dead, one sick
5—1 control mouse dead
6-21 no deaths
Immunized with 37 kDa protein: 10/10 survived
Controls with no protein: 2/10 survived (8/10 died)
Difference statistically significant: (p=0.0008) Rank sum test
Experiment No. 2
Twenty CBA/CaHΝ/J mice carrying the xid mutation were injected according to the following protocol:
1. All mice were bled prior to immunization to establish baseline immunity. Ten test mice were immunized subcutaneously in four sites with a total of 21 μg of 37 kDa protein antigen (pneumococcal fimbrial protein A) emulsified in Complete Freund's adjuvant (CFA) . Ten control mice were immunized identically with CFA and buffer substituting for the antigen.
2. Fourteen days later, the mice were boosted intraperitoneally (I.P.) with 100 μg of the 37 kDa protein antigen (test mice) or with buffer (controls) . No adjuvant was used with this booster immunization.
3. Eight days later, all mice were bled via the infraorbital sinus and the sera were collected and pooled into the two groups (immunized and controls) . At the same time, blood was collected from individual mice to assay for antibody responses.
4. One day later, two additional mice were injected I.O. with 0.1 ml of pooled immune sera to attempt to passively transfer immunity. Three additional mice were injected I.P. with 0.1 ml of pooled control mouse sera. (Only five mice were injected at this step because of the small amount of sera obtained from the immunized mice.)
5. One hour after the I.P. injections, these five mice were challenged intravenously (I.V.) with 140 colony-forming units (CFU) of a mid-log phase pneumococcal type 3 strain, WU2.
6. At the same time, the eighteen (8 test and 10 control)* mice were challenged I.V.with the same culture of WU2.
7. Deaths were tallied daily.
RESULTS: No. Dead/No. Challenged
Immunized with the 37 kDa protein: 0/8* Control mice: 10/10
Passive Protection:
Mice receiving immune sera: 0/2
Mice receiving control sera: 3/3
*Two of ten test mice died of other causes prior to challenged with WU2. Mice immunized with the 37 kDa protein were protected from fatal challenge with strain WU2, and this immunity could be passively transferred with sera from immunized mice.
Experiment No. 3
An enzyme-linked immunosorbent assay (ELISA) was developed using purified S. pneumoniae 37 kDa protein antigen as a capture for human antibodies. Paired sera were tested from children, less than 24 months of age, known to have pneumococcal pneumonia. Disease confirmation was determined by blood culture or antigen in the urine. It was found that 35% (9/26) had antibody titers greater than sera from non-ill children of the same age group, p=0.06. This illustrates that some of the children responded to the 37 kDa protein antigen after natural infection.
PREPARATION OF THE 37 kDa PROTEIN OR POLYPEPTIDE CONJUGATE Conjugates can be prepared by use of a carrier protein bound to the 37 kDa protein or polypeptides derived from 37 the kDa protein via a linker, to elicit a T cell dependent response. Such carrier proteins could be any immunogenic protein, for example, keyhole limpet hemocyanin, bovine serum albumin, tetanous toxoid, diphtheria toxoid, and bacterial outer membrane proteins. Examples of bacterial outer membrane proteins, useful as conjugates, include outer membrane proteins of Neisεeria meningitidis and Haemophiluε influenzae . Neiεεeria meningitidis can be an organism selected from Neiεεeria meningitidis , group A, B, or C.
In addition, the 37 kDa protein or polypeptides thereof can be used in a conjugate where the 37 kDa protein or polypeptides thereof are the T-cell dependent immunogenic carrier for polysaccharide antigens that are B-cell stimulators. This is based on the theory that polysaccharide antigens are B-cell stimulators and that protective immunity is usually generated by a combination of B-cell and T-cell stimulation. Protein antigens exhibit T-cell dependent properties; i.e., booster and carrier priming. T-cell dependent stimulation is important because children less than two years of age do not respond to T-cell independent antigens. The attachment or conjugation of antigens can be accomplished by conventional processes, such as those described in U.S. Patent No. 4,808,700, involving the addition of chemicals that enable the formation of covalent chemical bonds between the carrier immunogen and the immunogen.
In use, the 37 kDa protein antigen of this invention can be administered to mammals; e.g., human, in a variety of ways. Exemplary methods include parenteral (subcutaneous) administration given with a nontoxic adjuvant, such as an alum precipitate or peroral administration given after reduction or ablation of gastric activity; or in a pharmaceutical form that protects the antigen against inactivation by gastric juice (e.g., a protective capsule or microsphere) . The dose and dosage regimen will depend mainly upon whether the antigen is being administered for therapeutic or prophylactic purposes, the patient, and the patient's history. The total pharmaceutically effective amount of antigen administered per dose will typically be in the range of about 2μg to 50μg per patient.
For parental administration, the antigen will generally be formulated in a unit dosage in ectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles include water, saline. Ringer's solution, dextrose solution, and 5% human serum albumin. Non aqueous vehicles, such as fixed oils and ethyl oleate, may also be used. Liposomes may be used as vehicles. The vehicle may contain minor amounts of additives, such as substances which enhance isotonicity and chemical stability; e.g., buffers and preservatives.
All publications and patents mentioned herein above are hereby incorporated in their entirety by reference. Additionally, Russell et al. (Oct. 1990), J. of Clin. Microbiol. 28:2191-2195 is hereby incorporated in its entirety by reference.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT: Russell, Harold
(ii) TITLE OF INVENTION: PNEUMOCOCCAL FIMBRIAL PROTEIN A
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BIRCH, STEWART, KOLASCH &
BIRCH
(B) STREET: P.O. BOX 747
(C) CITY: FALLS CHURCH (D) STATE: VA
(E) COUNTRY: USA
(F) ZIP: 22040-0747
(V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version
#1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GERALD M. MURPHY, JR.
(B) REGISTRATION NUMBER: 28,977
(C) REFERENCE/DOCKET NUMBER: 1173-379P
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (703) 241-1300
28
SUBSTITUTESHEET (B) TELEFAX: (703) 241-0369
(C) TELEX: 248345
(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1175 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 243..1172
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CTCATCACAC CCGCTGCGAC AGCCTATCTC TATGCCAATA 40
GCCTCTGGTC CATGATGCTC CTTTCATCCG GATTAGGTGC 80
CCTAGCCTCT ATCCTAGGAC TCTTTATCGG CTACAGTTTC 120 AACATCGCCG TCGGGTCTTG TATCGTCCTC ACTTCTGCCA 160
TCTTCTTTCT CATCAGCTTC TTTATCGCTC CTAAGCAGAG 200
AAAGAATAAG CACGCTCTTT CACCTCATTA AAGGAGAAAC 240
AC ATG AAA AAA ATC GCT TCT GTC CTC GCC 269
Met Lys Lys lie Ala Ser Val Leu Ala 1 5
CTC TTT GTG GCG CTC TTG TTC GGC CTG TTG 299
Leu Phe Val Ala Leu Leu Phe Gly Leu Leu
10 15
GCC TGC AGC AAA GGC ACT TCT TCC AAG TCC 329
Ala Cys Ser Lys Gly Thr Ser Ser Lys Ser
20 25 TCA TCC GAT AAA TTG AAG GTG GTT ACC ACC 359
Ser Ser Asp Lys Leu Lys Val Val Thr Thr 30 35
AAC TCC ATC CTT GCC GAT ATC ACC AAA AAT 389 Asn Ser lie Leu Ala Asp lie Thr Lys Asn 40 45
ATC GCT GGG GAT AAA ATC GAG CTC CAC AGT 419 lie Ala Gly Asp Lys lie Glu Leu His Ser 50 55
29
SUBSTITUTE SHEET ATT GTA CCT GTC GGT CAA GAT CCC CAC GAG 449
He Val Pro Val Gly Gin Asp Pro His Glu 60 65
30
SUBSTITUTE SHEET TAC GAA CCG CTC CCA GAA GAT GTC AAA AAA 479
Tyr Glu Pro Leu Pro Glu Asp Val Lys Lys 70 75
ACT TCA CAA GCA GAC CTG ATC TTC TAC AAT 509
Thr Ser Gin Ala Asp Leu He Phe Tyr Asn 80 85 GGG ATC AAC CTC GAA ACG GGT GGC AAT GCT 539
Gly He Asn Leu Glu Thr Gly Gly Asn Ala 90 95
TGG TTT ACC AAA TTG GTC AAA AAT GCC AAT 579 Trp Phe Thr Lys Leu Val Lys Asn Ala Asn 100 105
AAA GTA GAA AAC AAG GAC TAT TTC GCT GCC 609
Lys Val Glu Asn Lys Asp Tyr Phe Ala Ala 110 115
AGC GAT GGC GTA GAG GTC ATC TAC CTG GAA 639
Ser Asp Gly Val Glu Val He Tyr Leu Glu 120 125
GGC CAA AAC CAA GCT GGA AAA GAA GAC CCT 669
Gly Gin Asn Gin Ala Gly Lys Glu Asp Pro 130 135 CAC GCT TGG CTC AAT CTC GAA AAC GGG ATT 699
His Ala Trp Leu Asn Leu Glu Asn Gly He 140 145
ATC TAC GCT AAA AAC ATT GCC AAA CAA TTA 729 He Tyr Ala Lys Asn He Ala Lys Gin Leu 150 155
ATC GCC AAA GAT CCA AAA AAT AAG GAC TTC 759
He Ala Lys Asp Pro Lys Asn Lys Asp Phe 160 165
TAC GAA AAA AAT CTA GCA GCC TAC ACT GAA 789
Tyr Glu Lys Asn Leu Ala Ala Tyr Thr Glu 170 175
AAA CTC AGC AAG CTA GAC CAA GAA GCC AAG 819
Lys Leu Ser Lys Leu Asp Gin Glu Ala Lys 180 185 CAA GCA TTC AAT AAC ATC CCA GCA GAG AAG 849
Gin Ala Phe Asn Asn He Pro Ala Glu Lys 190 195
AAG ATG ATC GTA ACC AGC GAA GGT TGC TTC 879 Lys Met He Val Thr Ser Glu Gly Cys Phe 200 205
31
SUBSTITUTE SHEET AAG TAC TTC TCC AAA GCC TAC GGC GTC CCA 909
Lys Tyr Phe Ser Lys Ala Tyr Gly Val Pro 210 215
TCT GCC TAT ATC TGG GAA ATC AAC ACT GAA 939
Ser Ala Tyr He Trp Glu He Asn Thr Glu 220 225 GTA GAA GGG ACA CCT GAA CAA ATC AAA ACG 969
Val Glu Gly Thr Pro Glu Gin He Lys Thr 230 235
CTG CTA GAG AAA TTG CGT CAA ACC AAA GTA 999 Leu Leu Glu Lys Leu Arg Gin Thr Lys Val 240 245
CCG TCC CTC TTT GTC GAA TCC AGT GTC GAT 1029
Pro Ser Leu Phe Val Glu Ser Ser Val Asp 250 255
GAG CGT CCT ATG AAA ACT GTG TCT AAG GAT 1059
Glu Arg Pro Met Lys Thr Val Ser Lys Asp 260 265
AGC AAT ATC CCT ATC TTT GCA AAG ATC TTT 1089
Ser Asn He Pro He Phe Ala Lys He Phe 270 275 ACT GAC TCG ATT GCC AAA GAA GGC GAA GAA 1119
Thr Asp Ser He Ala Lys Glu Gly Glu Glu 280 285
GGC GAC AGC TAC TAC AGC ATG ATG AAA TGG 1149 Gly Asp Ser Tyr Tyr Ser Met Met Lys Trp 290 295
AAT TTG GAG AAA ATC GCA GAA GGT TTG AAC 1179
Asn Leu Glu Lys He Ala Glu Gly Leu Asn 300 305
AAA TAA 1185
Lys
310
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 310 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
32
SUBSTITUTESHEET ( i) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :
Met Lys Lys He Ala Ser Val Leu Ala Leu
1 5 10
Phe Val Ala Leu Leu Phe Gly Leu Leu Ala
15 20
Cys Ser Lys Gly Thr Ser Ser Lys Ser Ser 25 30
Ser Asp Lys Leu Lys Val Val Thr Thr Asn
35 40 Ser He Leu Ala Asp He Thr Lys Asn He
45 50
Ala Gly Asp Lys He Glu Leu His Ser He
55 60
Val Pro Val Gly Gin Asp Pro His Glu Tyr
65 70
Glu Pro Leu Pro Glu Asp Val Lys Lys Thr
75 80
Ser Gin Ala Asp Leu He Phe Tyr Asn Gly
85 90 He Asn Leu Glu Thr Gly Gly Asn Ala Trp
95 100
Phe Thr Lys Leu Val Lys Asn Ala Asn Lys
105 110
Val Glu Asn Lys Asp Tyr Phe Ala Ala Ser
115 120
Asp Gly Val Glu Val He Tyr Leu Glu Gly 125 130
Gin Asn Gin Ala Gly Lys Glu Asp Pro His
135 140 Ala Trp Leu Asn Leu Glu Asn Gly He He
145 150
Tyr Ala Lys Asn He Ala Lys Gin Leu He
155 160
Ala Lys Asp Pro Lys Asn Lys Asp Phe Tyr
165 170
Glu Lys Asn Leu Ala Ala Tyr Thr Glu Lys 175 180
33
SUBSTITUTE SHEET Leu Ser Lys Leu Asp Gin Glu Ala Lys Gin
185 190
Ala Phe Asn Asn He Pro Ala Glu Lys Lys 195 200
Met He Val Thr Ser Glu Gly Cys Phe Lys
205 210 Tyr Phe Ser Lys Ala Tyr Gly Val Pro Ser
215 220
Ala Tyr He Trp Glu He Asn Thr Glu Val
225 230
Glu Gly Thr Pro Glu Gin He Lys Thr Leu
235 240
Leu Glu Lys Leu Arg Gin Thr Lys Val Pro
245 250
Ser Leu Phe Val Glu Ser Ser Val Asp Glu
255 260 Arg Pro Met Lys Thr Val Ser Lys Asp Ser
265 270
Asn He Pro He Phe Ala Lys He Phe Thr
275 280
Asp Ser He Ala Lys Glu Gly Glu Glu Gly
285 290
Asp Ser Tyr Tyr Ser Met Met Lys Trp Asn 295 300
Leu Glu Lys He Ala Glu Gly Leu Asn Lys
305 310
34
SUBSTITUTESHEET

Claims

WHAT IS CLAIMED IS:
1. A vaccine capable of providing protection against pneumococcal pneumonia comprising a pharmaceutically effective amount of pneumococcal fimbrial protein A (PfpA) or a polypeptide derived therefrom in pharmaceutically acceptable excipients.
2. A vaccine according to claim 1, wherein the pneumococcal fimbrial protein A or a polypeptide derived from the pneumococcal fimbrial protein A is part of a conjugate with a carrier protein.
3. A vaccine according to claim 2, wherein the carrier protein is selected from the group consisting of keyhole limpet of hemocyanin, bovine serum albumin, a toxoid and a bacterial outer membrane protein.
4. A vaccine according to claim 3, wherein the bacterial outer membrane is selected from the group consisting of outer membrane proteins of Neiεεeria meningitidis and Haemophilus influenzae.
5. A vaccine according to claim 1, additionally containing pharmaceutically acceptable adjuvants.
6. A vaccine according to claim 1, wherein the pneumococcal fimbrial protein A or a polypeptide derived therefrom is a carrier conjugated to a polysaccharide antigen.
7. A vaccine according to claim 3, wherin the toxoid is a member selected from the group consisting of tetanus toxoid and diphtheria toxoid.
8. A vaccine according to claim 1, which is capable of eliciting a combination of B-cell and T-cell stimulation.
9. A method of immunizing a mammal against pneumococcal pneumonia by administrating to said mammal a therapeutically effective amount of the vaccine of claim 1.
10. A method of immunizing a mammal against pneumococcal pneumonia by administrating to said mammal a therapeutically effective amount of the vaccine of claim 2.
11. A method of immunizing according to claim 9, wherein the mammal is a human.
12. A method of immunizing according to claim 10, wherein the mammal is a human.
13. A method of immunizing a mammal against pneumococcal pneumonia by administrating to said mammal a therapeutically effective amount of the vaccine of claim 6.
14. A method of immunizing according to claim 13, wherein the mammal is a human.
15. A method of immunizing a mammal against pneumococcal pneumonia by administrating to said mammal a therapeutically effective amount of the vacicne of claim 7.
16. A method of immunizing according to claim 15, wherein the mammal is human.
17. A method of immunizing a mammal against pneumococcal pneumonia by administrating to said mammal a therapeutically effective amount of the vaccine of claim 8.
18. A method of immunizing according to claim 17, wherein the mammal is human.
19. A DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to pneumococcal fimbrial protein A, or at least 5 contiguous amino acids thereof.
20. The DNA segment according to claim 1, wherein said DNA segment comprises the sequence shown in SEQ ID NO:l, allelic or species variation thereof, or at least 15 contiguous nucleotides thereof.
21. The DNA segment according to claim 2, wherein said DNA segment has the sequence shown in SEQ ID NO:l, allelic or species variation thereof.
22. The DNA segment according to claim 3, wherein said DNA segment has the sequence shown in SEQ ID NO:l.
23. The DNA segment according to claim 1, wherein said DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof.
24. The DNA segment according to claim 5, wherein said DNA segment encodes tfre amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof.
25. The DNA segment according to claim 6, wherein said DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2.
26. A polypeptide free of proteins with which it is naturally associated and comprising an amino acid sequence corresponding to pneumococcal fimbrial protein A, or at least 5 contiguous amino acids thereof.
27. The polypeptide according to claim 8, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof.
28. The polypeptide according to claim 9, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof.
29. The polypeptide according to claim 10, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
30. A polypeptide bound to a solid support and comprising an amino acid sequence corresponding to pneumococcal fimbrial protein A.
31. The polypeptide according to claim 12, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof.
32. The polypeptide according to claim 13, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof.
33. The polypeptide according to claim 14, wherein εaid polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
3I1. A recombinant DNA molecule comprising a vector and the DNA segment according to claim 1.
35. The molecule according to claim 16, wherein said molecule comprises pSTR3-l
36. A cell that contains the recombinant DNA molecule according to claim 16.
3γ A method of producing a polypeptide having an amino acid sequence corresponding to pneumococcal fimbrial protein A comprising culturing the cell according to claim 18 under conditions such that said DNA segment is expressed and εaid polypeptide thereby produced and isolating said polypeptide.
38. An antibody or binding fragment thereof having binding affinity to pneumococcal fimbrial protein A.
3g The antibody according to claim 20, wherein pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof.
1+0, The antibody according to claim 21, wherein pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO:2 or allelic or species variation thereof. ■ i . The antibody according to claim 22 , wherein pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO: 2.
k2. A hybridoma which produces a monoclonal antibody having binding affinity to pneumococcal fimbrial protein A, or binding fragment thereof.
k3. The hybridoma according to claim 24 , wherein pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO: 2 , allelic or -species variation thereof, or at least 5 contiguous amino acids thereof.
k k . The hybridoma according to claim 25 , wherein pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO: 2 or allelic or species variation thereof .
k ~~ . The hybridoma according to claim 26 , wherein pneumococcal fimbrial protein A has the amino acid sequence set forth in SEQ ID NO: 2.
ii 6 The hybridoma according to claim 24 , wherein said hybridoma comprises 1E7A3D7C2.
A diagnostic kit comprising: i) at least one monoclonal antibody according to claim 20, and ii) a conjugate comprising a binding partner of εaid monoclonal antibody and a label .
^ . A diagnostic kit comprising a conjugate comprising: i) at least one monoclonal antibody according to claim 20 , and ii) a label.
PCT/US1992/009522 1991-11-14 1992-11-16 Pneumococcal fimbrial protein a vaccines WO1993010238A1 (en)

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WO1995006732A3 (en) * 1993-09-01 1995-06-01 Univ Rockefeller Bacterial exported proteins and acellular vaccines based thereon
US5981229A (en) * 1993-09-01 1999-11-09 The Rockefeller University Bacterial exported proteins and acellular vaccines based thereon
WO1995006732A2 (en) * 1993-09-01 1995-03-09 The Rockefeller University Bacterial exported proteins and acellular vaccines based thereon
US5928900A (en) * 1993-09-01 1999-07-27 The Rockefeller University Bacterial exported proteins and acellular vaccines based thereon
WO1995016711A1 (en) * 1993-12-17 1995-06-22 Universidad De Oviedo Antibodies against pneumolysine and their applications
EP0912193A1 (en) * 1996-06-21 1999-05-06 Virginia Commonwealth University Vaccine to prevent streptococcal endocarditis
EP0912193A4 (en) * 1996-06-21 2000-11-02 Univ Virginia Commonwealth Vaccine to prevent streptococcal endocarditis
EP0837130A3 (en) * 1996-09-24 1999-11-03 Smithkline Beecham Corporation M protein analogue from Streptococcus pneumoniae
EP0837130A2 (en) * 1996-09-24 1998-04-22 Smithkline Beecham Corporation M protein analogue from Streptococcus pneumoniae
US6358704B1 (en) 1996-09-24 2002-03-19 Smithkline Beecham Corporation M protein
EP0846766A2 (en) * 1996-09-24 1998-06-10 Smithkline Beecham Corporation IgA Fc binding protein from Streptococcus pneumoniae
EP0834568A2 (en) * 1996-09-24 1998-04-08 Smithkline Beecham Corporation Novel saliva binding protein
EP0834568A3 (en) * 1996-09-24 1999-12-01 Smithkline Beecham Corporation Novel saliva binding protein
EP0846766A3 (en) * 1996-09-24 1999-11-24 Smithkline Beecham Corporation IgA Fc binding protein from Streptococcus pneumoniae
US7056510B1 (en) 1996-10-31 2006-06-06 Human Genome Sciences, Inc. Streptococcus pneumoniae SP036 polynucleotides, polypeptides, antigens and vaccines
US7141418B2 (en) 1996-10-31 2006-11-28 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
US8168205B2 (en) 1996-10-31 2012-05-01 Human Genome Sciences, Inc. Streptococcus pneumoniae polypeptides
EP1770164A3 (en) * 1996-10-31 2007-10-10 Human Genome Sciences, Inc. Streptococcus pneumoniae antigens and vaccines
WO1998018931A2 (en) * 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
WO1998018930A3 (en) * 1996-10-31 1998-10-08 Human Genome Sciences Inc Streptococcus pneumoniae antigens and vaccines
US6420135B1 (en) 1996-10-31 2002-07-16 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
WO1998018930A2 (en) * 1996-10-31 1998-05-07 Human Genome Sciences, Inc. Streptococcus pneumoniae antigens and vaccines
EP1400592A1 (en) * 1996-10-31 2004-03-24 Human Genome Sciences, Inc. Streptococcus pneumoniae polynucleotides and sequences
WO1998018931A3 (en) * 1996-10-31 1998-08-20 Human Genome Sciences Inc Streptococcus pneumoniae polynucleotides and sequences
US6676943B1 (en) 1997-04-24 2004-01-13 Regents Of The University Of Minnesota Human complement C3-degrading protein from Streptococcus pneumoniae
WO1999040200A1 (en) * 1998-02-03 1999-08-12 Center For Disease Control And Prevention RECOMBINANT LIPIDATED PsaA PROTEIN, METHODS OF PREPARATION AND USE
US7635486B1 (en) 1998-02-03 2009-12-22 The United States Of America As Represented By The Department Of Health And Human Services Recombinant lipidated PsaA protein, methods of preparation and use
US7501132B2 (en) 1998-03-02 2009-03-10 The United States Of America As Represented By The Department Of Health And Human Services Multiple antigenic peptides immunogenic against Streptococcus pneumonia
WO1999045121A1 (en) * 1998-03-02 1999-09-10 The Government Of The United States Of America, Represented By The Secretary Of The Department Health And Human Services EPITOPE PEPTIDES IMMUNOGENIC AGAINST $i(STREPTOCOCCUS PNEUMONIAE)
US8642048B2 (en) 1998-03-02 2014-02-04 The United States of America, as represented by the Secretary of the Department of Health and Human Services, Centers for Disease Control and Prevention Multiple antigenic peptides immunogenic against Streptococcus pneumonia

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