SK185199A3 - COMPOUNDS ENCODING THE PROTECTIVE M-LIKE PROTEIN OF STREPTOCOCCUSì (54) EQUI AND ASSAYS THEREFOR - Google Patents

Abstract

The molecular compounds they encode are described a type M protective protein from Streptococcus equi (SeM), an amino acid compound that is thus encoded, and a set of substances that include either the encoded substances or cellular components they encode. For example, the subject matter is the vaccines they use amino acid compounds or vectors and cellular the pathways required to form amino acid compounds. The following are methods for stimulating S. equispecific the horse's immune response and the way it allows detection of Streplococcus equi.

Description

Compounds encoding type M protective protein from Streptococcus equi and studies performed.

This application preferably claims the right to the original US patent application 60 / 050.577. registered June 24, 1997 .. ,, / • * 7

This invention has been developed with government grant support.

United States: USDA-NRICGP, No. 95-01837, and therefore the United States government may have certain rights in this invention.

TECHNICAL FIELD

The present invention provides methods for stimulating S. equi-specific, immune responses in horses.

BACKGROUND OF THE INVENTION

The present invention generally relates to molecular compounds that encode a type M protective protein from Streptococcus equi (SeM), an amino acid compound that is encoded therein, and a composition of substances that include either encoded compounds or cellular components that are encoded. For example, the present invention provides vaccines described herein using amino acid or vector based compounds and a cell pathway suitable for making amino acid compounds.

Streptococcus equi, Lancefield group C streptococci causes asphyxiating, highly contagious, infectious nasopharyngeal disease and drying of Equidae lymph nodes. The 28 kDa type M antifagocytic protein (SeM) is a major contagion factor and a protective antigen and a function limited by the deposition of C3b on the surface of the bacterium by directly bound fibrinogen. Boschwith and Timoney, 17 Microbiol. Pathogenesis 121 (1994) and Boschowith and Timotey, 62 Infect. Inrnun. 3515 (1994).

More recently, outbreaks of S. equi on horse farms have been prevented and treated with quarantine of suspicious animals, antiseptic food preparation, bedding and housing and antibiotics if indicated. Vaccines containing non-contagious S. equi or its fractions have been described, but the success factor was small. U.S. Pat. 5,183,659th discloses a vaccine that stimulates the nasopharyngeal antibody response in horses, but the vaccine has been limited, like many other such vaccines, by the risk of reversing a severe infection and the occasional development of inflammation in vaccinated horses.

S. equi is excreted in the nasal discharge and nodules from the lymph nodes of the affected animals. Common laboratory bacterial detection includes bacteriological cultures of nasal swabs, nasal washes and pus caused by inflammation, and is often difficult due to the presence of other contaminants, small numbers of organisms, or the presence of S. zooepidemicus and other β-hemolytic streptococci. The replenishment and identification of cultures usually takes 2 to 3 days, too long a period due to highly infectious choking and the need to quickly identify infected horses to be isolated at an early stage of infection.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide molecular compounds that encode SeM and a composition of substances that include either the encoded substances or the cellular components they encode.

It is therefore an object to present vectors, cell pathways and cell membrane products using the present compounds.

Another object is to provide a method for detecting Streptococcus equi.

Other objects and features of the present invention will be apparent from the following detailed description, examples, and requirements.

definition

The following terms should have the appropriate explanatory meaning. All other terms have the same meaning as commonly understood in the corresponding art in which these terms are used.

'Biological sample' means nasal or oral mucus or blood sample.

"Transformation" and "transfection" mean to take up the nucleic acid, enter the cell, whether or not it is incorporated into the genome.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in conjunction with the accompanying drawings, in which:

Figure 1 is the nucleotide and deduced amino acid sequence of SeM. The base and amino acid positions are shown on the left side of the figure. The putative promoter and ribosomal binding regions (RBS) are framed and the signal and membrane anchor sequences are shown in bold.

Figure 2 is the transfer of proteins utilizing specific antigen-antibody interactions, the so-called " An immunoblock presenting reactions of E. Coli BL21 lysate, SeM02, and S. equi mutanolysin extract with antisera 216 and 963 to SeM and recombinant SeM.

Figure 3 is an "immunoblock presenting reactions of mutanolysin extracts by a series of temporally and geographically separated S. equi isolates with antisera to the recombinant SeM. The estimated molecular weights are shown on the right side of the figure.

Figure 4 is an immunoblot presenting linear epitopes recognized by IgA in nasal washes of healing horses (8 weeks).

Figure 5 is a graph illustrating antibody-responsive regions of SeM in equine sera taken 8 weeks after healing from suffocation.

The present invention provides nucleic acid compounds consisting of compounds of the following sequence:

agctttctgtcacctgatggtccttatcaaatactgtaattgataacttcaaacagccct 61. gtagagattttactaacgacatagtatccatgctaagcgtcacccccttcataatcctca 121 cggtatcttattctatcttaaaatttaagaaaagcaaggatatgcacttataatgaaaaa 1.81 ATAGACÄTAAAAAACAATAATATACATTCTTGCTTATTAAATAAAAATGAC AATGTACTG 241 cataaagaagttcctgtcattaaaataaaagtgccatgäggttataatagtatggtaaaa

Caaaaaagtgtgcccataacgggtagagaggaattgacatatgtttttgagaaataacaa 301 361 421 gccaaaatttagcatcagáaaactaagtgccggtgcagcatcagtattagttgcaacaag TGTGTTGGGAGGGACAACTGTAAAAGCGAACTCTGAGGTTAGTCGTACGGCGACTCCAAG ÄJTATCGCGTGATTTAAAAAATAGATTAAGCGATATAGCCATAAGTGGAGATGCCTCATC 481 541 601 AGCCCAAAAAGTTCGAAAiyTTCTAAAAGGCGCCTCTGTTGGGGATTTACAGGCATTATT gagaggtcttgattcagcaagggctgcgtatggtagagatgattattacaatttattgat 661st gcacctttcatcgatgttaaatgataaacctgatggggatagaagacaattaagtttggc TTCATTACTTGTAGATGAAATTGAAAAGCGGATTGCTGATGGAGATAGGTATGCAAAACT 7 721 81 841 tcttgaggctaaacttgcagctattaaatctcaacaagaaatgcttagagaaagagattc CCAACTTCGAAATCTAGAGAAGGAGAAAGAACAAGAGCTCäCAAAAGCTAAAGATGAGCG

901 tcaagctcttaccGaatcattcaacaaaactttatcaagatcaacaaaagagtataataa

961 ACTAAAAAfcAGAACTTGCAAAAG AAAAAGAÁAAAGCAGCTAAGATG ACTAAGGÄATTAGC r- · .ľ.

1021 AGATAAGCTAAGCAATGCTGAAGCAAGTCGTGATÁAAGCCTTTGCAGTATCAAAAGATTT t

1081 agcagataaactaagtagtgctgaagcaagtcgtgátaaagcttttgcagtatcaaaaga

1141 tttagcagataaattggcagctaaaacagcagaagctcaaaagttaatggaaaacgttgg tagtctagaccgcttggtagagtctgcaaaacgtgaaatggctcaaaaattagcagaaat 1201 1261 1321 tgatcaattaactgctgataaggctaaggctgatgcagagcttgcacctgcaaatcacac CATTGCATCACTTCAAACAGAGCTAGAAAAAGCTAAGACAG AGCTTGCTGTTTCAGAGCG

1381 TTTGATTGAATCAGGCAAACGTGAAATTGCTGAGCTACAAAAACAAAAAGATGCTTCTGA

1441 TAAGGCTTTAGTAGAATCACAAGCTAATGTAGCAGAGCTTCAAAAACAAAAAGCAGCATC agatgctaaggtagcagagcttgaaaaagaagttgaagctgctaaagctgaggttgcag 1501 and 1561 tcttaaagcacaattagctaagaaagaagaagägcttgaagccgttaagaagg aaaaaga 1621 agcgcttgaagctaagattgaagagctcaaaaaagctcatcctgaggaactttcaaaact taaagaaatgcttgagaagaaagaccatgcaaatgcagatcttcaagcagaaatcaatcg 1681 1741 1801 ^ CTTGAAGCAAGAGCTAGCTGACAGGATTAAGTCATTGTCACAAGGTGGTCGTGCTTCACA acaaacccaggcactacaactgctaaagcaggtcaattgccatctactggtgagtctgc taacc'cattcttcactattgcagctcttactgtcatcgctggtgctggäatggctgtggt 1861 1921 1981 gtctcctaaacgcaaagaaaactaagctatttcctctttccccaatggacaatagccg aa ATAATAGAGCGACTATCGTTCTAACAC AAAAGCAACAGTCTCCTGTCTGTTGCTTTTTGT GATATTAGGGCTCATC AGTCTAGGCTAATGGTTTTCTGCGCTTTATCTGCA 2041 [SEQ ID No. (M-protein (SEM) gene of S. equi)]. The open reading frame starts at position 341 and ends on the basis of 1934. It is initially a compound of SEQ ID NO 1. The following are also compounds complementary to SEQ ID NO 1. The preferred portion of SEQ ID NO 1 is that described above as the base Nos. 341 to 1943, inclusive (SEQ ID NO 3). In addition, compounds defined by bases number 452 to about 1331 are also preferred (SEQ ID NO 4).

As can be appreciated by those skilled in the art, the examples of DNA compounds listed above can readily derive complementary RNK sequences. Each such RNK sequence is also considered to be within the scope of the present invention. In the above description, these DNA and RNK compounds are called "nucleic acid compounds".

In addition, the cells transferred by the nucleic acid compound (or multiple copies thereof) disclosed herein are presented herein. Preferably, the cell is transfected with SEQ ID NO 1 or a portion thereof. Such cells may be prokaryotic or euracryotic. Preferred cells include: E. coli, S. cerevisiae, and Salmonella spp. Also shown are vectors transfected with the nucleic acid compound (or multiple copies thereof) disclosed herein. Preferred vectors include varicella viruses, adenoviruses or other viral vectors.

Nucleic acid compounds may be obtained using PCR primers or from a gene bank, accession number U73162. Cell binding vectors can also be obtained based on experience in the art.

In addition, the following is an amino acid compound:

I

5 1 M F L R N 10 N R P R 1 ¹⁵ FSI · RRL 20 25 SAGAASVLVAT5 30 ' V L G 31 G T T V K A N S E V S R T A T PRLSRDLKNRLS D I A 61 I S G D A S S A Q K V R N L · L KGASVGDLQALL R G L 91 D S A R A A Y G R D Y Y N L L M H. L SSMLNDKP D G D 120 R R Q L S L A S L L V D E I E KRIADGDRYAKL LEA 151 K L A A I R S Q Q E M L R E R DSQLRNLEREKE Q E L 181 T C A K D E R Q A L T E S F N RTLSRSTKEYNK L K T 210 E L A K E K E K A A K M T R E LADKLSNAEASR D K A 241 F A V S R D L A D R L S E A A S .Ŕ DRAFAVSKD ICE 271 K L · A A K T A E A E K L M E N VGSLDRLVESAK R E M 302 A Q R L A E I D Q L T A D R A RADAELAAANDT I A S 335 L Q T E L E R A R T E L A V S ´ « ERLIESGKREIA E L Q 364 R Q R D A S D R A L V E S Q A NVAELEKQKAAS DAR 391 V A E L E REVE A A R A E V ADLXAQLAKKEE E L E 421 A V R R E REAL E A R I E E LKRAHAEELSKL K E M 452 L E R R D HANA D L Q A E X NRLKQELADRIK S L S 482 Q G G R A S Q T N P G T T T A KAGQLPSTGESA N P F

The present invention also encompasses portions of the aforementioned sequence. The most preferred sequence of the above sequence is: residues 37-33C (SEQ ID NO 5), but one skilled in the art can recognize that each special antigenic moiety is of commercial significance and is within the scope of the invention.

Amino acid compounds can be obtained either by multiple expression and purification in a microorganism, or in some cases by conventional peptide synthesis. The present invention provides a vaccine for S. equi. A vaccine that is administered by the nose or orally is preferred. The vaccine presented in the present invention may comprise all or part of SEQ ID NO 2.

The vaccines presented in this invention may be part of any pharmaceutically acceptable formulation. For example, SEQ ID NO 5 can be incorporated into bilayer cavities (liposomes) in an aqueous environment according to known procedures per se, as described in Debs et al., 265 J. Biol.Chem. 10189 (1990). Any achievable carrier or liposome-forming lipid can be used in any of the formulas that deliver the SeM antigen, for example, poly-DL-Lactide-co-glycolid can be used in a nasal spray that comprises SEQ ID NO 5. Formulas containing feed enhancing components an antigen to mucosa, such as a small amount of cholera toxin B-subunit, are also within the scope of the invention.

The present invention also provides methods that stimulate a S. equi specific immune response in horses comprising the present compound encoded by SEQ ID NO 2 or portions thereof. Nasal or oral administration of the application is preferred.

Finally, the present invention presents methods that determine the presence of S. equi in horses through a polymerization chain reaction. The diagnostic polymerization chain reaction assays of the present invention can be compared to known methods if the primers for the sequences disclosed herein are used as some of the starting materials. Methods for PCR can be found in many magazines and books, for example, PCR diagnostic methods can be performed according to Techniques in PCR, PCR, Current Protocols in Molecular Biology or Maniatis. As those skilled in the art will appreciate, those primers that contain at least 50% GC, preferably a size of 19 to 23 base pairs, and are not capable of duplicating portions of the target DNA in the priming step are preferred.

EXAMPLES OF THE INVENTION

Example 1: Cloning, Sequencing, and SeM Expression

The chromosomal DNA associated with S. equi CF32 was partially digested with Tsp 5091 (New England Biolabs Inc., Beverly MA) and the 3-8 kb fragments were ligated into λ ZAPII digested with EcoRI (Stratagene, LaJolla, CA). After digestion (Gaigapack II) (Stratagene, LaJolla, CA) and transfection into E. coli XLI-Blue MRF (Stratagene, LaJolla, CA), the library was seeded, propagated and stored at -70 ° C in 7% DMSO. The library was tested on duplicate nitrocellulose discs

ΙΟ use of rabbit antiserum 216 (diluted 1: 400) in the 41 kDa acid extract of the SeM fragment. Several reaction plaques were analyzed while all plaques showed a positive signal. The proteins in these phage lysates were separated by SDS PAGE and immunoblotted with serum 216. The plasmid containing the 3.5 kb fragment encoding SeM was removed from the positive phage and the resulting plasmid was designated pSeM01. Nucleotide sequencing was performed on HindIII, PvuII and HindIII-PvuII fragments of S. equi inserts inserted into pSK by automatic cyclic sequencing. Sequences were aligned, aligned and pooled using DNASIS (Hitachi Software Engineering America, Ltd., San Diego, CA). SeM without the signal sequence was subcloned into the BamHI site of the expression vector pET15b (Novagen, Madison, WT) using nucleotide chain amplification (PCR) methods with pSeMO1 as template and primers SeM-F (gcggatcCGAACTCTGAGGTTAGTCGT) (SEQ ID NO 6) and SeM-R (SeR-NO 6). gcggatccATAGCTTAGTTTTCTTTGCG) (SEQ ID NO 7). The resulting plasmid was designated pSeM02 and transformed into E. coli BL21 (DE3). Recombinant SeM was isolated from BL21 lysate by affinity chromatography on His-Bind Resin (Novagen, Madison, WI).

Sequence analysis revealed the presence of one open reading frame consisting of 1605 nucleotides encoding the seM gene (FIG. 1). The translation revealed a base (CH +4.5) preprotein consisting of 535 amino acids with a calculated molecular weight of 58.251 and a pI of 8.67. The N terminus of the amino acid sequence (residues 37-52) was identical to that obtained by direct micro-sequencing of the 41 kDa SeM fragment purified from S. equi acid extract. The predicted amino acid sequence properties show typical properties of streptococcal surface proteins. The signal sequence represents 36 residues. The N terminus of the stabilized protein has a positive charge. The anchor membrane of the interspersed region and terminal sequences are similar to those of the other groups A and C of the streptococcal sequences. Two straight repeats (21 residues) are located between residues 226 with 267. The other shorter straight repeats vary in length from 3 to 6 residues appearing in the carboxy terminal half of the molecule. Analysis of the secondary structure of the translated protein shows an extensive alpha helix region extending from residue 120 to 480. The predicted secondary structure exhibits bends near residues 120 and 480-500.

Example 2: Determination of amino acid sequence

The purified acid extract (2 mg), as described in Example 3, was inserted into a 2 cm wide 1.5 mm acrylamide gel groove for the prepared SDS PAGE. The gel was first subjected to a 40 minute wiring at 100 V s 0 prior to protein loading. , 1 mM thioglycolic acid. Electrophoresis followed and e. octrophoretic transfer to rmmobilon P (MjLipore), major 41 and 46 kDa SeM fragments were identified by ootioco, arbenia for 1 min. with .025% Coomassie blue in 40% methanol and 5% glacial acetic acid followed by 2 minutes. inkuoacioi. in 30% methanol and 5% glacial acetic acid. A large kDa fragment was excised and analyzed by sequencing on a 477A pulse liquid phase sequencer (Applied Bio Systems) at the Macromolecular Synthesis Laboratory at the University of Kentucky.

Example 3: Protein extraction

Type M protein was extracted from a culture of S. equi cultured overnight (18 h) by hot acid extraction (Lancefield and Perlmann. 96 J.Exp.Med. 71 (1952)) and adsorbed onto a column of hydroxyapatite in 10 mM phosphate buffer. solution with pH 7.2. Type M proteins were eluted into 0.2 M Na ₂ HPO ₄ , desalted on Sephadex G25, and lyophilized. The pellet was dissolved in 25% acetonitrile with 0.5% trifluoroacetic acid and loaded onto a reverse phase phenyl RP column (Bio-Rad, San Francisco CA) coupled to a Waters 650 protein purification system (Waters, Marlborough, MA). The protein was eluted using a linear gradient of 25 to 65% acetonitrile with 0.5% TFA. A spot immunoblot on nitrocellulose using specific rabbit antiserum SeM was used for peak analysis. The SeM-containing peak eluted at 42% acetonitrile. Positive peaks from several fractions were pooled and further purified on the same column. The purified protein was lyophilized, resuspended in PBS and stored in portions at -20 ° C. Mutanolysin extracts of bacterial strains of S. equi were treated as previously described (Galan and Timoney, 26 J. Clin. Microbiol. 1142 (1988)).

3

Example 4: Antisera

Antiserum was increased over purified SeM by a combination of hydroxyapatite and reverse phase chromatography. A white rabbit from New Zealand (216) was injected subcutaneously with 50 µg of SeM in complete Freund's adjuvant for another c? 3 weeks at intervals of 2 similar doses emulsified with incomplete Freund's adjuvant. Serum was collected at

8th week. Rabbit 963 was pre-immunized similarly to recombinant SeM from E. coli sonate. Rabbit Ec was immunized with an E. coli NovaBlue lysate containing plasmid pT7 Blue without insert. Adult ICR mice were immunized with 25 µg purified SeM from E. coli sonate by HIS-Tag chromatography. Purified SeM (25 µg) was mixed with 5 µg of mycolic dipeptide (MDP) and alhydrogel (30%) and transferred to a 100 ml subcutaneous dose. Two subcutaneous nasal doses containing 250 µg SeM but not MDP were dosed 10 and 20 days later. Mice were bled for 28 days. All antisera were stored at -70 ° C prior to use.

Example 5: "Immunoblotting

Streptococcal extracts or purified from E. coli sonicate were separated by SDS-10% polyacrylamide electrophoresis (PAGE) and the electrodes were circulated into a nitrocellulose gel membrane, incubated in a corresponding antiserum dissolved 1: 200 in PBS and then / peroxase G conjugated protein. 4000). Reactive fragments were visualized using 4-chloro 1-ontol substrate (0.5 mg / ml).

Rabbit antiserum 963 to recSeM reacted with a 58 kDa protein in S. Egui mutanolysin extract and a slightly larger 60 kDa protein expressed in E. coli BL21 containing pSeM02 (Fig. 2). The same protein fragments were recognized by rabbit antiserum 216 into a 41 kDa 5eM fragment. Figure 3 represents the "immunoblot *" profile of the mutalylated extracts of a series of 5th egui isolates taken at different times in the United States and Europe. Antisera against rec-seM recognized two protein fragments of 58 and 56 kDa in all extracts.

Example 6: ELISA

The SeM fragment (41 kDa large) extracted and purified by preparative agarose electrophoresis was used to coat the probes (2.5 mg / probe) from polystyrene ELISA plates (Costar, 25880, Corning Glass Company, Corning NY). After washing and incubating in 0.1 M phosphate buffered saline (PBS) containing 0.05 Tween 20 and 1% bovine serum albumin (BSA), mouse or rabbit sera were diluted 1:80 and 1: 200 in PBS and added. in triplicate to the probes (100 ml / probe). After 3 hours incubation at 37 ° C, binding to IgG was detected with either peroxidase conjugated protein G (1: 4000) or rabbit anti-mouse IgG followed by o-phenylenediamine solution (0.001 mM). Average OD values from three measurements were measured against antigen-containing probes. and PBS.

] 5

Example 7: Opsonic test

Equine neutrophils were separated from freshly drawn heparinized horse blood with a discontinuous Percoll gradient Pycock et al., 42 Res.Vet, Sci. 411 (1987). Neurophils from 7 ml of blood were resuspended in RPM1 medium (Gipco, Grand Island, NY) and 80 ml aliquots (6 x 10 5 cells) were added in triplicate to probes from 24 cell clusters (Costar, Cambridge, Mass). Each probe contained a round glass strip (12 mm in diameter). The cell pool was incubated for 2 hours. at 37 ° C in 5% CO? and cells were washed once with PBS to remove non-attached neurophiles. Test organisms (S. equi CF32 and S. zooepidemicus W60) were grown overnight at 37 ° C in THB with 0.2% fermentation extract to OD 0.6 . 20 ml culture was added to 25 ml serum and 450 ml RPMI was added. After the dish was shaken lightly for 30 min. at 37 ° C, the cover rings were washed again with PBS (pH 7.2), fixed in 10% formalin and stained with Giemsa. The number of neurophiles with associated streptococci per 100 cells was further budgeted for each serum and expressed as a percentage. All experiments were performed in triplicate. Differences in opsonic activity of immune and control sera were statistically evaluated by Student's t-test (unpaired study) based on the mean values obtained from three experiments.

Serum from mice immunized with purified recombinant SeM showed 15-fold greater opsonic activity for S. equi than non-immune mouse serum. These sera showed strong antibody responses by ELISA to a 41 kDa SeM fragment (Example 6).

Example 8: Study of bound fibrinogen

I

Horse fibrinogen (0.5 mb / probe) was bound to 96 polystyrene ELISA probes (Costar). After washing and incubation, recombinant SeM (0.4 mg / probe) was added in triplicate form to separate probes and incubated for 12 hours at 37 ° C. After washing, a rabbit anti-serum against a 41 kDa SeM fragment diluted 1:80 was added to the appropriate probes and incubated at 37 ° C for 2 hours. Control probes consisted of probes from which fibrinogen was omitted and probes treated with sera from the same rabbits as before immunization. A number of specific rabbit antibodies that bound the fixed SeM to fibrinogen were detected as described in the ELISA protocol.

The SeM showed strong binding to equine fibrinogen-cellified ELISA plate probes. The mean ELISA value (± SD) for fibrinogen-bound SeM after correction for unspecified bound protein to the probe surface was C, 9 ± 0.1. The corrected value was 0.1 ± 0.1 when the pre-immunized serum was used to study the binding of streptococcal proteins.

Example 9: Nucleotide sequence accession numbers

The genetic bank accession number for the nuk: eMe SeM sequence is U73162.

Example 10: Homology

With the exception of the signal and membrane anchor sequences, no SeM homology to group A or GM protein sequences was found in the gene bank database. SeM shows some homologies between the intrinsic signal (39% identity; and the anchor membrane (66% identity) with those listed in the database.

Example 11: Presence of SeM binding antibodies in healing horses

Figure 3 shows the regions of SeM reacting with nr Antibody.1 in horse sera collected 8 weeks after recovery from suffocation. Most horses showed epitope responses in the central SeM region (residues 170-2/0). Reactions of individual horses to third υ N-terminus and to region. the carbon end SeM were much more different. None of the horses responded to peptide 151-166 alone. Linear epitopes recognized by IgA in healing (8 weeks) nasal mucus can be found in the straight <.alpha. Area: reacting with antibody serum (Figure 4). Multiple epitopes are normalized and as in In the case of serum antibody, there is a marked difference in the response of individual horses.

Example 12: Immunization of Unexposed One Years. ponies

Two groups of three year old Walsh ponies were immunized using microencapsulated recombinant protein.

M-protein (SeM, amino acids 231 to 330) produced by E. coli BL21 and a microencapsulated extract of the E. coli host itself. The encapsulated protein (100 µg) was sprayed into the horse's nostril on the first day using a nasal sprayer. Carrier doses of 150 and 350 µg were administered Ί. and Day 42. Serum and nasal mucus were collected on days 1, 7, 21 and 42 and analyzed for SeM-specific IgG in serum and IgA in nasal mucus.

The specific mucosal IgA response to SeM was recognizable on day 21 in two of the three ponies and all ponies on day 49. None of the three control ponies immunized with E. coli extract alone responded to SeM. No reactions to antibody serum were recorded in any of the ponies. These studies demonstrate the ability to perform selectively-inducing specific mucosal responses to antibodies in horses using a microencapsulated streptococcal peptide.

Example 13: PCR diagnostic assay

Nasal swabs (Precision Dynamic Corp. San Fernardo, CA, Culturette, Baxter Healthcare Corp., Deerfield, IL) were collected from affected and uninsulated horses during the first to fifth day after clinical diagnosis of choking on farms A, B, C, and D. Some horses were inspected more than once within the next three weeks. Nasal swabs were collected from horses from UK farms on days 15-18 after mixing and revealing two horses with clinical suffocation. Both of these horses developed asphyxiation within 17 days after being subjected to mixing. The nasal swabs were collected by dropwise addition of 50 ml of phosphate buffer, pH 7.2) through a diameter of 8 mm wide latex tube, placed 15 cm into the nostril and centrifuged at 3000 g, and the pellet pieces were separated for culture and PCR. The swabs and nasal washes were cultured on Columbia CNA equine blood agar and incubated for 8 hours at 37 ° C. Beta hemolytic colonies were cultured. separately and their behavior was tested in fermentation on soils containing lactose, sorbitol and trehalose. Mucoid betaheramolytic colonies that did not ferment any of these sugars were identified as S. equi.

DNA for PCR from nasal swabs and washes was prepared as follows: swab tips were placed! to 300 μΐ of sterilized water, vortexed, the tips removed and centrifuged at 14000 g for 10 minutes. The sediments were resuspended in 20 μΐ K-buffer (1X Gen Amp buffer II, Perkin Elmer, 0.5% Tween 20, 100 g / ml proteinase K). Nasal wash pellets were resuspended in an equivalent volume of K-buffer. The suspensions were incubated at 55 ° C, boiled for 5 minutes and then centrifuged for 5 minutes at 14000 g. The PCR reaction mixture in a total volume of 30 μΐ was prepared in Gen Amp buffer II and contained 2 mM MgCl ₂ , 0, -2 mM dNTP, 0.5 units Taq polymerase, 0.25 μΜ SeM6 and SeM7 primers and 2-5 μΐ samples. The primer sequences were 5 'TGCATAAAGAAGTTCCTGTC (SeM7-forward (base 239-258) of SEQ ID NO 8) and 5'-GATTCGGTAAGAGCTTGACG (SeM back (base 899 to 918) of SEQ ID NO 9), Mineral oil (30µ) was added for that reason.

To seal the reaction well. The cycling was performed as follows: -92 ° C for 2 minutes, 92 ° C for 1 minute; 58 ° C for 1 minute (30 times); 72 ° C for 5 minutes; and finally 4 ° C. The PCR products were separated by agarose gel electrophoresis containing ethidium bromide. The SeM fragment amplified by the primers SeM6 and 7 was 679 bp in size.

A DNA fragment of 679 bp was detected by PCR in 37 samples including 14 out of 15 that were positive in culture. PCR sensitivity appears to be much higher than growth sensitivity.

It should be noted that, despite the full disclosure of the present invention, various changes and modifications are apparent to those skilled in the art. Such changes and modifications are intended to be within the scope of the present invention as defined by the appended claims.

SEQUENCE DESCRIPTION <110 »tJjnoney, John F.

Artiuehln, Sergey

Weivereity of Xentucky Reooarch Foundation <120> Compounds encoding Streptococcus equi type M protective protein and studies conducted <130 »P-1045 <140» filed herewlth <141 »1996-05-23 <150» 60 / 050,577 I <151 » 1997-06-24 <160 »9 <170» Patentin Ver. 2.0 <210 »1 <211» 2091 <212 »DNA <213» Streptococcus equi <400 »1. _____ agctttctgt cacctgatgg tccttatcaa atactgtaat tgataacttc aaacagccct 60 gtagagatt tactaacgac atagtatcca tgctaagcgt cacccccttc ataatcctca 120 cggtatctta ttctatctta aaatttaaga aaagcaagga tatgcactta taatgaaaaa 180 atagacataa aaaacaataa tatacattct tgcttattaa ataaaaatga caatgtactg 240 cataaagaag ttcctgtcat taaaataaaa gtgccatgag gttataatag tatggtaaaa 300 caaaaaagtg Igcccataac gggtogagag gaattgacat atgtttttga gaaataacaa ^360: gccaaaattt agcatcagaa aactaagtgc cggtgcagca tcagtattag ttgcaacaag 420 'tglSdggga gggacaactg taaaagcgaa ctctgaggtt agtcgtácgg cgaótččä'äg 480 altatcgcgt gatttaaaaa atagattaag cgatatagcc ataagtggag atgcctcatc 540 agcccaaaaa gttcgaaatc ttctaaaagg cgcctctgtt ggggatttac aggcattatt 600 gagaggtctt gattcagcaa gggctgcgta tggtagagat gattattaca atttattgat 660 gcacctttca tcgatgttaa atgataaacc tgatggggat agaagacaat taagtttggc 720 t tcattactt gtagatgaaa ttgaaaagcg gattgctgat ggagataggt atgcaaaact 780 (cttgaggct aaacttgcag ctattaaatc tcaacaagaa atgcttagag aaagagattc 840 ccaact tcga aatctagaga aggagaaaga acaagagctc acaaaagcta aagatgagcg 900 tcaagctctt accgaatcat tcaacaaaac tttatcaaga tcaacaaaag agtataataa 960 actaaaaaca gaacttgcaa aagaaaaaga aaaagcagct aagatgacta aggaattagc 1020 agataagcta agcaatgctg aagcaagtcg tgataaagcc tttgcagtat caaaagattt 1080 agcagataaa ctaagtagtg ctgaagcaag tcgtgataaa gcttttgcag tatcaaaaga 1140 tltagcagat aaattggcag ctaaaacagc agaagctgaa aagttaatgg aaaacgttgg 1200 tagtctagac cgcttggtag agtctgcaaa acgtgaaatg gctcaaaaat tagcagaaat 1260 tgatcaalta actgctgata aggctaaggc tgatgcagag cllgcagctg caaatgacac 1320 cattgcatca cttcaaacag agctagaaaa agctaagaca gagcttgctg ttlcagagcg 1380 tttgattgaa tcaggcaaac gtgaaattgc tgagctacaa aaacaaaaag atgcttctga 1440 laaggctlta gtagaatcac aagctaatgt agcagagctt gaaaaacaaa aagcagcatc 1500 agatgctaag gtagcagagc ttgaaaaaga agttgaagct gctaaagctg aggttgcaga 1560 tcttaaagca caatlagcta agaaagaaga agagcttgaa gccgttaaga aggaaaaaga 1620 agcgcttgaa gctaagattg aagagctcaa aaaagctcat gctgaggaac tttcaaaact 1680 taaagaaatg ctt gagaaga aagaccatgc aaatgcagat cttcaagcag aaatcaatcg 1740 cttgaagcaa gagctagctg acaggattaa gtcattgtca caaggtggtc gtgcttcaca 1800 aacaaaccca ggcactacaa ctgctaaagc aggtcaattg ccatctactg gtgagtctgc 1860 taacccaltc tlcactattg cagctcttac tgtcatcgct ggtgctggaa tggctgtggt 1920 gtctcctaaa cgcaaagaaa actaagctat ttcctctttc cccaatggac aatagccgaa 1980 ataatagagc gaclatcgtt ctaacacaaa agcaacagtc tcctgtctgt tgctttttgt 2040 gatattaggg, ctcatcagtc taggctaatg gttttctgcg ctttatctgc and 2091 <2IO> 2 | <211> 534 <212> PRT <213> Streptococcus cqui <400> 2 -...

Met Phe Leu Arg Asn Asn Lys For Lys Phe Ser Ile Arg Lys Leu Ser 15 10 15

Ala Gly Ala Ala Val Val Leu Val Val Leu Gly Gly Thr 20 25 30

Thr Val Lys Ala Asn Ser G, at Val Ser Arg Thr Ala Thr For Arg Leu 35 40 45

Ser Arg Asp Leu Lys Asn Arg Leu Ser Asp White Ala White Ser Gly Asp 50 55 60

Ala Ser Ser Ala Gin Lys Val Arg Asn Leu Leys Lys Gly Ala Ser Val 65 70 75 80

Gly Asp Leu Gin Ala Leu Leo Arg Gly Leu Asp Ser Ala Leo Ala 90 90 95

Tyr Gly Arg Asp Asp Tyr Tyr Asn Leu Met His Leu Ser Met Met 100 105 110

Leu Asn Asp Lys For Gly Asp Arg Arg Gin Leu Ser Leu Ala Ser 115 120 125

Leu Leu Val Asp Glu Ile Glu Lys Arg ile Ala Asp Gly Asp Arg Tyr 130 135 140

Ala Lys Leu Leu Glu Ala Lys Leu Ala Ala Ile Lys Ser Gin Gin 145 150 155 160

Met Leu Arg Glu Arg Asp Ser Gin Leu Arg Asn Leu Glu Lys 165 170 175

Glu Gin Glu Leu Tlir Lys Ala Lys

Ser Phe Asn Lys Leu Ser Arg Ser Thr Lys Leu 195 200 205

Lys Thr Glu Lys Glu Lys Glu Lys Glu Lys Ala Ala Lys Met Thr Lys 210 215 220

Glu Leu Ala Asp Lys Leu Ser Asn Ala Glu Ala Ser Arg Asp Lys Ala 225 230 235 240

Phe Ala Val Ser Ser Lys Asp Ser Lys Ser Ser Ala Glu Ala 245 250 255

Ser Asp Asp Lys Ala Phe Ala Val Ser Asp Lys Asp Asp Lys 260 265 | 270

Ala Glu Ala Glu Ala Glu Lys Leu Met Glu Asn Val Gly Ser 275 280 285

Leu Asp Arg Leu Val Glu Ser Ala Lys

Ala Glu White Asp Gin Leu Thr Ala Asp, Lys Ala Lys Ala Asp Ala Glu 305 310 315 320

Leu Ala Ala Asn Asp Thr Ala Ala Leu Gin Thr Glu Leu Glu 325 330 335

Llu Ala Llu Thr Glu Leu Ala Val Ser Glu Arg Leu White Glu Ser Gly 340 345 350

Lys Arg Glu White Ala Glu Leu Gin Lys Gin Lys Asp Ala Ser Asp Lys 355 360 365

Ala Leu Glu Ser Gin Ala Asn Val Glu Llu Glu Lys 370 375 380 |

Ala Ala Ser Asp Ala Lys Glu Leu Glu Lys Glu Glu Ala 385 390 395 400

Ala Lys Ala Glu Val Ala Asp Leu Lys Ala Gin Leu Ala Lys Lys Glu 405 410 415

Glu Glu Glu Leu Glu Glu Ala Glu Glu Ala Glu Glu Ala Glu Glu Ala Glu Ala Glu Glu Ala Glu Glu Ala Glu Glu Ala Glu Glu Ala

Glu Met Leu Glu Lys Lys Asp His Ala Asn Ala Asp Leu Gin Ala Glu 450 455 460 Asn Arg Leu

Gin Gly Gly Arg Ala Ser Gin Thr Asn Pro Gly Thr Thr Thr Ala Lys 485 490 495

Ala Gly Gin Leu Pro Ser Thr Gly Glu Ser Ala Asn Pro Plie Phe Thr 500 505 510 Ala AJ Ala Leu Thr Val Ala Ala Gly Ala Gly Met Ala Val Val Ser 515 520 525

The Lys Arg Lys Glu Asn 530 <2I0> 3 <211> 1603 <212> DNA <213> Streptococcus equi <400> 3 algtttttga gaaataacaa gccaaaattt agcatcagaa aactaagtgc cggtgcagca 60 tcagtattag ttgcaacaag tgtgttggga gggacaactg taaaagcgaa ctctgaggtt 120 agtcgtacgg cgactccaag attatcgcgt gatttaaaaa atagattaag cgatatagcc 180 ataagtggag atgcctcatc agcccaaaaa gttcgaaatc ttctaaaagg cgcctctgtt 240 ggggatttac aggcattatt gagaggtctt gatteageaa gggctgcgta tggtagagat 300 gatlattaca atttattgat gcacctttca tcgatgttaa atgataaacc tgatggggat 360 agaagacaat taagtttggc ttcattactt gtagatgaaa ttgaaaagcg gattgctgat 420 ggagataggt atgcaaaact tcttgaggct aaacttgcag ctattaaatc tcaacaagaa 480 atgcttagag aaagagatte ccaacttcga aatetagaga aggagaaaga acaagagctc 540 acaaaagcta aagatgagcg tcaagctctt accgaatcat tcaacaaaac tttatcaaga 600 tcaacaaaag agtataataa actaaaaaca gaacttgcaa aagaaaaaga aaaagcagct 660 aagatgacta aggaattagc agataageta agcaatgctg aagcaagtcg tgataaagcc 720 tttgcagtat caaaagattt agcagalaaa ctaagtagtg ctgaagcaag tcgtgtg tttgcag tatcaaaaga tttageagat aaatlggcag ctaaaacagc agaagelgaa 840 aagttaatgg aaaacgttgg tagtctagac egeltggtag agtctgcaaa acgtgaaatg 900 gctcaaaaat tagcagaaat tgatcaatta actgctgata aggctaaggc tgatgcagag 960 cttgcagctg caaatgacac cattgcatca cttcaaacag agctagaaaa agctaagaca 1020 gagcttgctg tttcagagcg tttgattgaa tcaggcaaac gtgaaattgc tgagctacaa 1080 aaacaaaaag atgcttctga taaggctlta gtagaatcac aagctaatgt agcagagctt 1140 gaaaaacaaa aagcagcatc agatgctaag gtagcagagc ttgaaaaaga agltgaagct 1200 gctaaagctg aggttgcaga tcttaaagca caattagcta agaaagaaga agagcttgaa 1260 gccgttaaga aggaaaaaga agcgcttgaa gctaagattg aagagctcaa aaaagctcat 1320 gctgaggaac tttcaaaact taaagaaatg cttgagaaga aagaccatgc aaatgcagat 1380 cttcaagcag aaatcaatcg cttgaagcaa gagctagctg acaggattaa gtcattgtca 1440 caaggtggtc gtgcttcaca aacaaaccca ggcactacaa ctgctaaagc aggtcaattg 1500 ccatctactg gtgagtctgc taacccattc ttcactattg cagctcttac tgtcatcgct 1560 ggtgctggaa tggctgtggt gtctcctaaa cgcaaagaaa act 1603 <210> 4 <211> 880 <212> DNA <21 3> Streptococcus equi <400> 4 tctgaggtta gtcgtacggc gactccaaga ttatcgcgtg atttaaaaaa tagattaagc 60 gatatagcca taagtggaga tgcctcatca gcccaaaaag ttcgaaatct tctaaaaggc 120 gcctctgttg gggatttaca ggcattattg agaggtcttg attcagcaag ggctgcgtat 180 ggtagagatg attattacaa tttattgatg cacctttcat cgatgttaaa tgataaacct 240 gatggggata gaagacaatt aagtttggct {cattacttg tagatgaaat tgaaaagcgg 300 attgctgatg gagataggta tgcaaaactt cttgaggcta aacttgcagc tattaaatct 360 caacaagaaa tgcttagaga aagagattcc caacttcgaa atctagagaa ggagaaagaa 420 caagagctca caaaagctaa agatgagcgt caagctctta ccgaatcatt caacaaaact 480 (tatcaagat caacaaaaga gtataataaa ctaaaaacag aacttgcaaa agaaaaagaa 540 aaagcagcta agalgactaa ggaattagca gataagctaa gcaatgctga agcaagtcgt 600 gataaagcct tlgcaglatc aaaagattta gcagataaac taagtagtgc tgaagcaagt 660 cgtgataaag ctlttgcagt atcaaaagat ttagcagata aattggcagc taaaacagca 720 gaagctgaaa agttaatgga aaacgttggt agtctagacc gcttggtaga gtctgcaaaa 780. cgtgaaatgg ctcaaaaatt agcagaaatt gatcaattaa ctgctgataa ggctaa ggct 840 galgcagagc (tgcagctgc aaatgacacc attgcatcac 880 <2IO> 5 <21I> 294 <2I2> PRT <213> Streptococcus equi <400> 5

Asn Ser Glu Ser Ser Tlir Ala Thr Pro Arg Leu Ser Arg Asp Leu 15 10 15

Lys Asn Arg Leu Ser Asp Ala Ala Ol Gly Asp Ala Ser Ser AJa 20 25 30

Gin Lys Val Arg Asn Leu Leu

40 45

Ala Leu Arg Gly Leu Asp Ser AJa Arg Ala Ala Tyr Gly Arg Asp 50 55 60

Asp Tyr Tyr Asn Leu Met His Leu Ser Ser Leu Asn Asp Lys 65 70 75 80

Pro Gly Asp Arg Arg Gin Leu Ser Leu Ala Ser Leu Leu Val Asp 85 90 95

Glu White Glu Lys Arg Ile Ala Asp Gly Asp Arg Tyr Ala Lys Leu Leu 100 105 110

Glu Ala Lys Leu Ala Ala Lys Ser Gin Gin Glu Met Leu Arg Glu 1 * 15 120 125

Arg Asp Ser Gin Leu Arg Asn Leu Glu Glu Lys Glu Glu Glu Leu 130 135 140

Thr Lys Ala Phys Asr Lys Asp Glu Arg Gin Ala Leu Thr Glu Ser Phe Asn Lys 145 150 155 160

Thr Leu Ser Thr Lys Glu Tyr Asn Lys Leu Lys Thr Glu Leu 165 170 175

Ala Lys Glu Lys Glu Lys | 190

Lys Leu Ser Asn A, and Glu Ala Ser Arg Lys Ala Phe Ala Val Ser 195 200 205

Lys Asp Leu Ala Asp Lys Leu Ser Ser Ala Ala Ser Arg Asp Lys 210, 215 220

Ala Phe Ala Val Ser Lys Asp Leu A ^ la Asp Lys Leu Ala Ala Lys Thr 225 230 235 240

Ala Glu Lys Leu Met Glu Asn Val Gly Ser Leu Asp 250 Leu

Val Glu Ser Ala Lys Arg Glu Met A, and Gin Lys Leu Ala Glu White Asp 260 265 270

Gin Leu Thr Ala Lys Ala Lys Ala Asp Glu Leu Ala Ala Ala 275 280 285

Asn Asp Thríl A, and Ser 290 <2IO> 6 <21I> 27 <2I2> DNA <213> Streptococcus equi <400> 6 gcggatccga actctgaggt tagtcgt <210> 7 <211> 28 <212> DNA <213> Streptococcus equi <400> 7 gcggatccat agcttagttt tctttgcg 28 <2I0> 8 <211> 20 <212> DNA <213> Streptococcus equi <400> 8 tgcataaa agttcctgtc 20 <2I0> 9 <211> 20 <212> DNA <213> Streptococcus equi < 400> 9 gattcggtaa gagcttgacg

Claims (14)

PATENT CLAIMS A compound comprising a compound of the formula: SEQ ID NO 1. The compound of claim 1 which is SEQ ID NO A prokaryotic cell comprising the compound of claim 1. A eukaryotic cell comprising the compound of claim 1. The cell of claim 3, which is E. coli The cell of claim 3, which is Salmonella spp A compound of the formula SEQ TD NO 2 or portions thereof. A vaccine comprising the compound of claim 7. The compound of claim 7, which is SEQ ID NO 5 The compound of claim 9, which is incorporated into the liposome. The compound of claim 10, further comprising a cholera toxin B subunit. 12. A method of inducing S. equi-specific immunity in a horse comprising the compound of claim 9. 13. A method for identifying a S. equ. characterized in that a biological sample is obtained from the prepared sample for PCR, performed by PCR using the corresponding primers for SEQ ID NO 1, electrophoresis of the final product, and the presence or absence of a fragment of the corresponding size is confirmed. Method according to claim 13, characterized in that primers SEQ ID NO 8 and SEQ ID NO 9 are used