Vaccine for streptococcus equi subsp. zooepidemicus

Abstract

Provided herein is a composition comprising a live strain of S. zooepidemicus and a pharmaceutically acceptable carrier, wherein the live strain of S. zooepidemicus contains a mutated M protein trans-acting positive regulator (MGA) gene that results in impaired DNA binding. Also provided are methods and uses to eliciting an immune response against an infection by S. zooepidemicus in a subject, comprising administering to the subject an effective amount of the composition described herein. Also provided is a method of generating strains of S. zooepidemicus with reduced virulence.

Description

VACCINE FOR STREPTOCOCCUS EQUI SUBSP. ZOOEPIDEMICUSRELATED APPLICATION

[0001] This disclosure claims benefit of United States Provisional Patent Application serial no. 63 / 442,307 filed January 31 , 2023, incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “13764- P68860PC00_SequenceListing.xml” (4,308,855 bytes), created on January 29, 2024, is herein incorporated by reference.FIELD:

[0003] The present disclosure relates to compositions comprising Streptococcus equi subsp. zooepidemicus strains; methods and uses of preventing and / or treating Streptococcus equi subsp. zooepidemicus infection in a subject; and methods of generating Streptococcus equi subsp. zooepidemicus strains with reduced virulence.BACKGROUND:

[0004] Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is considered an opportunistic pathogen of several warm-blooded hosts, including humans, equidae, camelidae, caninae and suidae (Cebra, Heidel, Cebra, Tornquist, & Smith, 2000; Corpa et al., 2018; Kernaghan, Bujold, & Maclnnes, 2012; Li et al., 2021 ; Priestnall & Erles, 2011). It is a Gram-positive, p-hemolytic coccus belonging to the Lancefield group C. Severe disease characterized by pneumonia, septicemia and meningitis has been associated with S. zooepidemicus (FitzGerald et al., 2017; Pelkonen et al., 2013). Historically, this bacterium has been suggested as a normal inhabitant of the palatine tonsils of pigs, being detected by both culture and high-throughput sequencing in samples collected from healthy animals (Kernaghan et al., 2012). However, virulent strains of S. zooepidemicus reportedly killed over 300,000 pigs in China in the 1970s, associated with high-mortality outbreaks of sudden death and respiratory disease (Feng & Hu, 1977). Early in 2019, the first outbreaks of sudden deaths, increased mortality and increased abortion rates associated with S. zooepidemicus in pigs housed in commercial facilities in North America were reported (Costa & Lage, 2020). Since then, the pathogen was also isolated from outbreaks of septicaemic disease and increased mortality throughout thenorthern United States of America, Canada, New Zealand (D. Lawton, personal communication), and the Netherlands (Chen et al., 2020; M. Houben, 2021 ; Sitthicharoenchai et al., 2020). Interestingly, S. zooepidemicus sequence type 194 (ST- 194) was associated with all these outbreaks, except for the one in the Netherlands where a new sequence type was identified (M. Houben, 2021 ).

[0005] An initial study described the experimental infection of finisher pigs and sows with S. zooepidemicus isolates obtained from different hosts, including ST-194 (Hau et al., 2021). Clinical disease progression, cytokine response, gross and microscopic lesions were described, as well as the lack of cross-protection between isolates obtained from horses and ST-194. Currently, there are no commercial vaccines available for this pathogen. Until 2019, control and prevention methods were not applied given its commensal nature, the lack of evidence of disease in North America and the knowledge gap regarding the transmission routes and pathogen shedding patterns in pigs.

[0006] US6682745 discloses the use of live attenuated bacteria, including from S. zooepidemicus, for the manufacture of vaccine for submucosal administration in horse.

[0007] US9987342 discloses compositions comprising polypeptides or proteins ofStreptococcus equi subsp. equi or subsp. zooepidemicus that are able to elicit an immunogenic response when administered to a non-human mammal such as pigs.

[0008] US7455844 discloses methods against infections caused by Streptococcus zooepidemicus in equine, camelid, canine and human using compositions comprising polypeptides of Streptococcus equi that are able to elicit an immune response when administered.

[0009] There remains a need for methods that control and / or prevent disease caused by S. zooepidemicus.SUMMARY:

[0010] The present disclosure relates to compositions comprising a live strain of S. zooepidemicus and a pharmaceutically acceptable carrier, wherein the live strain of S. zooepidemicus contains a mutated M protein trans-acting positive regulator (MGA) gene that encodes an MGA protein with impaired DNA binding.

[0011] In some embodiments, the mutated MGA gene comprises a frameshift mutation. In some embodiments, the mutated MGA gene comprises a deletion.

[0012] In some embodiments, the mutated MGA gene comprises a frameshift mutation at nucleotide position 84, 85 and / or 86 relative to the full length sequence of the MGA gene, for example, as shown in SEQ ID NO: 2. In some embodiments, the mutated MGA gene comprises a deletion of nucleotides adenine and thymine at positions 84 and 85 relative to the full length sequence of the MGA gene, for example, as shown in SEQ ID NO: 2.

[0013] In some embodiments, the mutated MGA gene has a sequence as shown in SEQ ID NO: 3.

[0014] In some embodiments, the live strain of S. zooepidemicus is Strain H-1 deposited in the International Depositary Authority of Canada under Accession No. 260123-01.

[0015] In some embodiments, the composition further comprises one or more further agents for treating a S. zooepidemicus infection.

[0016] In some embodiments, the one or more further agents comprise penicillin, ampicillin and / or tilmicosin.

[0017] Also disclosed are methods of eliciting an immune response against an infection by S. zooepidemicus in a subject, comprising administering to the subject an effective amount of a composition disclosed herein. Also disclosed are uses of a composition disclosed herein to elicit an immune response against an infection by S. zooepidemicus in a subject. Further disclosed is a composition disclosed herein for use to elicit an immune response against an infection by S. zooepidemicus in a subject. Even further disclosed is a use of a composition disclosed herein in the manufacture of a medicament for eliciting an immune response against an infection by S. zooepidemicus.

[0018] In some embodiments, the methods and uses are for preventing S. zooepidemicus infection. In some embodiments, the methods are for treating S. zooepidemicus infection.

[0019] In some embodiments, the infection is an infection by S. zooepidemicus sequence type 194 (ST-194), optionally ATCC 35246, or a virulent strain thereof having at least 95% sequence identity thereto.

[0020] In some embodiments, the infection is an infection by S. zooepidemicus sequence type 326 (ST-326), as shown in SEQ ID NO: 1 , ora virulent strain thereof having at least 95% sequence identity thereto.

[0021] In some embodiments, the composition is for use in a single dose. In some embodiments, the composition is for use in two doses, optionally wherein the two doses are 21 days apart.

[0022] In some embodiments, the composition is for use nasally or orally.

[0023] In some embodiments, the composition is for use at 104- 1011cfu / mL, 105-1011cfu / mL, 106- 1011cfu / mL, 107- 1011cfu / mL, 107- 1010cfu / mL, or 108- 1010cfu / mL.

[0024] In some embodiments, the composition is for use at 107- 1010cfu / mL.

[0025] In some embodiments, the composition is for use at a volume of 0.5 mL to2.0 mL.

[0026] In some embodiments, the subject is an animal, optionally the subject is a livestock animal, optionally the subject is a Camelidae, Caninae, Felidae, Bovidae, Suidae, or Equidae.

[0027] In some embodiments, the subject is a pig.

[0028] Also disclosed are methods for generating a S. zooepidemicus with reduced virulence, comprising mutating a M protein trans-acting positive regulator (MGA) gene in a virulent S. zooepidemicus strain, wherein the mutation encodes an MGA protein with impaired DNA binding.

[0029] In some embodiments, mutating the MGA gene comprises introducing a frameshift mutation.

[0030] In some embodiments, mutating the MGA gene comprises introducing a deletion mutation.

[0031] In some embodiments, mutating the MGA gene comprises introducing a frameshift mutation at nucleotide position 84, 85 and / or 86, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

[0032] In some embodiments, mutating the MGA gene comprises a deletion of nucleotides adenine and thymine at positions 84 and 85, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

[0033] In some embodiments, the mutation that encodes the MGA protein with impaired DNA binding has a sequence as shown in SEQ ID NO: 3.

[0034] In some embodiments, the method further comprises testing the S. zooepidemicus strain for reduced virulence.

[0035] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the description as a whole.BRIEF DESCRIPTION OF THE DRAWINGS:

[0036] For a better understanding of the embodiments described herein and to show more clearly how they may be carried into effect, reference will now be made, by way of example only, to the accompanying drawings which show at least one exemplary embodiment, and in which:

[0037] Figure 1 is a graph showing rectal temperature of vaccinated (Vax, n=6) and control (Control, n=5) pigs after each dose of vaccination with strain H-1 (1 mL oral, 1 mL nasally delivered 35 and 14 days prior to inoculation with the virulent strain ST194). The dotted line is the fever threshold in pigs (40°C). AM - Morning clinical check. PM - Afternoon clinical check.

[0038] Figure 2 is a graph showing rectal temperature of vaccinated (Vax- challenge, n=6) and control (Control - challenge, n=5) pigs following 2 doses of the vaccine strain H-1 (1 mL oral, 1 mL nasally delivered 35 and 14 days prior to inoculation with the virulent strain ST194). The dotted line is the fever threshold in pigs (40°C). AM - Morning clinical check. PM - Afternoon clinical check.

[0039] Figure 3 illustrates results from bacterial culture from control and vaccinated (vax) pigs. Score 0 = no Streptococcus detected, 1 = mild number of Streptococcus, 2 = moderate, 3 = severe, 4 = too many to count. Tonsil colonization is normal regardless of animal status. Vax - vaccinated.

[0040] Figure 4 is a graph showing results of qPCR detection of S. zooepidemicus on nasal swabs following vaccination of pigs (n=6) with H-1 strain.

[0041] Figure 5 is a graph showing rectal temperatures of pigs following the 1stdose and vaccinated orally (live oral, n=5), nasally (live nasal, n=5) or unvaccinated (control, n=3). AM - Morning clinical check. PM - Afternoon clinical check.

[0042] Figure 6 is a graph showing rectal temperatures of pigs following the 2nd dose and vaccinated orally (live oral, n=5), nasally (live nasal, n=5) or unvaccinated (control, n=3). AM - Morning clinical check. PM - Afternoon clinical check.

[0043] Figure 7 is a graph showing rectal temperatures of pigs following the infection challenge. Pigs were vaccinated orally (live oral, n=5), nasally (live nasal, n=5) or unvaccinated (control, n=3). AM - Morning clinical check. PM - Afternoon clinical check.

[0044] Figure 8 illustrates results from bacterial culture from control and vaccinated pigs via oral (n=5) or nasal (n=5) routes. Score 0 = no Streptococcus detected, 1 = mild number of Streptococcus, 2 = moderate, 3 = severe, 4 = too many to count. Tonsil colonization is normal regardless of animal status. Controls were mock vaccinated.

[0045] Figure 9 is a graph showing average rectal temperatures of pigs following infection challenge. Pigs were vaccinated with H-1 and challenged with ST-326 (1507, n=4), vaccinated with H-1 and challenged with ST-194 (n=4) or unvaccinated and challenged with ST-326 (1507, n=4).

[0046] Figure 10 is a graph showing survival of pigs following infection challenge. Pigs were vaccinated with H-1 and challenged with ST-326 (1507, n=4), vaccinated with H-1 and challenged with ST-194 (n=4) or unvaccinated and challenged with ST-326 (1507, n=4).

[0047] Figure 11 is a graph showing average rectal temperatures of pigs following infection challenge. Pigs were vaccinated with a single dose of H-1 and challenged with ST-194 (n=4), or unvaccinated and challenged with ST-194 (n=4).

[0048] Figure 12 is a graph showing survival of pigs following infection challenge. Pigs were vaccinated with a single dose of H-1 and challenged with ST-194 (n=4), or unvaccinated and challenged with ST-194 (n=4).

[0049] Figure 13 shows MGA sequence alignment of a virulent wildtype strain (ATCC 35236) and the strain H-1. Shaded text denotes the deletion in the H-1 sequenceand the conserved, functional sequence in the wildtype strain. *-denotes matching nucleotide.

[0050] Figure 14 shows loss of HTH-motif in the H-1 MGA protein (dark), when compared to the wildtype virulent strain (light, ATCC 35246). Beta helix are lost in the H- 1 protein, giving place to beta strands (arrows, highlighted in the circle).

[0051] Figure 15 shows that the H-1 vaccine reduces wildtype shedding in challenged animals. The boxes on the top of the graph depict the number of animals shedding the wild type (challenge) strain in a given day. The top number reflects the number of control pigs (unvaccinated), and the bottom umber reflects the number of vaccinated pigs.DETAILED DESCRIPTION OF THE DISCLOSURE:

[0052] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0053] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.I. Definitions

[0054] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0055] As used herein, the term “immune response” means development of a humoral and / or cellular immune response in a subject to an antigen, for example, a pathogen. A pathogen can include, for example, a virulent strain of S. zooepidemicus. An immune response can include, for example, one or more of: production of antibodies, activation of B cells, activation of T cells etc.

[0056] The term “unattenuated” as used herein refers to a pathogen that has not been manipulated or weakened to reduce its virulence. An unattenuated pathogen can for example be a natural isolate or a strain that resulted from attenuation of another strain but that is not further attenuated.

[0057] The term “attenuated” as used herein refers to a pathogen that has been manipulated or weakened to reduce its virulence.

[0058] The term “ST-194” or “sequence type 194” as used herein refers to a group of strains similar to the initial strain, based on multi-locus sequence typing, including, without limitation the strain of S. zooepidemicus first recovered from pigs in Sichuan Province, China in 1975 that led to the death of over 300,000 pigs in a 2-week period. In one embodiment, the ST-194 strain is Streptococcus equi subsp. Zooepidemicus, ATCC 35246, having the reference sequence GenBank GCA_000219765.1 . In another embodiment, the ST-194 strain is a genetic variant of ST-194 that causes disease in pigs. For example, a strain that comprises carbamate kinase (arcC), ribonucleosidediphosphate reductase (nrdE), prolyl-tRNA synthetase (proS), signal peptidase I (spi), thymidylate kinase (tdk), triosephosphate isomerase (tpi), acetyl-CoA acetyltransferase (yqi L) , or functional variants thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to the gene sequences found in ATCC 35246 strain.

[0059] The term “ST-326” or “sequence type 326” as used herein refers to the strain of S. zooepidemicus first identified in the United Kingdom, which is known to cause disease in pigs. In one embodiment, the ST-326 strain is defined in SEQ ID NO: 1. In another embodiment, the ST-326 strain is a genetic variant of ST-326 that causes disease in pigs. For example, a strain that comprises carbamate kinase (arcC), ribonucleoside-diphosphate reductase (nrdE), prolyl-tRNA synthetase (proS), signal peptidase I (spi), thymidylate kinase (tdk), triosephosphate isomerase (tpi), acetyl-CoA acetyltransferase (yqi L) , or functional variants thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to the gene sequences found in SEQ ID NO: 1.

[0060] The term “Strain H-1” or “H-1” as used herein refers to a S. zooepidemicus strainthat was created as described in the Examples. The strain has 99.85% sequence identity with ATCC 35246. Sample cultures of Strain H-1 have been deposited with the International Depositary Authority of Canada Collection (1015 Arlington Street, Winnipeg, Canada, R3E 3R2) under Accession No. 260123-01 on January 26, 2023. A copy of the Deposit Receipt and Statement of Viability is provided in Appendix A. The parental strain was originally isolated from pigs and humans who consumed raw pork in Southeast Asia. The H-1 strain was created by attenuation by starvation through passaging the parental strain in brain-heart infusion broth, followed by 32 days of incubation at 20°C on Dorset Egg media, and another passage on brain-heart infusion broth. As contemplated for use herein, the H-1 strain itself is administered or used as is, i.e. , unattenuated, and thus, can be considered use of a live, unattenuated H-1 strain, available as Accession No. 260123- 01.

[0061] The term “M protein trans-acting positive regulator” or “MGA” as used herein refers to a DNA-binding protein which regulates gene expression, including activating expression of virulence genes. The MGA regulon is comprised of MGA and other downstream genes (of the M family of proteins, emm, mrp, arp, enn), C5a peptidase (scpA), MSCRAMMs (fba, sof), collagen-like protein (scl1 / sclA), and secreted inhibitor of complement (sic)) whose products are critical for bacterial adherence, internalization, and host immune evasion. The MGA wild-type gene may be from Streptococcus equi subsp. zooepidemicus, ATCC 35246. For example, the wildtype MGA gene from S. zooepidemicus may be as shown in SEQ ID NO: 2. The MGA gene may contain a mutation which impairs DNA binding. For example, the deletion may be a frameshift mutation, such as a nucleotide insertion or a deletion, relative to the wildtype MGA gene. The frameshift mutation may be at nucleotide positions 84, 85 and / or 86, relative to the wildtype MGA gene. The MGA gene may have a deletion of nucleotides adenine and thymine at positions 84 and 85 relative to the full length sequence of the MGA gene, optionally as shown in SEQ ID NO: 3.

[0062] The term “mutation” as used herein refers to insertions or deletions of one or more nucleotides within a sequence, or substitutions of one or more nucleotides within a sequence. An insertion or deletion mutation may cause a frameshift mutation. A substitution may be a nucleotide change that results in a stop codon.

[0063] The term “frameshift mutation” as used herein refers to an insertion or deletion of one or more nucleotides that is not 3, or not a multiple of 3. Accordingly, the frameshift mutation disrupts the reading frame in such a manner that alters the translation of all subsequent codons.

[0064] The term “sequence identity” as used herein refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , % identity = [number of identical overlapping positions] I [total number of positions] X 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. T o obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0065] The term "pharmaceutically acceptable carrier" refers to any carrier, agent, excipient, or vehicle that are not biologically or otherwise undesirable. Except where the carrier, agent, excipient or vehicle is incompatible with the active ingredient, its use in the therapeutic formulations is contemplated. The use of such pharmaceutically acceptable carrier is well known in the art.

[0066] The term “effective amount” as used herein refers to any amount of a compound or a composition that is sufficient to generate a desired response, including but not limited to preventing or reducing the chance of disease onset, slowing disease progression, and alleviating symptoms. As used herein, the term can for example refer to an amount that is sufficient to elicit an immune response in a subject.

[0067] The term "treating" or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. For example, asubject with a S. zooepidemicus infection can be treated with a composition described herein to reduce mortality rate.

[0068] The term “prevention” or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a disease, disorder or condition or manifesting a symptom associated with a disease, disorder or condition. The disease, disorder or condition can for example be an infection of a virulent strain of S. zooepidemicus.

[0069] As used herein, the term “subject” refers to all members of the animal kingdom, and suitably refers to humans. For example, members of the animal kingdom may include all mammals, such as livestock. Subjects may include mammals of the order Primate, or mammals of the family Camelidae, Caninae, Bovidae, Felidae, Suidae, and Equidae. The mammals may further be of the genera Camelus, Canis, Bos, Felis, Sus, Equus and ovine.

[0070] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0071] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0072] All numerical values herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0073] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate themeaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0074] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0075] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0076] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0077] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

[0078] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.II. Compositions and Methods for Use

[0079] It is demonstrated herein that, a specific strain of S. zooepidemicus can protect against virulent strains of S. zooepidemicus when used or administered live to a subject.

[0080] Accordingly, in one aspect, there is provided a composition comprising a live strain of S. zooepidemicus and a pharmaceutically acceptable carrier, wherein the live strain of S. zooepidemicus contains a mutated M protein trans-acting positive regulator (MGA) gene that encodes an MGA protein with impaired DNA binding.

[0081] In an embodiment, the mutated MGA gene comprises a frameshift mutation.

[0082] In one embodiment, the mutated MGA gene comprises a deletion.

[0083] In another embodiment, the mutated MGA gene comprises a frameshift mutation at nucleotide position 84, 85 and / or 86, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

[0084] In an embodiment, the mutated MGA gene comprises a deletion of nucleotides adenine and thymine at positions 84 and 85 relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

[0085] In one embodiment, the mutated MGA gene has a sequence as shown in SEQ ID NO: 3.

[0086] In one embodiment, the live strain of S. zooepidemicus is Strain H-1 deposited in the International Depositary Authority of Canada under Accession No. 260123-01.

[0087] Sample cultures of Strain H-1 have been deposited with the International Depositary Authority of Canada Collection (1015 Arlington Street, Winnipeg, Canada, R3E 3R2) under Accession No. 260123-01 on January 26, 2023. A copy of the Deposit Receipt and Statement of Viability is provided in Appendix A.

[0088] In an embodiment, the composition further comprises one or more further agents for treating a S. zooepidemicus infection.

[0089] In an embodiment, the one or more further agents comprise penicillin, ampicillin and / or tilmicosin.

[0090] The composition disclosed herein can be used as a vaccine or immunogenic composition, which is typically administered to or used in healthy subjects to, for example, prevent and / or reduce the likelihood of disease onset caused by an infection and / or to reduce severity of the disease in case of infection. The composition can also be used as a treatment. For example, the composition can be administered to or used in subjects having or suspected of having a S. zooepidemicus infection.

[0091] The composition described herein can be prepared by any known methods for the preparation of pharmaceutically acceptable compositions such that an effective amount can be administered to or used in a subject such that an effective quantity of theactive substance is combined in a mixture with a pharmaceutically acceptable carrier to for example, elicit a prophylactic or therapeutic immune response.

[0092] Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient. In an embodiment, the composition disclosed herein further comprises other agents suitable for preventing and / or treating a S. zooepidemicus infection. Suitable agents include, without limitation, penicillin, ampicillin and tilmicosin.

[0093] In an embodiment, the pharmaceutically acceptable carrier is water or saline.

[0094] In another embodiment, the composition is formulated for administration or use in drinking water.

[0095] In another aspect, there is provided a method of eliciting an immune response against an infection by S. zooepidemicus in a subject, comprising administering to the subject an effective amount of a composition disclosed herein. Also provided is a composition disclosed herein for use to elicit an immune response against an infection by S. zooepidemicus in a subject. Further provided is a use of a composition disclosed herein for eliciting an immune response against an infection by S. zooepidemicus. Even further provided is a use of a composition disclosed herein in the manufacture of a medicament for eliciting an immune response against an infection by S. zooepidemicus.

[0096] When administered to or used in a healthy subject, eliciting of the immune response can prevent or reduce the likelihood of disease onset, and / or reduce the severity of illness and / or mortality rate if the subject subsequently develops a disease caused by an infection by a virulent strain of S. zooepidemicus. When administered to or used in a subject having or suspected of having a disease caused by infection by a virulent strainof S. zooepidemicus, eliciting of the immune response can for example lead to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, etc.

[0097] Accordingly in an embodiment, the method or use is for preventing S. zooepidemicus infection. In another embodiment, the method or use is for treating S. zooepidemicus infection.

[0098] The immune response can be elicited against a virulent strain of S. zooepidemicus. In one embodiment, the immune response is elicited against S. zooepidemicus sequence type 194 (ST-194), optionally ATCC 35246, or a virulent strain thereof having at least 95% sequence identity thereto. In another embodiment, the immune response is elicited against S. zooepidemicus sequence type 326 (ST-326), as shown in SEQ ID NO: 1 , or a virulent strain thereof having at least 95% sequence identity thereto.

[0099] Methods to determine whether an immune response is elicited are known to those skilled in the art. For example, where an immune response is against a viral pathogen, viral titer can be measured in a subject that has been administered with the composition and compared with a control subject. A control subject can be a subject that has not been administered the composition prior to being exposed to the pathogen.

[0100] The composition for use with the methods and uses described herein can be formulated for any suitable route of administration and using any suitable dosage form, for example, by parenteral, intravenous, subcutaneous, intramuscular, intraperitoneal, inhalation or spray (e.g. via aerosol), mucosal administration, rectal administration, vaginal administration, skin patch or skin application or oral administration, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. For example, the compositions can be formulated for mucosal administration (such as intranasal, oral, ophthalmic, rectal, vaginal, sublingual) or for injection (such as subcutaneous, intramuscular, intravenous).

[0101] Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20thedition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0102] In an embodiment, the composition is formulated for nasal administration or use. In an embodiment, the composition is formulated for oral administration or use. In an embodiment, the composition is formulated for administration or use by drenching. In another embodiment, the composition is formulated for administration or use in drinking water.

[0103] In an embodiment, a strain of S. zooepidemicus, optionally Strain H-1 , is grown in suitable culture media and used directly for administration. If to be used directly for administration, the culture media contains only pharmaceutically acceptable components. Suitable culture media include for example brain-heart infusion media enriched with 1 % glucose.

[0104] In another embodiment, the composition disclosed herein is administered or formulated as a spray. For example, the composition can be administered to the surface of the nasal cavity by using an atomizer. The composition can also be sprayed on a subject to allow the composition to come into contact with the eye, nasal cavity, and / or mouth of the subject.

[0105] In one embodiment, the composition is administered or formulated as a nasal spray.

[0106] The effective amount to be administered or used can be determined by conventional methods known to a person skilled in the art. An effective amount can for example be determined by starting with a low dose and incrementally increased until a desirable outcome is reached.

[0107] For example, the composition can be administered or used at 104- 1011cfu / mL at a volume of 0.5-2 mL orally or nasally.

[0108] In an embodiment, the composition is administered or used at 104- 1011cfu / mL. In an embodiment, the composition is administered or used at 105- 1011cfu / mL.In an embodiment, the composition is administered or used at 106- 1011cfu / mL. In an embodiment, the composition is administered or used at 107- 1011cfu / mL. In an embodiment, the composition is administered or used at 107- 1010cfu / mL. In an embodiment, the composition is administered or used at 108- 1011cfu / mL. In an embodiment, the composition is administered or used at 108- 1010cfu / mL.

[0109] In an embodiment, the composition is administered or used at 104cfu / mL. In an embodiment, the composition is administered or used at 105cfu / mL. In an embodiment, the composition is administered or used at 106cfu / mL. In an embodiment, the composition is administered or used at 107cfu / mL. In an embodiment, the composition is administered or used at 108cfu / mL. In an embodiment, the composition is administered or used at 109cfu / mL. In an embodiment, the composition is administered or used at 1010cfu / mL. In an embodiment, the composition is administered or used at 1011cfu / mL.

[0110] In an embodiment, the composition is administered or used at a volume of 0.5 mL. In an embodiment, the composition is administered or used at a volume of 0.6 mL. In an embodiment, the composition is administered or used at a volume of 0.7 mL. In an embodiment, the composition is administered or used at a volume of 0.8 mL. In an embodiment, the composition is administered or used at a volume of 0.9 mL. In an embodiment, the composition is administered or used at a volume of 1.0 mL. In an embodiment, the composition is administered or used at a volume of 1.1 mL. In an embodiment, the composition is administered or used at a volume of 1.2 mL. In an embodiment, the composition is administered or used at a volume of 1.3 mL. In an embodiment, the composition is administered or used at a volume of 1.4 mL. In an embodiment, the composition is administered or used at a volume of 1.5 mL. In an embodiment, the composition is administered or used at a volume of 1.6 mL. In an embodiment, the composition is administered or used at a volume of 1.7 mL. In an embodiment, the composition is administered or used at a volume of 1.8 mL. In an embodiment, the composition is administered or used at a volume of 1.9 mL. In an embodiment, the composition is administered or used at a volume of 2.0 mL.

[0111] In an embodiment, the composition is administered or used at a dose of 0.5mL per nasal cavity. In an embodiment, the composition is administered or used at a dose of 1 mL orally. In an embodiment, the composition is administered or used at a dose of 2mL orally.

[0112] The composition disclosed herein may be administered or used according to any suitable schedule, for example as a single dose, or multiple doses (e.g. two doses), separated by a suitable period of time (e.g. about one day), or any other suitable number of doses and / or period of time, depending for example on the formulation, and / or route of administration.

[0113] In an embodiment, the composition is administered or used in a single dose. In an embodiment, the composition is administered or used in a 2-dose regimen, with at least 21 days between the 2 doses.

[0114] In an embodiment, the composition is administered or used in 2 doses with 21 days apart.

[0115] The methods and uses disclosed herein can further comprise administering or using one or more further agents suitable for treating a S. zooepidemicus infection. Suitable agents for treating a S. zooepidemicus infection include without limitation, penicillin, ampicillin and tilmicosin.

[0116] In an embodiment, the one or more further agents are selected from the group consisting of penicillin, ampicillin and tilmicosin.

[0117] The one or more agents can be co-administered or used with the composition disclosed herein. For co-administration or use, the one or more agents and the composition disclosed herein can be formulated separately or combined in a single formulation if suitable as determined by a skilled person. Accordingly, the present description provides a single unit dosage form comprising the composition described herein, a therapeutic agent, and a pharmaceutically acceptable carrier. The term “coadministration” shall mean that at least two compounds or compositions are administered to or used in the animal such that effective amounts or concentrations of each of the two or more compounds may be found in the animal at a given point in time. Although compounds according to the present disclosure may be co-administered to or used in an animal at the same time, the term embraces both administration or use of two or more agents at the same time or at different times, provided that effective concentrations of all co-administered compounds or compositions are found in the animal at a given time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering or using the two substances within a few hours of each other, or even administering or using one substance within 24 hours of administration or use of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for one skilled in the art. In particular embodiments, two substances will be administered or used substantially simultaneously, i.e., within minutes of each other, or in a single composition that containsboth substances. In other embodiments, the combination of agents is administered or used in a non-contemporaneous fashion.

[0118] In an embodiment the subject is an animal. In an embodiment the subject is a livestock animal. In an embodiment, the subject is a Primate. In another embodiment, the subject is a Camelidae, Caninae, Felidae, Bovidae, Suidae, or Equidae. In a particular embodiment the subject is a pig.

[0119] In a further aspect, there is provided a method for generating a S. zooepidemicus strain with reduced virulence, comprising mutating a M protein transacting positive regulator (MGA) gene in a virulent S. zooepidemicus strain, wherein the mutation encodes an MGA protein with impaired DNA binding.

[0120] In an embodiment, mutating the MGA gene comprises introducing a frameshift mutation.

[0121] In an embodiment, mutating the MGA gene comprises introducing a deletion mutation.

[0122] In an embodiment, mutating the MGA gene comprises introducing a frameshift mutation at nucleotide position 84, 85 and / or 86, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO:2, or an equivalent position in an MGA gene having at least 95% sequence identity thereto.

[0123] In an embodiment, mutating the MGA gene comprises introducing a deletion of nucleotides adenine and thymine at positions 84 and 85, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

[0124] Methods of introducing a mutation are known to those skilled in the art. For example, by use of CRISPR / Cas systems, serial passaging under laboratory conditions, selective pressure through starvation, chemical or physical mutagenesis, insertional mutagenesis, transposon mutagenesis, site-directed mutagenesis, sequence saturation mutagenesis, signature tagged mutagenesis, or knock-out / knock-in genetic manipulation.

[0125] In an embodiment, the method further comprises testing the S. zooepidemicus strain for reduced virulence.

[0126] Methods of testing virulence are known to those skilled in the art. For example, the lethal dose required to kill 50% of infected hosts (LDso), cell viability andcytotoxicity assays, bioassay, or quantification of virulence factors. A further example would be determining whether the S. zooepidemicus with reduced virulence elicits an immune response, as measured in a subject or system (e.g. cell model) that has been administered the S. zooepidemicus strain for reduced virulence and compared with a control. The control can be a subject or system (e.g. cell model) that has been administered the virulent parental strain.

[0127] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.EXAMPLESExample 1 : Materials and Methods

[0128] Strain H-1 is a gram positive cocci identified as Streptococcus equi subsp. Zooepidemicus that induces strong beta-haemolysis in blood agar, but does not grow as mucoid colonies (different from the virulent strain ST194). Strain H-1 was grown initially by plating into a Columbia blood agar (5% sheep blood). Isolated colonies were transferred to BHI (brain-heart infusion enriched with 1 % glucose) media for 12h at 37°C and 5% CO2. After this period, BHI broth cultures were transferred to a containment vessel and transported to a BSL-2 room. Broth was vigorously shaken and a nasal atomizer and 1 mL syringe were used for vaccination. Nasal and oral vaccination were performed, 0.5mL per nostril and 1 mL oral, in a 2 dose regimen 21 days apart, at 108cfu / mL or 1010cfu / mL. 14 days after the second dose, pigs were challenged with a virulent strain ST- 194. Each pig was challenged with 1 mL of the challenge strain orally, and 1 mL nasally (106cfu / ml), once, 14 days after the second dose. Vaccinated pigs (vax, n=6) and mock vaccinated (control, n=5) were monitored for clinical signs, rectal temperature, and changes in behaviour twice daily. Nasal swabs were collected after each vaccine dose, as well after challenge, for 7 days.Example 2: Trial 1

[0129] The procedures described in Example 1 were used to test the effect of H-1 vaccination in pigs at a 1010cfu / ml concentration. On day 5 post-challenge, 3 / 5 control pigs had to be euthanized due to welfare concerns. None of the vaccinated pigs were euthanized prior to the end of the trial period or had poor clinical prognosis that warranted intervention. Post-mortem bacteriology results revealed widespread presence of Streptococcus zooepidemicus in internal organs of the control group pigs, while vaccinated animals were free of bacterial growth in internal organs (Figure 3). Tonsils from all pigs are positive for Streptococci on CNA agar - which is expected as this is a contaminated site where many bacteria are present, unrelated to disease. Tonsil samples tested negative for the challenge strain by PCR. Shedding of S. zooepidemicus in nasal secretions was evaluated by qPCR (Figure 4). By 5 days after the first dose, an average of less than 2 genome copies / swab was detected. Less than 10 genome copies / swab were detected following the 2nddose in vaccinated animals.

[0130] This data suggests that H-1 vaccination is protective against challenge with ST-194 in pigs.Example 3: Trial 2 - Oral vs nasal vaccination

[0131] The same procedures described in Example 1 were repeated, with the difference that 5 pigs were vaccinated orally, 5 pigs were vaccinated nasally, and 3 pigs were unvaccinated, and the H-1 dose used was 108cfu / mL.

[0132] All unvaccinated pigs developed fever and had to be euthanized prior to the end of the experimental trial (7 days). Vaccinated pigs had rectal temperatures below the fever threshold following each vaccine dose (Figures 5 and 6). Following challenge, on average, orally vaccinated pigs had fever during less than 24 hours, while nasally vaccinated pigs had fever for less than 12 hours. Control pigs developed fever 24 hours post infection, did not return to normal temperatures and were euthanized (Figure 7). With the exception of one pig vaccinated nasally, bacterial culture results presented evidence that Streptococcus zooepidemicus did not lead to septicaemic disease in vaccinated animals (Figure 8).Example 4: Inactivated Strain H-1

[0133] Pigs (n=12) were vaccinated either orally (n=6) or nasally (n=6) with a preparation of inactivated (or killed) Strain H-1 (109cfu / ml). Both preparations were adjuvanted (Montanide Gel 01 for nasal, Montanide GR for oral). Animals were given 2 doses 21 days apart, and then challenged similarly to the other trials. All animals developed clinical signs and had to be euthanized within 5 days of inoculation due to the severity of clinical signs. S. zooepidemicus was isolated from multiple sites.Example 5: Vaccination with H-1 cross-protects against other S. zooepidemicus types

[0134] Pigs (n=12) from a high-health herd free of S. zooepidemicus and other swine pathogens were acclimated for 7 days in a BSL-2 facility. Pigs were randomly allocated into one of the three groups: A- Unvaccinated, challenged with ST-326 (1507); B- Unvaccinated, challenged with ST-194; C- Vaccinated, challenged with ST-326 (1507, n=4 / group). At day 0 post vaccination (DPV), pigs in the vaccinated group C received one dose (oral) of the vaccine strain (107CFU / mL, 2 mL). An identical booster dose was given on 21 DPV. Animals were challenged on 37 DPV with 2x106CFU / mL of either ST-194 (B) or ST-326 (A and C).

[0135] ST-194 challenged (B) pigs developed high fever (>41 °C) 24 hours postchallenge (Figure 9). All pigs in this group were euthanized by day 3 post challenge due to welfare concerns (Figure 10). Gross lesions were present in all of the pigs in this group (Table 1).

[0136] 1507 / ST-326 unvaccinated pigs developed mild, persistent fever that lasted over 7 days in 2 / 4 pigs (Figure 9). Fever was detected in 30 / 62 (49%) of the measuring events (statistically different from 1507 / ST-326 vaccinated group, P<0.001). A longer incubation period than ST-194 was observed. On days 6 and 7 post challenge, 1 pig / day had to be euthanized due to welfare concerns (Figure 10, P=0.0012). Overall, disease observed in this group was less severe than that observed in the ST-194 group. Gross lesions were present in 50% (2 / 4) of the pigs in this group, but were still significant (T able 1).

[0137] 1507 / ST-326 vaccinated pigs developed transient, mild fever (<24h duration). Vaccinated pigs had a statistically reduced number of fever episodes (8 / 64, 12%), when compared to the unvaccinated 1507 / ST-326 pigs (Figure 9, P<0.001). Nopigs in this group were euthanized or died during the studied period (Figure 10). No gross lesions were identified upon necropsy in this group (Table 1 ).Table 1 : Post-mortem lesions identified at necropsyExample 6: A single dose of H-1 vaccine

[0138] Pigs from a high-health herd free of S. zooepidemicus and other swine pathogens were acclimated for 7 days in a BSL-2 facility. Pigs were randomly allocated into one of the two groups: A- Unvaccinated, challenged with ST-194; B- Vaccinated with one dose, challenged with ST-194 (1507, n=4 / group). At day 0 post vaccination (DPV), pigs in the vaccinated group B received one dose (oral) of the vaccine strain (107CFU / mL,2 mL). Animals were challenged on 14 days post vaccination (DPV) with 2x106CFU / mL of ST-194.

[0139] Unvaccinated, challenged (group A) pigs developed high fever (>41 °C) 24 hours post-challenge (Figure 11 ). All pigs in this group were euthanized by day 3 post challenge due to welfare concerns (Figure 12). Gross lesions were present in all of the pigs in this group.

[0140] Pigs vaccinated with 1 dose of the H-1 vaccine (group B) developed fever between 24h (3 / 4) and 48h (1 / 4) post challenge. By day 5 post-challenge, 2 / 4 pigs continued with fever and had to be euthanized due to welfare concerns, including lameness. The remaining 2 / 4 pigs resolved fever by day 5 post-challenge and showed no other clinical signs until the end of the studied period (Figure 11). Survival of 50% (2 / 4) of the pigs in this group was significantly different from the unvaccinated group (Log-rank test, P=0.008).Example 7: Mutation of MGA impairs DNA binding

[0141] MGA is a large DNA-binding protein composed of approximately 1500 bp, 500 amino acids and a molecular weight of 62 kDa. The exact mechanism through which MGA regulates transcription remains unclear, but it is very effective at binding to a diverse pool of promoter sites. It plays a major role on how the bacteria colonize and successfully elicit infection in humans and animals.

[0142] A frameshift deletion mutation immediately after the promoter region at nucleotide position 84_85delAT (amino acid position Leu28fs) was induced in the MGA gene from Streptococcus equi subsp. zooepidemicus isolate (H-1 ) as shown in SEQ ID NO: 3. Alignment of the H-1 MGA gene sequence (SEQ ID NO: 3) with a wildtype, virulent strain (ATCC 35246; SEQ ID NO: 2) demonstrated the deletion of 2 bases (AT) at the position mentioned above (Figure 13).

[0143] The frameshift deletion mutation radically affected protein conformation. Using Alphafold2 (Jumper et al., 2021 ), the structure of the strain with this mutation was predicted.

[0144] An overlay of the wildtype MGA protein (light; from S. zooepidemicus, ATCC 35246) and the H-1 strain (dark) is shown in Figure 14. The Helix-turn-helix (HTH) motif was lost (circle). The HTH motif is a DNA-binding motif, it recognizes DNA, binding MGA to the double-helix structure and plays a role in gene expression regulation. This is key to the regulatory function of MGA.

[0145] Another virulent strain, also ST-194, phylogenetically closely related to H-1 , was isolated from pigs in Alberta, Canada. This strain, 2230328_9, as shown in SEQ ID NO: 4 does not contain the MGA frameshift mutation. Accordingly, this strain was virulent and caused illness and lethality in pigs.

[0146] Without wishing to be bound by theory, it was hypothesized that impairing functionality of the MGA gene will lead to strains that are not capable of inducing disease due to an uncoordinated expression of virulence factors. However, since those factors are still present, they are recognized by the host and induce an immune response.

[0147] To evaluate the efficacy of this method, and underscore the significance of the MGA gene, the H-1 strain, containing the mutated MGA gene, was used as a vaccine in pigs as shown in the above Examples.Example 8: Shedding Trial

[0148] A Shed-Spread Study (nose-to-nose, nose-to-anus confirmation) was conducted by housing vaccinated (n=6) and unvaccinated (n=6) pigs in the same room, 1 meter apart, for 19 days following the second dose (oral). One pig in the unvaccinated control group died suddenly following challenge. The number of pigs shedding / day and the load of bacterial shedding was significantly reduced in vaccinated pigs, when compared to unvaccinated animals, and only transiently detectable in 16% of the pigs in the first 3 days following the second dose. Control pigs did not have detectable levels up to 19 days post-second vaccine dose (Figure 15).

[0149] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.

[0150] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and / or biomarker sequences (e.g. protein and / or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.SEQUENCESSEQ ID NO: 2: Wildtype MGA sequenceSEQ ID NO: 3: Mutated MGA sequenceAppendix AInternational Form IDAC / BP / 4RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT (issued pursuant to Rule 7 1 of the Budapest Treaty Regulations)International Form IDAC / BP / 9CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATIONCebra CK, Heidel JR, Cebra ML, Tornquist SJ, Smith BB. Pathogenesis of Streptococcus zooepidemicus infection after intratracheal inoculation in llamas. Am J Vet Res. 2000;61 (12):1525-9.Chen X, Resende-De-Macedo N, Sitthicharoenchai P, Sahin O, Burrough E, Clavijo M, et al. Genetic characterization of Streptococcus equi subspecies zooepidemicus associated with high swine mortality in the United States. Transbound Emerg Dis. 2020;67(6):2797- 808.Corpa JM, Carvallo F, Anderson ML, Nyaoke AC, Moore JD, Uzal FA. Streptococcus equi subspecies zooepidemicus septicemia in alpacas: three cases and review of the literature. J Vet Diagnost Invest. 2018;30(4):598-602.Costa MO, Lage B. Streptococcus equi subsp. zooepidemicus-Associated Sudden Deaths in Swine, Canada Emerg Infect Dis. 2020;26(10):812636.Feng Z, Hu J. Outbreak of swine streptococcosis in Sichan province and identification of pathogen. Anim Husbandry Vet Med Lett. 1977;2:7-12.FitzGerald W, Crowe B, Brennan P, Cassidy JP, Leahy M, McElroy MC, et al. Acute fatal haemorrhagic pneumonia caused by Streptococcus equi zooepidemicus in greyhounds in Ireland with subsequent typing of the isolates. Vet Rec. 2017; 181 (5): 119.Hau SJ, Buckley A, Brockmeier SL. Bacterin Vaccination Provides Insufficient Protection Against Streptococcus equi Subspecies zooepidemicus Infection in Pigs. Front Vet Sci. 2022;9:827082.Hau SJ, Lantz K, Stuart KL, Sitthicharoenchai P, Macedo N, Derscheid RJ, et al. Replication of Streptococcus equi subspecies zooepidemicus infection in swine. Vet Microbiol. 2021 :109271.M. Houben MOC, L. Peeters, T. Geudeke, J. van Helmond, J. Kwinten, E. van Engelen, K. Junker, B. van der Putten., editor Streptococcus equi subsp. zooepidemicus, an emerging pig pathogen? European Symposium of Swine Health Management; 2021 April 14th-17th; Online.Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, CowieA, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021 Aug;596(7873):583-589. doi: 10.1038 / S41586-021-03819-2. Epub 2021 Jul 15. PMID: 34265844; PMCID: PMC8371605.Kernaghan S, Bujold AR, Maclnnes JI. The microbiome of the soft palate of swine. Anim Health Res Rev. 2012; 13(1 ): 110-20.Li J, Zhao Y, Gao Y, Zhu Y, Holyoak GR, Zeng S. Treatments for Endometritis in Mares Caused by Streptococcus equi Subspecies zooepidemicus: A Structured Literature Review. J Equine Vet Sci. 2021 ;102:103430.Pelkonen S, Lindahl SB, Suomala P, Karhukorpi J, Vuorinen S, Koivula I, et al. Transmission of Streptococcus equi subspecies zooepidemicus infection from horses to humans. Emerg Infect Dis. 2013;19(7):1041-8.Priestnall S, Erles K. Streptococcus zooepidemicus: an emerging canine pathogen. Vet J. 2011 ;188(2):142-8.Sitthicharoenchai P, Derscheid R, Schwartz K, Macedo N, Sahin O, Chen X, et al. Cases of high mortality in cull sows and feeder pigs associated with Streptococcus equi subsp. zooepidemicus septicemia. J Vet Diagnost Invest. 2020;32(4):565-71.

Claims

CLAIMS:1 . A composition comprising a live strain of S. zooepidemicus and a pharmaceutically acceptable carrier, wherein the live strain of S. zooepidemicus contains a mutated M protein trans-acting positive regulator (MGA) gene that encodes an MGA protein with impaired DNA binding.

2. The composition of claim 1 , wherein the mutated MGA gene comprises a frameshift mutation.

3. The composition of claim 1 or 2, wherein the mutated MGA gene comprise a deletion.

4. The composition of any one of claims 1 to 3, wherein the mutated MGA gene comprises a frameshift mutation at nucleotide position 84, 85 and / or 86, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

5. The composition of any one of claims 1 to 4, wherein the mutated MGA gene comprises a deletion of nucleotides adenine and thymine at positions 84 and 85, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

6. The composition of any one of claims 1 to 5, wherein the mutated MGA gene has a sequence as shown in SEQ ID NO: 3.

7. The composition of any one of claims 1 to 6, wherein the live strain of S. zooepidemicus is Strain H-1 deposited in the International Depositary Authority of Canada under Accession No. 260123-01.

8. The composition of any one of claims 1 to 7, wherein the composition further comprises one or more further agents for treating a S. zooepidemicus infection.

9. The composition of claim 8, wherein the one or more further agents comprise penicillin, ampicillin and / or tilmicosin.

10. A composition as defined in any one of claims 1-9, for use to elicit an immune response against an infection by S. zooepidemicus in a subject.11 . The composition for use of claim 10, for preventing S. zooepidemicus infection.

12. The composition for use of claim 10, for treating S. zooepidemicus infection.

13. The composition for use of any one of claims 10 to 12, wherein the infection is an infection by S. zooepidemicus sequence type 194 (ST-194), optionally ATCC 35246, or a virulent strain thereof having at least 95% sequence identity thereto.

14. The composition for use of any one of claims 10 to 12, wherein the infection is an infection by S. zooepidemicus sequence type 326 (ST-326), as shown in SEQ ID NO: 1 , or a virulent strain thereof having at least 95% sequence identity thereto.

15. The composition for use of any one of claims 10 to 14, wherein the composition is for use in a single dose.

16. The composition for use of any one of claims 10 to 14, wherein the composition is for use in two doses, optionally wherein the two doses are 21 days apart.

17. The composition for use of any one of claims 10 to 16, wherein the composition is for use nasally or orally.

18. The composition for use of any one of claims 10 to 17, wherein the composition is for use at 104- 1011cfu / mL, 105- 1011cfu / mL, 106- 1011cfu / mL, 107- 1011cfu / mL, 107- 1010cfu / mL, or 108- 1010cfu / mL.

19. The composition for use of any one of claims 10 to 18, wherein the composition is for use at 107- 1010cfu / mL.

20. The composition for use of any one of claims 10 to 19, wherein the composition is for use at a volume of 0.5 mL to 2.0 mL.21 . The composition for use of any one of claims 10 to 20, wherein the subject is an animal, optionally the subject is a livestock animal, optionally the subject is a Camelidae, Caninae, Felidae, Bovidae, Suidae, or Equidae.

22. The composition for use of any one of claims 10 to 21 , wherein the subject is a pig.

23. Use of a composition as defined in any one of claims 1 to 9, to elicit an immune response against an infection by S. zooepidemicus in a subject24. Use of a composition as defined in any one of claims 1 to 9, in the manufacture of a medicament to elicit an immune response against an infection by S. zooepidemicus in a subject.

25. The use of claim 23 or 24, for preventing S. zooepidemicus infection.

26. The use of claim 23 or 24, for treating S. zooepidemicus infection.

27. The use of any one of claims 23 to 26, wherein the infection is an infection by S. zooepidemicus sequence type 194 (ST-194), optionally ATCC 35246, or a virulent strain thereof having at least 95% sequence identity thereto.

28. The use of any one of claims 23 to 26, wherein the infection is an infection by S. zooepidemicus sequence type 326 (ST-326), as shown in SEQ ID NO: 1 , or a virulent strain thereof having at least 95% sequence identity thereto.

29. The use of any one of claims 23 to 28, wherein the composition is for use in a single dose.

30. The use of any one of claims 23 to 28, wherein the composition is for use in two doses, optionally wherein the two doses are 21 days apart.31 . The use of any one of claims 23 to 30, wherein the composition is for use nasally or orally.

32. The use of any one of claims 23 to 31 , wherein the composition is for use at 104- 1011cfu / mL, 105- 1011cfu / mL, 106- 1011cfu / mL, 107- 1011cfu / mL, 107- 1010cfu / mL, or 108- 1010cfu / mL.

33. The use of any one of claims 23 to 32, wherein the composition is for use at 107- 1010cfu / mL.

34. The use of any one of claims 23 to 33, wherein the composition is for use at a volume of 0.5 mL to 2.0 mL.

35. The use of any one of claims 23 to 34, wherein the subject is an animal, optionally the subject is a livestock animal, optionally the subject is a Camelidae, Caninae, Felidae, Bovidae, Suidae, or Equidae.

36. The use of any one of claims 23 to 35, wherein the subject is a pig.

37. A method for generating a S. zooepidemicus strain with reduced virulence, comprising mutating a M protein trans-acting positive regulator (MGA) gene in a virulent S. zooepidemicus strain, wherein the mutation encodes an MGA protein with impaired DNA binding.

38. The method of claim 37, wherein mutating the MGA gene comprises introducing a frameshift mutation.

39. The method of claim 37 or 38, wherein mutating the MGA gene comprises introducing a deletion mutation.

40. The method of any one of claims 37 to 39, wherein mutating the MGA gene comprises introducing a frameshift mutation at nucleotide position 84, 85 and / or 86, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

41. The method of any one of claims 37 to 40, wherein mutating the MGA gene comprises a deletion of nucleotides adenine and thymine at positions 84 and 85, relative to the full length sequence of the MGA gene, as shown in SEQ ID NO: 2.

42. The method of any one of claims 37 to 41 , wherein the mutation that encodes the MGA protein with impaired DNA binding has a sequence as shown in SEQ ID NO: 3.

43. The method of any one of claims 37 to 42, further comprising testing the S. zooepidemicus strain for reduced virulence.

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