The recombinant production of Gap4, a chimeric GapC plasmin binding protein comprising the entire amino acid sequence of the Streptococcus dysgalactiae GapC protein in addition to unique amino acid sequences from the Streptococcus parauberis and Streptococcus agalactiae GapC proteins, is described. Also described is the use of Gap4 chimeric GapC protein in vaccine compositions to prevent or treat streptococcal infections in general and mastitis in particular.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides GapC multiple epitope fusion proteins and polynucleotides encoding the same. In one embodiment, the invention is directed to a multiple epitope fusion polypeptide comprising the general structural formula (I): (A).sub.x--(B).sub.y--(C).sub.z (I) wherein (I) is a linear amino acid sequence; B comprises an amino acid sequence containing at least five amino acids which amino acids correspond to an antigenic determinant of a GapC protein; A and C each comprise an amino acid sequence that is (i) different from B, (ii) different from the other, and (iii) an amino acid sequence containing at least five amino acids, which amino acid sequence corresponds to an antigenic determinant of a GapC protein wherein said antigenic determinant is not adjacent to B in nature; y is an integer of 1 or more; and x and z are each independently integers wherein x+z is 1 or more.

In certain embodiments, the multiple epitope fusion polypeptide further comprises a signal sequence and/or a transmembrane sequence. Further, A, B, and/or C of the multiple epitope fusion polypeptide may linked by one or more spacer sequences, wherein the spacers (i) are amino acid sequences of from 1 to 1,000 amino acids, inclusive; (ii) can be the same or different as A, B, or C; and (iii) can be the same or different as each other.

In certain embodiments, A, B, and C each comprise epitopes from one or more species of bacteria, such as from one or more bacterial species of the genus Streptococcus, including but not limited to one or more bacterial species selected from the group consisting of Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis, Streptococcus parauberis, and Streptococcus iniae.

In yet another embodiment, A, B, and C each comprise amino acid sequences selected from the group consisting of

(a) the amino acid sequence shown at about amino acid positions 61 to 81, inclusive, of FIGS. 1 through 5 (see Original Patent), or any amino acid sequence having at least about 80% identity thereto;

(b) the amino acid sequences shown at about amino acid positions 102 to 112, inclusive, of FIGS. 1 through 5, or any amino acid sequence having at least about 80% identity thereto;

(c) the amino acid sequences shown at about amino acid positions 165 to 172, inclusive, of FIGS. 1 through 5, or any amino acid sequence having at least about 80% identity thereto;

(d) the amino acid sequences shown at about amino acid positions 248 to 271, inclusive, of Figures through 5, or any amino acid sequence having at least about 80% identity thereto; and

(e) the amino acid sequences shown at about amino acid positions 286 to 305, inclusive, of FIGS. 1 through 5, or any amino acid sequence having at least about 80% identity thereto.

In another embodiment, the multiple epitope fusion polypeptide comprises the amino acid sequence depicted in FIG. 6 (SEQ ID NO:22, see Original Patent).

In yet further embodiments, the invention is directed to polynucleotide sequences encoding the multiple epitope fusion polypeptide sequence described above or compliments thereof, as well as recombinant vectors comprising the polynucleotide, host cells comprising the recombinant vectors and methods of recombinantly producing the polypeptides.

In another embodiment, the invention is directed to a vaccine composition comprising a pharmaceutically acceptable vehicle and a multiple epitope fusion polypeptide as described above. In certain embodiments, the vaccine compositions comprise an adjuvant.

In still a further embodiment, the invention is directed to a method of producing a vaccine composition comprising the steps of

(1) providing the multiple epitope fusion polypeptide; and

(2) combining the polypeptide with a pharmaceutically acceptable vehicle.

In another embodiment, the invention is directed to a method of treating or preventing a bacterial infection in a vertebrate subject comprising administering to the subject a therapeutically effective amount of a vaccine composition as described above.

In certain embodiments, the bacterial infection is a streptococcal infection. Further, the bacterial infection may cause mastitis.

In yet another embodiment, the invention is directed to a method of treating or preventing a bacterial infection in a vertebrate subject comprising administering to the subject a therapeutically effective amount of a polynucleotide as described herein.

In certain embodiments, the bacterial infection is a streptococcal infection. Further, the bacterial infection may cause mastitis.

In further embodiments, the invention is directed to antibodies directed against the above multiple epitope fusion polypeptides. The antibodies may be polyclonal or monoclonal.

In another embodiment, the invention is directed to a method of detecting Streptococcus antibodies in a biological sample, comprising:

(a) reacting said biological sample with a multiple epitope fusion polypeptide under conditions which allow said Streptococcus antibodies, when present in the biological sample, to bind to said sequence to form an antibody/antigen complex; and

(b) detecting the presence or absence of said complex, and thereby detecting the presence or absence of Streptococcus antibodies in said sample.

In still a further embodiment, the invention is directed to an immunodiagnostic test kit for detecting Streptococcus infection. The test kit comprises a multiple epitope fusion polypeptide as described herein and instructions for conducting the immunodiagnostic test.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second Edition (1989); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

General Overview of the Invention

Central to the present invention is the discovery that the GapC protein is capable of eliciting an immune response in a vertebrate subject. Experiments performed in support of the present invention have demonstrated that immunization of dairy cattle with the GapC protein of S. dysgalactiae conferred protection against experimental infection with this organism, and furthermore, conferred cross-protection against infection by S. uberis.

GapC is produced by a number of different streptococcus species. With the exception of several localized variable regions, the amino acid sequences of the GapC proteins produced by those strains are highly conserved. Therefore, it is desirable to construct multiple epitope GapC fusion proteins comprising antigenic determinants taken from both the highly conserved regions of GapC, and the unique regions of GapC proteins from several streptococcal species. Experiments performed in support of the present invention have demonstrated that such a protein is capable of eliciting broad immunity against a variety of streptococcal infections while providing the additional economic advantage of minimizing the number of antigens present in the final formulation, and concomitantly reducing the cost of producing that formulation.

The GapC multiple epitope fusion proteins of the present invention are described by the general structural formula (A).sub.x--(B).sub.y--(C).sub.z representing a linear amino acid sequence. B is an amino acid sequence of at least five and not more than 1,000 amino acids of an antigenic determinant from a GapC protein, and y is an integer of 2 or more. A and C are each different from B, as well as being different from each other, and are independently an amino acid sequence of an antigenic determinant containing at least five and not more than 1,000 amino acids not immediately adjacent to B in nature. x and z are each independently an integer of 0 or more, wherein at least one of x and z is 1 or more.

Typically, A, B, and C are antigenic determinants from the GapC proteins of one or more bacterial species. In a preferred embodiment, A, B, and C are amino acid sequences comprising one or more antigenic determinants from the GapC protein of one or more of the following species of streptococcus: S. dysgalactiae; S. agalactiae; S. uberis; S. parauberis, and S. iniae.

In this regard, FIGS. 9 through 13 (see Original Patent) show plots of the following for the streptococcal GapC proteins employed by the present invention: Kyte-Doolittle hydrophathy, averaged over a window of 7; surface probability according to Emini; chain flexibility according to Karplus-Schulz; antigenicity index according to Jameson-Wolf; secondary structure according to Garnier-Osguthorpe-Robson; secondary structure according to Chou-Fasman; and predicted glycosylation sites. FIGS. 15 through 19 (see Original Patent) show plots of secondary structure according to Chou-Fasman for the aforementioned proteins. One of skill in the art can readily use the information presented in FIGS. 9 through 13 and 15 to 19 (see Original Patent) in view of the teachings of the present specification to identify antigenic regions which may be employed in constructing the chimeric protein of the present invention.

Most preferably, A, B, and/or C include one or more variable regions of the GapC proteins from more than one streptococcus species. In this regard, FIGS. 8A-8C (see Original Patent) show an amino acid sequence alignment which illustrates regions of homology and variability that exist among GapC proteins from S. dysgalactiae (SEQ lID NO:12). S. agalactiae (SEQ ID NO:14) S. uberis (SEQ ID NO:16) S. parauberis (SEQ ID NO:18) and S. iniae (SEQ ID NO:20). Amino acid sequences for the GapC proteins of S. pyogenes (SEQ ID NO:24). and S. equisimilis (SEQ ID NO:26) are also included. In particular, several variable regions are located at amino acid positions 62 to 81; 102 to 112; 165to 172; 248 to 271; and 286 to 305.

The multiple epitope fusion protein of the present invention may also include spacer sequences interposed between A, B, and/or C. The spacer sequences are typically amino acid sequences of from 1 to 1,000 amino acids, may be the same or different as A, B, or C, and may be the same or different as each other.

The present invention may also include a signal sequence and/or a transmembrane sequence. Examples of suitable signal sequences include the E. coli LipoF signal sequence, and the OmpF signal sequence. Examples of suitable transmembrane sequences include those associated with LipoF and OmpF.

An especially preferred embodiment of the present invention is the multiple epitope fusion protein Gap4. The amino acid sequence of Gap4 (SEQ ID NO:22), a representative multiple epitope GapC fusion protein, is shown in FIGS. 6A-6C (see Original Patent), as is the polynucleotide sequence which encodes it (SEQ ID NO:21). Gap4 is a 47.905 kDa chimeric protein of 448 amino acids. Residues 1 to 27 are identical to amino acid residues 1 to 27 of the E. coli LipoF signal sequence. Residues 28 to 123 are identical to residues 1 to 96 of the S. dysgalactiae GapC protein. Residues 124 (leucine) and 125 (glutamic acid) are spacer amino acids. They are followed by residues 126 to 165, which are identical to residues 56 to 95 of S. parauberis as well as to the same residues of S. uberis. Residue 166 (isoleucine) is a spacer amino acid. Residues 167 to 208 are identical to residues 55 to 96 of the S. agalactiae GapC protein. Residues 209 (threonine) and 210 (serine) are spacer amino acids. Residues 211 to 448 are identical to residues 99 to 336 of the S. dysgalactiae GapC protein.

As expressed, Gap4 has a cysteine residue present at the amino terminal end of the mature protein. The LipoF signal sequence and cysteine residue are present to ensure that the chimeric molecule is efficiently secreted from the bacterial host cell and becomes bound to the host cell membrane via the lipid-moiety. The protein may then be extracted from the cell surface via differential solubilization with a detergent such as Sarkosyl or TritonX-100.RTM. (see Example 5 infra).

The GapC chimeric proteins of the present invention or antigenic fragments thereof can be provided in subunit vaccine compositions. In addition to use in vaccine compositions, the proteins or antibodies thereto can be used as diagnostic reagents to detect the presence of infection in a vertebrate subject. Similarly, the genes encoding the proteins can be cloned and used to design probes to detect and isolate homologous genes in other bacterial strains. For example, fragments comprising at least about 15-20 nucleotides, more preferably at least about 20-50 nucleotides, and most preferably about 60-100 nucleotides, or any integer between these values, will find use in these embodiments.

The vaccine compositions of the present invention can be used to treat or prevent a wide variety of bacterial infections in vertebrate subjects. For example, vaccine compositions including GapC multiple epitope fusion proteins comprising antigenic determinants from S. dysgalactiae, S. uberis, S. parauberis, S. iniae, and/or group B streptococci (GBS) such as S. agalactiae, can be used to treat streptococcal infections in vertebrate subjects that are caused by these or other species. In particular, S. uberis and S. agalactiae are common bacterial pathogens associated with mastitis in bovine, equine, ovine and goat species. Additionally, group B streptococci, such as S. agalactiae, are known to cause numerous other infections in vertebrates, including septicemia, meningitis, bacteremia, impetigo, arthritis, urinary tract infections, abscesses, spontaneous abortion etc. Hence, vaccine compositions containing chimeric GapC proteins will find use in treating and/or preventing a wide variety of streptococcal infections.

Similarly, GapC multiple epitope fusion proteins comprising antigenic determinants derived from other bacterial genera such as Staphylococcus, Mycobacterium, Escherichia, Pseudomonas, Nocardia, Pasteurella, Clostridium and Mycoplasma will find use for treating bacterial infections caused by species belonging to those genera. Thus, it is readily apparent that chimeric GapC proteins can be used to treat and/or prevent a wide variety of bacterial infections in numerous species.

The GapC multiple epitope fusion proteins of the present invention can be used in vaccine compositions either alone or in combination with other bacterial, fungal, viral or protozoal antigens. These other antigens can be provided separately or even as fusion proteins comprising the GapC chimeric protein fused to one or more of these antigens. For example, other immunogenic proteins from S. uberis, such as the CAMP factor, hyaluronic acid capsule, hyaluronidase, R-like protein and plasminogen activator, can be administered with the chimeric GapC protein. Additionally, immunogenic proteins from other organisms involved in mastitis, such as from the genera Staphylococcus, Corynebacterium, Pseudomonas, Nocardia, Clostridium, Mycobacterium, Mycoplasma, Pasteurella, Prototheca, other streptococci, coliform bacteria, as well as yeast, can be administered along with the GapC fusion proteins described herein to provide a broad spectrum of protection. Thus, for example, immunogenic proteins from Staphylococcus aureus, Str. agalactiae, Str. dysgalactiae, Str. zooepidemicus, Corynebacterium pyogenes, Pseudomonas aeruginosa, Nocardia asteroides, Clostridium perfringens, Escherichia coli, Enterobacter aerogenes and Klebsiella spp. can be provided along with the GapC plasmin-binding proteins of the present invention.

Production of GapC Multiple Epitope Fusion Proteins

The above-described chimeric proteins and active fragments and analogs derived from the same, can be produced by recombinant methods as described herein. These recombinant products can take the form of partial protein sequences, full-length sequences, precursor forms that include signal sequences, or mature forms without signals.

The GapC plasmin-binding protein DNA sequences used to construct the chimeric proteins of the present invention can be isolated by a variety of methods known to those of skill in the art. See, e.g., Sambrook et al., supra. Methods for isolating, cloning and sequencing the gene sequences encoding GapC proteins from S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis, and S. iniae are detailed in Examples 1, 2 and 3, infra.

After isolating and cloning the desired GapC protein sequences, polynucleotide sequences encoding the chimeric proteins are constructed using standard recombinant techniques including PCR amplification, restriction endonuclease digestion and ligation. See, e.g., Sambrook et al., supra. Methods for constructing Gap4, an especially preferred embodiment of the present invention, are detailed in Example 4, infra.

Alternatively, the DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned. The DNA sequences can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311.

Once coding sequences for the desired proteins have been prepared, they can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage .lamda. (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B. Perbal, supra.

The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. If a signal sequence is included, it can either be the native, homologous sequence, or a heterologous sequence. For example, the LipoF signal sequence is added to the amino-terminal region of the chimeric protein Gap4 to permit secretion of the protein after expression. See Examples 4E and 5, infra. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

Other regulatory sequences which allow for regulation of expression of the protein sequences relative to the growth of the host cell may also be desirable. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. It may also be desirable to produce mutants or analogs of the GapC plasmin-binding protein. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromycesfragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins of the present invention are produced by culturing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into the growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

The proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis. Chemical synthesis of peptides may be preferable if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.

The chimeric GapC plasmin-binding proteins of the present invention, or their fragments, can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen of the present invention, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known procedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.

Monoclonal antibodies to the chimeric GapC plasmin-binding proteins and to the fragments thereof, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by using hybridoma technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against the chimeric GapC plasmin-binding proteins, or fragments thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against. Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with subunit vaccine preparations to enhance the immune response. Polyclonal and monoclonal antibodies are also useful for diagnostic purposes.

Vaccine Formulations and Administration

The GapC multiple epitope fusion proteins of the present invention can be formulated into vaccine compositions, either alone or in combination with other antigens, for use in immunizing subjects as described below. Methods of preparing such formulations are described in, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18 Edition, 1990. Typically, the vaccines of the present invention are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in or suspension in liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. The active immunogenic ingredient is generally mixed with a compatible pharmaceutical vehicle, such as, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.

Adjuvants which enhance the effectiveness of the vaccine may also be added to the formulation. Adjuvants may include for example, muramyl dipeptides, avridine, aluminum hydroxide, dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art.

The chimeric GapC plasmin-binding protein may be linked to a carrier in order to increase the immunogenicity thereof. Suitable carriers include large, slowly metabolized macromolecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.

The chimeric GapC plasmin-binding proteins may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone. A sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3-(4-dithiopyridyl propionate. Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.

Other suitable carriers for the chimeric GapC plasmin-binding proteins of the present invention include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U.S. Pat. No. 5,071,651, incorporated herein by reference. Also useful is a fusion product of a viral protein and the subject chimeric proteins made by methods disclosed in U.S. Pat. No. 4,722,840. Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the proteins of the present invention may be coupled to erythrocytes, preferably the subject's own erythrocytes. Methods of coupling peptides to proteins or cells are known to those of skill in the art.

Furthermore, the chimeric GapC plasmin-binding proteins (or complexes thereof) may be formulated into vaccine compositions in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Vaccine formulations will contain a "therapeutically effective amount" of the active ingredient, that is, an amount capable of eliciting an immune response in a subject to which the composition is administered. In the treatment and prevention of mastitis, for example, a "therapeutically effective amount" would preferably be an amount that enhances resistance of the mammary gland to new infection and/or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered somatic cell count in milk from the infected quarter. For example, the ability of the composition to retain or bring the somatic cell count (SCC) in milk below about 500,000 cells per ml, the threshold value set by the International Dairy Federation, above which, animals are considered to have clinical mastitis, will be indicative of a therapeutic effect.

The exact amount is readily determined by one skilled in the art using standard tests. The chimeric GapC plasmin-binding protein concentration will typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. With the present vaccine formulations, 5 to 500 .mu.g of active ingredient per ml of injected solution should be adequate to raise an immunological response when a dose of 1 to 3 ml per animal is administered.

To immunize a subject, the vaccine is generally administered parenterally, usually by intramuscular injection. Other modes of administration, however, such as sub-cutaneous, intraperitoneal and intravenous injection, are also acceptable. The quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. The subject is immunized by administration of the vaccine in at least one dose, and preferably two doses. Moreover, the animal may be administered as many doses as is required to maintain a state of immunity to infection.

Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, aerosol, intranasal, oral formulations, and sustained release formulations. For suppositories, the vehicle composition will include traditional binders and carriers, such as, polyalkaline glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.

Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

Controlled or sustained release formulations are made by incorporating the protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel.RTM. copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. The chimeric GapC plasmin-binding proteins can also be delivered using implanted mini-pumps, well known in the art.

The chimeric GapC plasmin-binding proteins of the instant invention can also be administered via a carrier virus which expresses the same. Carrier viruses which will find use with the instant invention include but are not limited to the vaccinia and other pox viruses, adenovirus, and herpes virus. By way of example, vaccinia virus recombinants expressing the novel proteins can be constructed as follows. The DNA encoding the particular protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the instant protein into the viral genome. The resulting TK recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

An alternative route of administration involves gene therapy or nucleic acid immunization. Thus, nucleotide sequences (and accompanying regulatory elements) encoding the subject chimeric GapC plasmin-binding proteins can be administered directly to a subject for in vivo translation thereof. Alternatively, gene transfer can be accomplished by transfecting the subject's cells or tissues ex vivo and reintroducing the transformed material into the host. DNA can be directly introduced into the host organism, i.e., by injection (see International Publication No. WO/90/11092; and Wolff et al. (1990) Science 247:1465-1468). Liposome-mediated gene transfer can also be accomplished using known methods. See, e.g., Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol. 4:206-209; Brigham et al. (1989) Am. J. Med. Sci. 298:278-281; Canonico et al. (1991) Clin. Res. 39:219A; and Nabel et al. (1990) Science 249:1285-1288. Targeting agents, such as antibodies directed against surface antigens expressed on specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to specific tissues and cells susceptible to infection.

 

Claim 1 of 12 Claims

1. A method of detecting Streptococcus antibodies in a biological sample, comprising: (a) reacting said biological sample with a multiple epitope fusion polypeptide comprising more than one Streptococcus GapC epitope from more than one Streptococcus species, under conditions which allow said Streptococcus antibodies, when present in the biological sample, to bind to said sequence to form an antibody/antigen complex, wherein said fusion protein comprises epitopes that correspond to: (i) the amino acid sequences shown at amino acid positions 62 to 81, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and 20; (ii) the amino acid sequences shown at about amino acid positions 102 to 112, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and 20; (iii) the amino acid sequences shown at about amino acid positions 165 to 172, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and 20; (iv) the amino acid sequences shown at about amino acid positions 248 to 271, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and 20; and (v) the amino acid sequences shown at about amino acid positions 286 to 305, inclusive, of SEQ ID NOS: 12, 14, 16, 18 and 20; and (b) detecting the presence or absence of said complex, and thereby detecting the presence or absence of Streptococcus antibodies in said sample.

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