IES76925B2 - Subunit Vaccine for Streptococcus Equi - Google Patents

Abstract

The invention relates to a fibrinogen-binding protein of Streptococcus equi, to a DNA fragment encoding this protein and to the use of the purified fibrinogen-binding protein or suitable truncate in the preparation of a vaccine against S. equi infection, also known as Strangles, in horses.

Description

Subunit Vaccine for Streptococcus Eaui The invention relates to a fibrinogen-binding protein of Streptococcus equi and to the use of the purified protein in the preparation of a vaccine against 5. equi infection in horses.

Strangles is one of the most important infectious diseases affecting horses. It is of major economic importance to the racing and thoroughbred industry. Strangles is caused by the bacterium Streptococcus equi also known as Streptococcus equi subsp. equi (a group C streptococcus), affects the upper respiratory tract and is highly contagious. Horse to horse spread often leads to large outbreaks with many animals infected.

Even horses with mild forms of the disease must be isolated and removed from training, stud or other heavy work for up to three months. Existing vaccines have poor efficacy (Yelle, 1987; Timoney, 1988). The present invention involves the development of a novel and efficacious vaccine against the disease and is based on a novel, high molecular-weight (Mf) protective antigen which has fibrinogen-binding properties i.e. an M-like protein.

The main protective antigen of 5. equi is thought to be an M protein. The best characterized M proteins are those of the group A streptococcus, Streptococcus pyogenes. M proteins are members of a broad family of proteins from Gram-positive bacteria. These proteins are associated with the cell wall and are thus generally called cell-wallassociated proteins. They are surface exposed and usually interact with various factors present in the extracellular matrix of mammalian tissues and in body fluids. These factors include collagen, complement components, antibodies, fibrinogen (Fg), fibronectin, kininogen, laminin, 2-macro-globulin, plasmin, prothrombin and salivary glycoproteins.

Cell-wall-associated proteins, although quite distinctive, share some common features. Firstly, these proteins are usuallyo^-helical coiled-coiled dimers, the N-terminal halves of which project from the cell as fibrillar-type structures. Secondly, the N-terminal signal sequences usually show significant homologies. In addition, the C-terminal regions which are responsible for anchoring these proteins to the bacterial cell wall and membrane also show some homology. For example, there is usually a proline-rich region followed by a highly conserved Lys-Pro-X-Thr-Gly-X (LPXTGX) motif, a stretch of about 20 hydrophobic residues and a short tail S 76 925 - 2 of predominantly charged residues. Finally, many of these proteins possess tandemly repeated sequences. Where a protein shows a number of distinct repeats, these are normally distinguished by letters e.g. A repeats, B repeats etc. (Fischetti, 1991; Goward et al., 1993).

Historically, M protein was a name given to a group of proteins of 5. pyogenes. These proteins conferred on the bacterium the ability to resist phagocytic killing and they were also thought to be involved in adhesion to host surfaces. These two properties are thought to be due in part to two characteristic binding functions of M proteins i.e. the ability to bind fibrinogen and complement factor H. In addition to these binding functions, M proteins also undergo phase and antigenic variation, and up to 80 different serotypes have been discerned (Fischetti, 1991).

Protective M proteins have also been detected in 5. equi. All the available evidence suggests that immunity to this protein(s) may be important in protection (Galan and Timoney, 1985). Several different methods have been used to extract the M-protein from 5. equi cells (Boschwitz et al., 1991). These methods include hot acid treatment of whole cells, hot alkaline treatment of whole cells, treatment of whole cells with 4% sodium dodecyl sulphate, ammonium sulphate precipitation of culture supernatants and mutanolysin extraction of bacterial protoplasts. These preparations have been analysed in Western immunoblotting experiments using convalescent horse serum, nasopharyngeal mucus, antiserum to mutanolysin-extracted protein and antiserum to a 41-kDa fragment from acid extracts (Galan & Timoney, 1985; Timoney & Trachman, 1985; Galan & Timoney, 1988; Boschwitz, 1991; Timoney & Mukhtar, 1993). All extracts contain multiple reactive protein bands in the molecular mass range 25-140 kDa. In acid extracts, the most immunologically reactive bands have molecular masses of 46 kDa, 41 kDa and 29 kDa. In mutanolysin extracts, there are two reacting bands in the 58-kDa region and a higher molecular mass band of 120 kDa. The 58-, 46-, 41- and 29-kDa proteins possess the same N-terminal amino acid sequence (data not shown in Timoney & Mukhtar, 1993). Timoney's group suggest that the 58-kDa protein is the native M-protein, that the lower molecular weight reacting bands are peptide fragments i.e. degradation products of M-protein, and that the high molecular mass protein of 120-140 kDa is M-protein complexed to the cell wall (Galan & Timoney, 1985; Timoney et al., 1991; Timoney & Mukhtar, 1993). - 3 Like M-protein of 5. pyogenes, the M-protein of 5. equi has been implicated in inhibition of phagocytosis. Recent evidence suggests that resistance to phagocytosis may be related to the ability of M-protein to interfere with the deposition of equine complement on the cell surface (Boschwitz & Timoney, 1994a). These authors have also provided some additional evidence that binding of fibrinogen to 5. equi cells has some anti-phagocytic activity. From a study of non-isogenic strains, they further suggest that the M-protein may bind fibrinogen since a strain of 5. equi expressing low levels of M-protein bound 64% less fibrinogen than another strain expressing normal M-protein levels (Boschwitz & Timoney, 1994b). However, binding of fibrinogen to a defined molecular species has not been shown.

A mouse model has also been used to test the protective potential of streptococcal extracts and of immunoglobulin preparations. Acid-extracted M-protein (containing the mixture of immunologically-reactive species described above) and monoclonal antibodies generated to this preparation have been shown to afford 60-80% protection in a mouse model (Timoney & Trachman, 1985; Jean-Francois et al., 1991). Efforts were also undertaken to clone the gene encoding M-protein (Galan & Timoney, 1987, Timoney et al., 1991). Galan & Timoney, 1987 isolated clones from a recombinant phage bank which reacted with opsonic and anti-M protein antiserum and expressed proteins of Mf 58,000, 53,000 and 50,000. In another publication (Timoney et al., 1991), two plasmid subclones from the phage library were described and about 550 bp of DNA sequence was published.

At this stage it is perhaps worth reemphasizing that the principle group working in the field of M-proteins of 5. equi (i.e. that of Timoney) considers the 58-kDa protein antigen to be the native M-protein.

In contrast to the M-protein of 5. pyogenes a variety of evidence suggests that there is only one serotype of M-protein from S. equi. This includes evidence from precipitin and passive protection experiments, sensitivity to bactericidal serum, immunoblot and M analysis, DNA restriction analysis and Southern blot analysis (see Galan & Timoney, 1988) Existing vaccines against strangles are based on crude bacterins (heat-killed 5. equi) or acid extracts enriched in the M-protein. U.S. Patent No. 3,852,420 is concerned with extracting immunity-provoking - 4 antigen by hot acid treatment of cells. U.S. Patent No. 4,582,798 of Brown et al., is concerned with an alternative technique for extracting the antigen of interest by treatment of the cells with mutanolysin and an anionic detergent. Existing vaccines include Equibac (Fort Dodge Labs., Iowa), Strangles vaccine (Commonwealth Serum Labs., Melbourne), Strepguard (Haver Labs., Kansas), Strepvax II (Cooper Animal Health, Kansas) and Stranglevac (Bayer Animal Health, Kansas/Miles Inc., Slough, U.K.). These vaccines are administered intramuscularily with adjuvant. They cause some side effects and more importantly afford little protection (Yelle, 1986; Timoney, 1988). Galan & Timoney (1985) showed that there was no correlation between protection and the levels of serum bactericidal antibodies directed against S. equi whole cells. The authors hypothesize that immunity to infection is mediated by locally produced mucosal nasopharyngeal antibodies and that the lack of efficacy of existing vaccines is due to the failure to stimulate this kind of response. Indeed, Brown & Bryant, 1990 (U.S. Patent No. 4,944, 942) provide convincing evidence that intranasal immunization can protect horses from experimental challenge with 5. equi. The antigenic material to be used in this invention can be inactivated whole organisms or appropriate extracts prepared from these organisms or recombinant DNA or synthetic peptides. It is stated that the preferred technique for preparing antigenic material is by enzyme extraction using enzymes such as pepsin, lysozyme or mutanolysin and to follow the enzyme extraction with treatment with an anionic detergent such as SDS.

The object of the present invention is to provide a cheap, effective vaccine against 5. equi infection or strangles of horses.

A further object is to provide a defined subunit vaccine against strangles. It is also an object to isolate a protective cell-wallassociated protein from 5. equi, and to purify this protein to homogeneity. An additional objective is to isolate the gene encoding the said protein.

According to the present invention there is provided a fibrinogenbinding protein (FgBP) isolated from S. equi subspecies equi having the following characteristics:(1) it migrates on Laemmli SDS-PAGE gels with an apparent molecular weight - 5 of approximately 220,000 D, (2) it binds horse fibrinogen, and (3) it protects against 5. equi infection (Strangles) in horses.

The fibrinogen-binding protein is preferably associated with the cell wall of 5. equi.

Preferably the fibrinogen-binding protein comprises an amino acid sequence selected from (1) NSEVSRTATPRL and (2) LQKAKDERQALTESFNKTLS.

The fibrinogen-binding protein may comprise the nucleotide sequence as shown in Figure 15.

The invention further provides a DNA fragment as deposited at The National Collections of Industrial and Marine Bacteria, St. Machar Drive, Aberdeen, Scotland under the Accession Number NCIMB 40807 on 25th June 1996, encoding the S. equi fibrinogen-binding protein or a fragment substantially similar thereto also encoding S. equi fibrinogen-binding protein and/or Strangles protective activity.

The invention further relates to a host cell comprising a DNA fragment as described above and to a method of producing a fibrinogen-binding protein comprising culturing a host cell containing the said DNA fragment and isolating the protein from the culture.

In a further aspect the invention provides a method of producing an 5. equi fibrinogen-binding protein comprising the steps:(1) genetically engineering a host cell containing the DNA fragment defined above to overexpress and/or secrete the fibrinogen-binding protein (or suitable truncate) into the culture supernatant, (2) isolating the fibrinogen-binding protein in a cell-free supernatant fraction, and (3) purifying the fibrinogen-binding protein. - 6 By suitable truncate is meant a fibrinogen-binding protein derivative which is lacking the C-terminal segment responsible for anchoring the protein to the cell envelope. Such a truncate would not be capable of binding to the cell wall since it would be lacking its cell wal1/membrane anchor domains, and would thus be secreted into the supernatant, simplifying the purification procedure.

The fibrinogen-binding protein or truncate may be isolated in a cellfree supernatant fraction by either (a) lysis of the host cell followed by centrifugation or by any of a number of filtration methods known in the art, or by (b) isolation of the culture supernatant by centrifugation or by any of a number of filtration methods known in the art.

The fibrinogen-binding protein or truncate may be purified by fibrinogen- affinity chromatography or other chromatographic procedures known in the art.

The invention further provides a vaccine comprising a fibrinogenbinding protein as defined above or whenever produced by a method as described above.

The invention also provides an S. equi fibrinogen-binding protein as defined above for use in the preparation of a vaccine against Strangles infection in horses.

By substantially similar herein is meant DNA fragments encoding fibrinogen-binding activity which have sufficient sequence identity or homology to the deposited DNA fragment, by virtue of the degeneracy of the genetic code or by virtue of mutation, to hybridise therewith and to bind fibrinogen and to protect against 5. equi infection or Strangles in horses.

The invention will be described further with reference to the drawings in which there is shown: Figure 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and fibrinogen-affinity blotting analysis of proteins released from purified cell walls of 5. equi following treatment with mutanolysin. Samples were boiled at 100°C in Laemmli sample buffer and - 7 analysed by SDS-PAGE using a 12.5% (wt/vol) polyacrylamide separation gel (Laemmli, 1970). Lane 1A and IB, cell envelopes following extraction with 2% (wt/vol) SDS; lane 2A and 2B, supernatant fraction from purified cell walls following incubation with mutanolysin; lane 3A and 3B, supernatant fraction from purified cell walls incubated without mutanolysin. The SDS-gel in Panel A was stained with Coomassie-brilliant blue. Panel B shows an identical gel electrotransferred onto nitrocellulose and affinity probed with horseradish peroxidase conjugated-horse fibrinogen. Positions and molecular masses of mutanolysin-extracted proteins and of mutanolysin are indicated at the right of panel A. The position of the fibrinogen-reacting 220-kDa protein is indicated by arrowhead at the right of Panel B. Molecular masses were determined from the relative mobilities of the following standard molecular mass marker proteins: rabbit myosin (205 kDa), B-galactosidase (116 kDa), phosphorylase b (94 kDa), bovine serum albumin (66.2 kDa), catalase (61 kDa), glutamate dehydrogenase (55.4 kDa), fumarase (48.5 kDa), alcohol dehydrogenase (41 kDa), Omp F protein of E. coli (36.5 kDa), carbonic anhydrase (30 kDa), chymotrypsinogen (25.1 kDa), trypsin inhibitor (20.1 kDa), lysozyme (14.3 kDa).

Figure 2. Analysis by SDS-PAGE of the purification of the FgBP from 5. equi. Samples solubilized at 100°C in Laemmli sample buffer and analysed by SDS-PAGE using a 12.5% (wt/vol) polyacrylamide separating gel (Laemmli, 1970). Lane 1, cell envelope fraction; lane 2, cell envelope fraction following incubation with mutanolysin; lane 3, pellet obtained following incubation of cell envelopes with mutanolysin; lane 4, supernatant fraction obtained following incubation of cell envelopes with mutanolysin; lane 5, proteins eluted unbound from the fibrinogen affinity column; lane 6, FgBP purified by fibrinogen-affinity chromatography. The position of the FgBP is indicated. Indicated to the left of the SDS-gel are the positions (in kilodaltons) to which standard molecular mass marker proteins migrated.

Figure 3. Protective effect of FgBP against lethal 5. equi infection in mice, (a) Twenty one, and seven days prior to challenge, a group of 11 mice were immunized (subcutaneously) with partially purified FgBP emulsified in MPL+S-TDCM Ribi adjuvant. A control group of mice were immunized (subcutaneously) with MPL+S-TDCM Ribi adjuvant emulsified in phosphate-buffered saline (PBS). All mice were subsequently challenged with 3 x 10$ colony forming units (CFU) of 5. equi cells, (b) Forty - 8 two and fourteen days prior to challenge, a group of 10 mice were immunized (subcutaneously) with affinity-purified FgBp emulsified in MPL+S-TDCM Ribi adjuvant. Control mice were immunized with PBS only or with MPL+S-TDCM Ribi adjuvant emulsified with PBS. All mice were subsequently challenged ς with 1.5 x 10 CFU of 5. equi cells. Mice were sacrificed if considered terminally ill with body temperatures below 32°C. □ controls; Φ' vaccinates.

Figure 4. Serum IgG response, as monitored by ELISA, of mice vaccinated with FgBP and of control unvaccinated mice. The log of serum dilutions versus the mean absorbance readings of the vaccinated (φ) group of mice and of the control ( Θ) group of mice are shown.

Figure 5. Numerical clinical scoring system used in the equine trials.

Figure 6. Total daily clinical scores during equine trial. Days of vaccination (V) and challenge (C) are indicated. 0 denotes horse Hl; O denotes horse H2; denotes horse H3.

Figure 7. Daily temperature score during equine trial. Days of vaccination (V) and challenge (C) are indicated. [D denotes horse Hl; O denotes horse H2; V denotes horse H3.

Figure 8. Daily fibrinogen levels and white blood cell counts for horse Hl during equine trial. Days of vaccination (V) and challenge (C) are indicated. 0 denotes fibrinogen level; φ denotes white blood cell counts.

Figure 9. Daily fibrinogen levels and white blood cell counts for horse H2 during equine trial. Days of vaccination (V) and challenge (C) are indicated. □ denotes fibrinogen level; φ denotes white blood cell counts.

Figure 10. Daily fibrinogen levels and white blood cell counts for horse H3 during equine trial. Days of vaccination (V) and challenge (C) are indicated. Q denotes fibrinogen level; φ denotes white blood cell counts.

Figure 11. Serum IgG titres against FgBP for horses Hl, H2 and H3 taken on - 9 days post primary vaccination. Days of vaccination (V) and challenge (C) are indicated. /3 denotes horse Hl; O denotes horse H2; M denotes horse H3.

Figure 12. Nasal mucosa (secretory) IgG titres against FgBP for horses Hl H2 and H3 taken on days post primary vaccination. Days of vaccination (V) and challenge (C) are indicated. Q denotes horse Hl; O denotes horse H2; β denotes horse H3.

Figure 13. Oligonucleotide probe used in Southern blots to screen for the gene encoding the FgBP. The corresponding amino acid sequence is shown above in single letter code.

Figure 14. Genotypes of bacterial strains.

Figure 15. Nucleotide and deduced amino acid sequence of the 5' region of the gene encoding the FgBP of S. equi. The probable start of the signal sequence is arrowed. The sequences corresponding to that determined by direct amino acid sequence analysis of the V8 protease fragments are underlined.

Figure 16. Partial restriction map of pFBP200. The open box represents the multiple cloning site in lacZ of pGEM7. The thin line represents GEM11 DNA. The thicker line represents 5. equi DNA. The region which has been sequenced has a bar beneath it.

Results Isolation of cell wall-associated proteins In order to identify candidate cel1-wal1-associated proteins from 5. equi, it was first necessary to separate these proteins from other membrane proteins. This involved purification of cell-wall material, degradation of cell-wall polymers and isolation of proteins specifically released from the degraded cell wall. These steps are outlined below. 5. equi cells were grown to late logarithmic phase in Todd-Hewitt broth supplemented with 0.2% (wt/vol) yeast extract. Usually about 6 litres of cells were grown at a time, the yield of cells being about 2 g wet weight per litre of broth. Bacterial cells were harvested by centrifugation - 10 (16,000 x g for 15 min), washed once in lOmM Tris-HCl buffer pH 7.2 (Tris buffer) and finally resuspended in 120 ml Tris buffer containing DNase (50yg/ml), RNase (50 pg/ml) and protease inhibitors (phenylmethylsulfonylfloride [2 mM], and benzamidine hydrochloride [2 mM]. The bacteria were ο then lysed by two passages through a French pressure cell (32,000 lb/in ). Unlysed cells were removed from the cell lysate by centrifugation (3,000 x g for 10 min). Cell wall-membranes (cell envelopes) were pelleted from the cleared lysate by centrifugation (45,000 x g for 1 h) and washed three times in Tris buffer. The cell envelopes were then resuspended in 36 ml of Tris buffer containing 2% (wt/vol) sodium dodecyl sulphate (SDS) and incubated at 20°C for 1 h. Purified cell-wall material (plus associated polymers and proteins) was obtained as an SDS-insoluble (pellet) fraction following centrifugation (45,000 x g for 1 h). The SDS-extraction was repeated once more as outlined above. The SDS-insoluble pellet was washed 5 times in Tris buffer, once in 10 mM sodium phosphate buffer, pH 7.2, and finally resuspended in 9 ml of 10 mM sodium phosphate buffer, pH 7.2. The purified cell wall was then digested by incubation for 18 h at 37°C with mutanolysin (Sigma; 18,000 U), in the presence of protease inhibitors (NX-p-tosyl-L-lysinechloromethylketone [2mM], phenylmethylsulfonyl- floride [2 mM], and benzamidine hydrochloride [2 mM]). The extract was centrifuged (45,000 x g for 1 h) and material released from the digested cell wall was obtained in the supernatant fraction. These various fractions were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that several proteins were specifically released from the digested peptidoglycan. These proteins included one major protein of apparent Mr220,000. Other proteins of apparent Mr94,000, 74,000 and 56,000 and minor species of Mr44,000, 38,000 and 29,000 were also detected (Figure 1, lane 2A). It can be concluded that these proteins are likely to be associated with the cell wall.

Fibrinogen-binding ability of the 220-kDa protein An affinity blotting assay of the above preparation of cell-wallassociated proteins was performed using horse fibrinogen which had been labelled with horseradish peroxidase.

Removal of contaminating fibronectin and IgG from horse fibrinogen Prior to conducting the affinity blotting assay the major - 11 contaminating serum proteins (fibronectin and immunoglobulin G) were first removed from the preparation of horse fibrinogen by gelatin affinity chromatography (Mosher et al., 1980) and by protein G affinity chromatography (Pierce Chemical Co.). In these procedures, contaminating fibronectin was first removed using a gelatin-affinity column (Mosher etl al., 1980) prepared by covalently linking gelatin to CnBr-activated TM Sepharose 4B using the method recommended by the manufacturer (Pharmacia LKB Biotechnology). Horse fibrinogen (10 mg/ml in 50 mM sodium phosphate, 1 mM ethylenediaminetetraacetate (EDTA) and 0.5M NaCl, pH 7.5) was then applied to the gelatin column. Fibronectin remained bound to the column, whereas fibrinogen was eluted as unbound material. The eluate was then dialysed against 100 mM sodium acetate (pH 5.0), and a precipitate of lipoproteins removed by centrifugation (16,000 x g for 15 min). Contaminating horse IgG was then removed from the supernatant fraction using a protein G/agarose affinity column and following the procedure recommended by the manufacturer (Pierce Chemical Co.). Horse IgG remained bound to the column whereas fibrinogen was eluted as unbound material. Purified fibrinogen (free of fibronectin and IgG) was then dialysed against distilled water and lyophilised.

Conjugation of fibrinogen with horseradish peroxidase Purified fibrinogen was labelled with horseradish peroxidase based on a method described by Winston et al. (1995). This entailed firstly treatment of horseradish peroxidase (5 mg/ml) with 40 mM NalO^ in the dark for 30 min at 20°C. Ethylene glycol was then added to a final concentration of 0.64 M and the solution incubated for a further 1 h before dialysis versus 1 M sodium acetate buffer (pH 4.4). Lyophilised fibrinogen (25 mg) was the dissolved in the above solution of horseradish peroxidase. The pH of the solution was adjusted to 9.5 using 100 mM sodium carbonate buffer (pH 9.5) and the mixture was incubated in the dark for lh at 20°C. 0.1 ml of aqueous NaBH^ (4 mg/ml) was added and the solution incubated for a further 1 h in order to reduce reactive groups. Peroxidase-labelled fibrinogen was then dialysed extensively against phosphate buffered saline (pH 7.2; PBS) and finally stored at -20°C.

Horse fibrinogen affinity-blotting assay Affinity blotting assays were performed in a similar manner to that - 12 described for Western immunoblotting (Caffrey et al., 1988). 2% (wt/vol) dried skimmed milk was used as a blocking reagent. Bound peroxidaseconjugated horse fibrinogen was visualized with 4-chloro-l-naphthol. The results of the affinity blot showed that the protein of apparent Mf of 220,000 bound horse fibrinogen (Figure IB).

Purification of the fibrinogen-binding protein The fibrinogen-binding protein (FgBP) of apparent Mf 220,000 was purified to homogeneity using fibrinogen-affinity chromatography. The purification steps are outlined below and an SDS polyacrylamide gel of the various purification steps are shown in Figure 2. Bacterial cell envelopes (i.e. cell wall-membranes which had not been extracted in SDS) were isolated by a procedure outlined in the Section entitled Isolation of cell wall-associated proteins. Isolated cell envelopes were then washed three times in Tris buffer, once in 10 mM sodium phosphate buffer (pH 6.8) and finally resuspended in 12 ml of sodium phosphate buffer containing mutanolysin (9600 U) and protease inhibitors (Nok-p-tosyl-L-lysinechloromethylketone [2mM], phenyl-methylsulfonylfloride [2 mM], and benzamidine hydrochloride [2 mM]). The suspension was extracted at 37°C for 18 h and then centrifuged (45,000 x g for 1 h). Material released from the digested cell envelope and thus obtained in the supernatant fraction was analysed by SDS-PAGE (see Figure 2). The profile of proteins, which were released into the supernatant fraction, was identical to that released following mutanolysin extraction of purified cell walls (see Section entitled Isolation of cell wal1-associated proteins and Figure 1). The supernatant fraction of the mutanolysin extract was then incubated, with shaking, for 2 h with approximately 7 ml of fibrinogen-sepharose 4B prepared by covalently linking horse fibrinogen to CnBr-activated Sepharose 4B™ by a method recommended by the manufacturer (Pharmacia LKB Biotechnology). The fibrinogen-sepharose 4B slurry was poured into a 1 x 9 cm chromatography column, and eluted with 10 mM sodium phosphate buffer (pH 6.8) until the θθ280ηιη aPProac^ec* zero· 0.2 M glycine (pH 2.5) was used to elute bound FgBP. Fractions were collected into 1M Tris-HCl (pH 8.00) and analysed by SDS-PAGE. Those containing FgBP were pooled, dialysed against PBS and lyophilised. - 13 Assessment of the protective potential of FgBP in a mouse model To assess the vaccinogenic potential of the FgBP, two separate protection experiments were performed in a mouse model. . equi cells used for challenge . equi cells used for challenge of the mice were grown as described in the Section entitled Isolation of cell wal1-associated proteins. Harvested cells were washed twice in sterile PBS (1/5 culture volume), resuspended in PBS (1/100 culture volume) and suspensions were stored in aliquots at -70°C. 5. equi cells stored in this manner maintained viability for several months. On the day of challenge, an aliquot of frozen cells was thawed and diluted appropriately in PBS.

Mouse challenge experiments In the first of these experiments, the protective immunogen was a preparation of FgBP which had been isolated following mutanolysin extraction of purified cell walls and further purified (to approximately 80% homogeneity) by gel filtration chromatography. Eleven mice were immunized subcutaneously on days 0 and 14 with 100 pg protein emulsified in MPL+S-TDCM Ribi adjuvant (active ingredients monophosphoryl lipid A and trehalose dimycolate; RIBI Immunochem Research, Inc.). Ten control mice were immunized with adjuvant emulsified in PBS only. All mice were c challenged on day 21 by intraperitoneal injection of 3 x 10 colony forming units (CFU) of virulent 5. equi and were monitored for 32 days post challenge. In this study, mortality of vaccinated mice was reduced by 50% and moreover, the mean time to death was extended from 3.5 days for unvaccinated mice to 10.5 days for vaccinated mice (see Figure 3a).

In the second protection study, 10 mice were immunized subcutaneously on days 0 and 28 with 50 pg of affinity-purified FgBP (see Section entitled Purification of the fibrinogen-binding protein) emulsified in MPL+S-TDCM Ribi adjuvant. 5 control mice were immunized with PBS only and 5 mice were immunized with MPL+S-TDCM Ribi adjuvant emulsified in PBS. All mice were challenged on day 42 with 1.5 x 105 CFU of 5. equi. In this experiment, all vaccinates were protected following challenge of a lethal dose of 5. equi (see Figure 3b). Furthermore, at no stage did any - 14 vaccinated mouse show any clinical signs of illness. In contrast, mortality in the control group of mice was 80%.

Serum IgG response to FgBP Enzyme-linked immunosorbent assays (ELISA) were performed on sera taken from vaccinated and control mice prior to challenge in order to determine the serum IgG response to FgBP. Assays were carried out using standard procedures (Newell et al., 1988). Briefly, microtitre plates were filled with 50 pi volumes of purified FgBP solution (50.4 ng/ul of 100 mM sodium carbonate buffer, pH 9.6) and incubated overnight at 20°C. Coatedwells were blocked by incubation for 45 min with 50 mM Tris acetate buffer pH, 7.4 containing 1% (wt/vol) bovine serum albumin, 0.05% (vol/vol) Tween 20 and 0.9% (wt/vol) NaCl and washed three times with 0.05% (vol/vol) Tween 20 in 0.9% (wt/vol) NaCl (wash buffer). The wells were incubated for 2 h with serial two-fold dilutions of sera prediluted 1:250 in blocking solution and then washed three times in wash buffer. The wells were incubated for a further 1 h with 1:2000 dilution of peroxidase-labelled affinity-purified anti-mouse IgG (H+L) and washed again in wash buffer. S.S'S.S'-tetramethylbenzidine was used as substrate and absorbance readings were measured at 450nm. All assays were performed in duplicate.

Background readings were determined from the use of uncoated wells which were processed in an identical fashion to those coated with FgBP.

Results (Figure 4) show that the vaccinated mice had a high level of serum antibody to the 220-kDa FgBP whereas the control mice did not. This was confirmed by Western immunoblots conducted using the above sera (data not shown).

These data show that purified FgBP offers significant protection to mice against lethal challenge by 5. equi.

Vaccinogenic potential of FgBP against S. equi infection in horses In view of the encouraging results obtained in the mouse challenge experiments, the vaccinogenic potential of purified FgBP was tested in the host species for 5. equi, the horse. - 15 Vaccine FgBP was purified using fibrinogen-affinity chromatography as described in the Section entitled Purification of the fibrinogen-binding protein.

Challenge The challenge organism, 5. equi, was grown, washed and stored as described in the Section entitled Assessment of the protective potential of FgBP in a mouse model.

Experimental animals Two thoroughbred male horses (2-3 years) and one male pony (12 years) were used in the trial. None had a previous history of strangles.

Vaccination and challenge Two horses, termed Hl (a thoroughbred) and H2 (the pony), were vaccinated with FgBP intranasally three times at approximately three week intervals i.e. on days 0, 21 and 42. Each vaccination consisted of 500 |jg of FgBP in 4 ml PBS. One control horse, termed H3 (a thoroughbred) was immunized by the same protocol using PBS alone. Nineteen days after the final booster (day 61), all three horses were challenged intranasally with 1 x 107 CFU of 5. equi in 4 ml of PBS.

Intranasal vaccination and challenge were administered using a dog urinary catheter (50 cm length x 2.0 mm diameter) attached at the proximal end to a 20-ml syringe. The distal end of the dog catheter was heat-sealed and about 15-20 small holes (<1 mm in diameter) were made around its circumference. The entire length of the catheter was inserted into the horse's nose and the fluid sprayed into the tonsillar region. A 2-ml volume was delivered in this manner up each nostril.

Evaluation of response to vaccination and challenge The clinical response of the horses to vaccination and challenge was determined on a daily or every-other-day basis, using the clinical scoring - 16 system outlined in Figure 5. Blood samples and two nasal swabs were taken at weekly intervals. Blood samples were subject to routine haematological and biochemical tests as well as ELISA tests to monitor serum IgG to FgBP. In addition, nasal swabs were analyzed for microbial flora and by ELISA for secretory IgG and IgA to FgBP.

Haematology, blood biochemistry and microbiology Haematological, biochemical and microbiological tests were performed by the Irish Equine Centre, Johnstown, Co. Kildare, Ireland. Blood haematology tests included those for red blood cell, packed cell volume, haemoglobin, mean cell volume, mean corpuscular haemoglobin content, platelets, white blood cell, fibrinogen, neutrophils, lymphocytes, monocytes and eosinophils. Blood biochemical tests included those for total protein, albumin, aspartate aminotransferase, creatinine kinase, gamma glutamyltransferase, total bilirubin, sodium and potassium levels. Microbiological analysis included routine tests for the presence of Staphlycoccus aureus, -haemolytic streptococci, B-haemolytic streptococci, Neisseria sp., Bacteroides sp., Clostridium sp., non-haemolytic Escherichia coli and Streptococcus equi.

Processing of nasal swabs for ELISA assays Nasal swabs were individually immersed in 1 ml of saline for 18 h at 4°C. Samples were then vortexed vigorously for 2 min and the saline removed. The swab was then vortexed in a further 0.5 ml of saline. The final volume of the combined nasal swab washings was then adjusted to 1.5 ml, the washings were centrifuged at 13,000 x g for 10 minutes and the supernatant fluids were stored at -20°C until use.

ELISA of serum and secretory immunoglobulin to FgBP ELISA assays were carried out by standard procedures essentially as described in the Section entitled Assessment of the protective potential of the FgBP in the mouse model. After coating with FgBP and washing, wells were incubated for 2 h with serial two-fold dilutions of either sera (prediluted 1:5000 in blocking buffer) or nasal swab washings. After rinsing the wells in wash buffer, the wells were incubated for 1 h with either a 1:2000 dilution of peroxidase-labelled affinity-purified - 17 anti-horse IgG (H+L) or a 1:2000 dilution of peroxidase-labelled affinity-purified anti-horse IgA (a-chain specific). 3,3'5,5'-tetramethylbenzidine was used as substrate and absorbance readings were measured at 450nm. All assays were performed in triplicate. The ELISA endpoint titre was regarded as the reciprocal of the serum or nasal swab washing which gave an θθ45θηιη of 0.1 and was calculated graphically. Background readings were determined from the use of uncoated wells which were processed in an identical fashion to those coated with FgBP. Titres obtained from the use of nasal swab washings were normalised with respect to total protein.

Results The total clinical scores of the three horses are shown in Figure 6.

Of the 16 individual clinical symptoms incorporated in the total clinical scores (Figure 5), temperature is considered to be the best indicator of forthcoming illness and is highlighted in Figure 7. Two other relevant (blood) parameters i.e. fibrinogen levels and white blood cell counts, which are indicative of infection and inflammation are shown in Figures 8, and 10. The immunoglobulin titres of the sera and nasal swab washings are shown in Figures 11 and 12, respectively. (a) Prechallenge and general observations Of the three horses, Hl showed a higher base-line total daily clinical score than either of the other two (Figure 6), a feature caused principally by slightly enlarged lymph glands (i.e. a lymph palpatation clinical score of 2 as compared to a score of 0 for both H2 and H3) and which may have been due to an underlying chronic respiratory disease. Throughout the trial, the vaccinated pony (H2) exhibited very erratic fibrinogen levels (Figure 9), a feature caused not by infection/inflammation but by fluctuations in physical activity as evidenced by dramatic changes in the levels of two muscle enzymes (aspartate aminotransferase and creatinine kinase; data not shown). It is apparent from a study of individual clinical indices and the total daily clinical score prior to challenge that there were no adverse reactions to the vaccination regimen.

In response to vaccination, there was a significant increase (up to 35-fold) in the titre of serum IgG against FgBP in horse H2, but only a marginal increase in horse Hl. As expected the unvaccinated control H3 showed baseline serum IgG titres prior to challenge (Figure 11). Similar profiles were obtained for secretory IgG (Figure 12). (b) Post-challenge observations All three horses were challenged on day 61, nineteen days after the final vaccination (day 42). All horses remained stable for at least 11 days post-challenge (day 72). The vaccinated pony (H2) never showed any signs of illness over the full course of the trial. There was no significant increase in clinical scores (Figures 6 and 7) and 5. equi was never isolated from nasal swabs. Eleven days post-challenge, the vaccinated horse Hl developed an increase in temperature (increase in score from 0 to 4) followed by an increase in nasal discharge (increase in score from 0 to 2; see Figures 6 and 7). Its total daily clinical score increased over two-fold to a maximum score of 13 and, there was an increase in levels of fibrinogen and white blood cells (see Figure 8). That the infection was strangles was confirmed by the isolation of 5. equi from the nasal discharge. After 11 days, the horse had recovered.

Twenty days post-challenge (day 81), the temperature of unvaccinated control horse, H3, started to increase (Figure 7). This was followed by an increase in throat swelling (increase in score from 0 to 4) and excessive nasal discharge (increase in score from 0 to 4). Significantly, the clinical symptoms of disease of this horse were much more severe than that evidenced in either of the vaccinates. The total daily clinical score of H3 increased seven-fold to a maximum score of 19. Together with obvious clinical signs of illness and distress there was also a clear increase in the levels of fibrinogen and white blood cells (Figure 10), the isolation of 5. equi from nasal discharges confirming the aetiology of disease.

After 9 days of severe infection (day 90), the condition of H3 improved and the horse was fully recovered by day 101 (see Figures 6 and 7).

Neither of the vaccinations showed significant increase in serum IgG to FgBP following challenge. Whereas, there was a marked (up to 10-fold) increase in serum IgG levels during the convalescent period for unvaccinated control H3 (Figure 11). In contrast, all three horses showed some increase in the levels of secretory IgG following challenge (Figure 12). - 19 Three important conclusions can be drawn from this equine trial. Firstly, intranasal vaccination with purified FgBP in PBS does not cause any discernable local or systemic side effects. Secondly and more significantly, vaccination of horses with purified FgBP, in the absence of adjuvants or transdermal penetrants, affords protection against experimental 5. equi infection. Thus, the vaccinated pony (H2) did not show any clinical symptoms of disease, whereas the other vaccinated horse (Hl) showed a much milder form of the disease than that evidenced by the unvaccinated control horse (H3). Finally, in view of the above, purified FgBP has considerable potential as a constituent of a subunit vaccine against strangles.

Cloning of the gene encoding the FgBP The general strategy for cloning the gene encoding the FgBP involved: (a) the design of a degenerate oligonucleotide probe made from a knowledge of amino acid sequence; (b) Southern blotting experiments to identify a positive clone in a 5. equi chromosomal gene library; (c) subcloning and sequencing of positive-reacting restriction fragments.

Design of oligonucleotide probe To facilitate the design of a suitable oligonucleotide, N-terminal amino acid sequence analysis of defined peptides fragments of FgBP was undertaken. Peptide fragments were generated as follows. Mutanolysinsoluble extracts of purified cell-wall material from 5. equi were electrophoresed on SDS-polyacrylamide gels containing 12.5% [wt/vol] polyacrylamide. The SDS-gels were stained with Coomassie-brilliant blue and bands corresponding to FgBP were excised from the gel. These acrylamide strips, containing about 5-10 pg protein, were then equilibrated in Laemmli running buffer, inserted into wells of a new SDS-gel (12.5% [wt/vol] polyacrylamide) and overlayed with Laemmli sample buffer containing staphylococcal V8 protease. Samples were electrophoresed briefly (20 min, 10 mA) then in situ proteolysis allowed to proceed for one hour. Electrophoresis was then continued in the standard fashion.

Analysis revealed that FgBP could be degraded to several peptides with apparent Ms 3,000-14,000. These peptides were transblotted onto TM ’ Problot and N-terminal amino acid sequence analysis was performed directly on three of these peptides using an Applied Biosystems 447A pulsed- 20 liquid protein sequencer. Analysis yielded two distinct sequences. These are given as follows in standard single letter code (1) NSEVSRTATPRL; and (2), LQKAKDERQALTESFNKTLS. A suitable degenerate oligonucleotide (Figure 13) was then made from a consideration of amino acid sequence No. 2 above.

Isolation of 5. equi chromosomal DNA Genomic DNA was isolated from 5. equi using a modification of the method described by Lindberg et al. (1972). Bacteria were cultured in 150 ml of broth as described in the section entitled Isolation of cell wall-associated proteins. Bacteria were then harvested, washed once in 50 mM Tris-malate (pH 7.0) containing 10 mM MgC^. once in 100 mM Tris-HCl (pH 7.5) containing 10 mM EDTA, 120 mM NaCl and finally resuspended in 6 ml of 100 mM Tris-HCl (pH 7.5) containing 10 mM EDTA, 120 mM NaCl and incubated for 30 min at 37°C with mutanolysin (8,000 U) and lysozyme (50 mg). Isolation of DNA then followed the published procedure (Lindberg et al., 1972).

Construction of gene bank of S. equi DNA A gene bank in the lambda replacement phage,/(GEM11, was made essentially as described by the suppliers (Titus, 1991a). 5. equi genomic DNA was partially cleaved with the restriction endonuclease, Sau3M, in order to generate fragments in the size range 15-20 kb. These were then partially filled-in with dATP and dGTP using DNA polymerase 1 Klenow fragment and were ligated with the left and right arms of the A.GEM11, which had been precleaved with ΧΛοΙ and partially filled-in with dATP and dGTP. Size selection of inserts is achieved during packaging as only/^arms containing inserts of between 9 and 23 kb are encapsidated. The titre of the resultant library was 2 x 10θ. '-end labelling of degenerate oligonucleotide The degenerate oligonucleotide (Figure 13) was labelled with P dATP using T4 polynucleotide kinase as described by S. Tabor (1995). The reaction mixture containing forward exchange buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl9, 5 mM dithiotreitol [DTT] and 0.1 mM spermidine), 10 pmol degenerate oligonucleotide, 50 pCi r dATP (3000 Ci/mmol j and T4 polynucleotide kinase (10 U) was incubated at 37°C for 50 min. The reaction was terminated by addition of 25 mM EDTA (pH 8.0) and the volume of the reaction mixture was increased to 100 pi. Unincorporated dATP was removed from the labelled probe using a Sephadex G-25 column (NAP-5) as described by the supplier (Pharmacia LKB Biotechnology).

Plaque hybridizations Plaques of the A gene library were propagated on E. coli LE392 (about 500 plaques per plate; Figure 14) using the overlay method outlined in Titus (1991a). Plaque hybridizations were based on the method of Southern (1975) as outlined by O'Reilly et al. (1988) with the following modifications. Plaque blots were hybridized at 50°C for 2 h in prehybridization solution (5X SSC [saline sodium citrate contains 0.17 M sodium citrate and 0.15 M NaCl], 0.5% [wt/vol] SDS and 5X Denhardts solution) and hybridized at 50°C for 18 h in prehybridization solution containing 1 pmol labelled oligonucleotide probe. Blots were then washed at 37°C for 15 min in 5X SSC, for 15 min in 2X SSC and at 42°C for 15 min in IX SSC. The blots were then sealed into plastic bags and exposed to X-ray film at -70°C for 3-7 days.

The above hybridization experiments identified one plaque (ASE12.1) which reacted positively with the labelled oligonucleotide.

This plaque was picked, resuspended in phage buffer (20 mM Tris-HCl, pH 7.4 containing 0.1 M NaCl and 10 mM MgSO^) and propagated to homogeneity.

Isolation of DNA from λ SE12.1 DNA was isolated from ASE12.1 by a method which involved purification and concentration of phage particles, lysis of phage capsids and purification of released DNA. Firstly, a high titre plate stock of ASE12.1 (3 x 10^1) plaque forming units [PFU] were prepared as detailed by O'Toole & Foster (1988). This plate stock was then used to prepare a high titre liquid lysate stock of A-SE12.1 as described by O'Toole & Foster (1988) with the following modifications. 500 jjI of an 18-h culture of E. coli LE392 grown in L-broth containing 2% (wt/vol) maltose and 10 mM MgSO^, was incubated at 37°C for 15 min with 5 x 10® PFU of ASE12.1 in the presence of 10 mM CaCl2 and 10 mM MgCl2· This mixture was then added to 250 ml of phage medium (1% casamino acids, 1 x M9 salts, 0.4% (wt/vol) glucose, 0.4% (wt/vol) maltose, 5 mM MgCl2, 0.1 mM CaCl2 and 30 pg/ml - 22 tryptophan) in a 2-litre baffled flask and incubated at 37°C for about 6 h at 300 rpm. 5 ml of chloroform was then added to the lysate in order to kill any remaining bacteria. Phage particles were precipitated by addition of NaCl (to a final concentration of 0.5 M) and PEG 6000 (to a final concentration of 10% [wt/vol]) to the lysate and purified to homogeneity on caesium chloride gradients following the procedure outlined by O'Toole & Foster (1988). DNA was isolated from the purified particles as detailed by Maniatis et al. (1982).

Southern hybridization experiments Phage DNA was digested with various restriction enzymes using conditions described by the manufacturers and the restriction digests were 32 analysed by Southern blotting experiments with the P-labelled oligonucleotide probe (Southern, 1975). Prehybridization, hybridization and stringency of washings were performed as described in the section entitled Plaque hybridizations. The results of the Southern blotting experiments showed that the recombinant phage possessed a 5. equi DNA insert of about 15 kb. A 1.8 kb Sacl restriction fragment which was located adjacent to one of the phage arms reacted with the oligonucleotide probe.

Cloning of 1.8 kb Sacl restriction fragment Sacl restriction fragments of λSE12.1 was ligated to Sacl-digested plasmid pGEM-7 Zf(+) and transformed into E. coli XLl-Blue (Figure 14) as described by Titus (1991b) and Seidman et al., 1995. Transformants were plated onto L-agar plates containing ampici11 in (lOOjjg/ml, 5-bromo-4-chloro-3-indolyl B-D-galactopyranoside (X-gal; 40 pg/ml) and isopropyl-B-D-thiogalactopyranoside (IPTG; 40 μΜ). Plasmid DNA was isolated from several transformants (Birnboim, 1983; Birnboim & Doly, 1979) and analyzed in Southern hybridization experiments using the labelled oligonucleotide probe. One recombinant of pGEM7 containing a positive-reacting 1.8 kb insert was identified and the recombinant plasmid is termed pFBPlOO.

DNA sequencing of Sacl restriction fragment The nucleotide sequence of most of the Sacl insert was determined. - 23 This involved preparation of plasmid DNA, generation of nested deletions of the plasmid and DNA sequence analysis of 300-400 bp stretches of the said deletions.

Plasmid DNA was purified from broth-grown E. coli (pFBPlOO) by a modified alkaline lysis method as outlined by Feliciello & Chinali (1993). pg of the purified plasmid DNA was cleaved to completion with two restriction enzymes Sphl and fcoRI, which generates exonuclease III resistant and exonuclease III sensitive sites, respectively. 200-300 bp nested deletions of the digested plasmid were generated using exonuclease TM III (Erase-a-Base system) as described by the manufacturer (Titus, 1991c). The deleted plasmid DNA was then treated with Klenow DNA polymerase, ligated with T4 DNA ligase and transformed into E. coli DH5K (Figure 14). Transformed cells were plated onto L-agar containing ampicillin (100 jig/ml). Plasmid DNA was isolated from several of the resultant recombinants from each time point and analysed by restriction digestion and agarose gel electrophoresis.

For DNA sequence analysis, plasmid DNA with overlapping deletions was isolated by the method of Feliciello and Chinali (1993). Automated DNA sequence analysis of the overlapping deletions was performed using an Applied Biosystems 373A DNA sequencer and Taq DyeDeoxy terminator chemistry (DNA sequencing service, King's College School of Medicine and Dentistry, London). DNA sequence was determined using the M13 forward primer 24mer (5' CGCCAGGGTTTTCCCAGTCACGAC). 1400 bp of nucleotide sequence was determined (Figure 15).

Translation, to amino acids, of the reverse complement of the 1400 bp sequence resulted in the identification of two stretches of amino acid sequence corresponding to those determined from direct amino acid sequence analysis of V8 protease fragments of the protein itself (Figure 15 and Section entitled Amino acid sequence analysis of FgBP). Immediately preceding one of these amino acid sequences (sequence No. 1) is a stretch of 36 amino acids which shows homology with signal sequences of other streptococcal cell wall proteins. The results of the nucleotide sequencing experiments have identified an open reading frame encoding for the 5' end of the FgBP. - 24 Cloning of a 6 kb Sphl-Sfil restriction fragment Experiments were performed to isolate a DNA fragment containing the entire gene encoding the FgBP. This involved digestion of /(SE12.1 with various restriction enzymes, analysis of the digests in Southern blotting experiments using the Sad fragment as a probe and cloning of an appropriately sized restriction fragment into a plasmid vector.

The 1.8kb Sad fragment, purified from an agarose gel, was random *5 Ο TM labelled witho(- P dATP using the Prime-a-gene labeling system supplied by Promega (Titus, 1991d). The labelled fragment was used, in Southern blotting experiments, to screen ASE12.1 which had been digested with various restriction enzymes. The basic method of Southern blotting was as described in the Section entitled Southern blotting experiments with the following modifications. Prehybridizations and hybridizations were carried out a 65°C. The hydridization solution contained 5ng of labelled probe. After hybridization, blots were washed as follows: twice for 2 min at 65°C in a solution containing 2X SSC and 0.5% (wt/vol) SDS, twice for 15 min at 65°C in a solution containing 2X SSC and 0.1% (wt/vol) SDS and twice for 15 min at 25°C in a solution containing IX SSC and 0.1% (wt/vol) SDS. The results of the Southern blots showed that a Sphl-Sfil restriction fragment of approximate size 6kb reacted with the labelled gene probe.

This Sphl-Sfil fragment was cloned into the plasmid pGEM-7 Zf(+) (Promega) as follows. Phage DNA was digested to completion with Sfil and Sphl. Digested phage DNA (200 ng) was then filled-in with dNTPs using DNA polymerase 1 Klenow fragment, ligated to 150 ng of Smal-digested pGEM-7 Zf(+) and transformed into E. coli DH5o((see Section entitled Cloning of Sacl restriction fragment). Southern blotting experiments identified a recombinant plasmid (pFBP200) which contained the Sfil/Sphl restriction fragment (Figure 16). The recombinant E. coli strain is termed E. coli MM2 and was deposited at the National Collections of Industrial and Marine Bacteria under the Accession Number NCIMB 40807 on 25th June 1996.

Suppliers of reagents and chemicals TM CnBr-activated Sepharose was obtained from Pharmacia LKB - 25 Biotechnology, Milton Keyes, U.K. ImmunoPure^Immobi 1 ized protein G was obtained from Pierce Chemical Co., IL, USA. 96-well ELISA plates were obtained from Sarsdedt Ltd, Wexford, Ireland. Horse fibrinogen, horseradish peroxidase and peroxidase-labelled affinity-purified anti-mouse IgG (H+L) were obtained from Sigma Chemical Co., Dorset, U.K. Restriction enzymes and other molecular biology reagents were obtained from Promega Corporation, WI, USA and New England Biolabs Inc., MA, USA. The TM TM Erase-a-Base system and Prime-a-gene labeling system were obtained from Promega Corporation, WI, USA. Peroxidase-labelled affinity-purified goat anti-horse IgG (H+L) was obtained from ICN Biomedicals Inc., CA, USA. Peroxidase-labelled affinity-purified goat anti-horse IgA (a-chain specific) was obtained from Bethyl Laboratories Inc., TX. MPL+S-TDCM Ribi adjuvant was obtained from RIBI Immunochem Research Inc., MT, USA.

Standard Amino Acid Abbreviations Amino acid Single letter code Alanine A Arginine R Asparagine N Aspartic acid D Cysteine C Glutamine Q Glutamic acid E Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenyalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V - 26 References Birnboim, H.C. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol 100:243-255.

Birnboim, H.C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acid Res. 7:1513-1523.

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Fischetti, V.A. 1991. Streptococcal M protein. Sci. Am. 264:32-39. - 27 Galan, J.E. and J.F. Timoney. 1988. Immunologic and genetic comparison of Streptococcus equi isolates from the United States and Europe. J. Clin. Microbiol. 26:1142-1146.

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Claims (5)

1. A fibrinogen-binding protein isolated from 5. equi subsp. equi having the following characteristics:(1) it migrates on Laemmli SDS-PAGE gels with an apparent molecular weight of approximately 220,000 D, (2) it binds horse fibrinogen, and (3) it protects against 5. equi infection in horses (Strangles), or a truncate thereof.

2. A DNA fragment as deposited at The National Collections of Industrial and Marine Bacteria under the Accession Number NCIMB 40807, encoding the 5. equi fibrinogen-binding protein or a fragment substantially similar thereto also encoding 5. equi fibrinogenbinding protein and/or Strangles protective activity.

3. A method of producing an 5. equi fibrinogen-binding protein comprising the steps:(1) genetically engineering a host cell containing the DNA fragment of claim 2 to overexpress and/or secrete the fibrinogen-binding protein (or suitable truncate) into the supernatant, (2) isolating the fibrinogen-binding protein or truncate in a cell-free supernatant fraction, and (3) purifying the fibrinogen-binding protein or truncate.

4. A fibrinogen-binding protein substantially as described herein with reference to the Examples and/or the accompanying drawings.

5. An 5. equi fibrinogen-binding protein whenever prepared by a process as claimed in claim 3.