mRNA vaccine for rhodococcal foal pneumonia

Abstract

The present invention pertains to a ribonucleic acid (RNA)-based vaccine for immunizing equine subjects, particularly foals, against Rhodococcus equi (RE) pneumonia (referred to herein as a RE vaccine). More specifically, the present invention provides for a mRNA vaccine that contains an open reading frame (ORF) encoding an immunogenic or antigenic bacterial peptide or polypeptide, such as virulence-associated protein A (VapA), which, when administered to an equine subject, will induce a humoral response and / or a cell-mediated immune (CMI) response against the respiratory pathogen RE.

Description

[0001] mRNA VACCINE FOR RHODOCOCCAL FOAL PNEUMONIA

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 696,604, filed September 19, 2024, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

[0004] STATEMENT REGARDING SEQUENCE LISTING

[0005] The Sequence Listing for this application is labeled “Seq-List.xml” which was created on September 17, 2025 and is 7,982 bytes. The entire content of the sequence listing is incorporated herein by reference in its entirety.

[0006] BACKGROUND OF THE INVENTION

[0007] Rhodococcus equi (RE) is a facultative intracellular pathogen that is widely distributed in the soil and environment of horses worldwide. It is common for foals at horse breeding farms to become infected, with a proportion of those infected foals developing severe pneumonia, while many other foals resolve infection without developing clinical signs. Those that do not succumb to disease develop lifelong immunity, and active infection is rare in adult horses. Nonetheless, RE infection has a major detrimental impact on the equine breeding industry for several reasons: it is nearly ubiquitous in the soil at horse farms, the disease is recurrent at many breeding farms, and recovered foals may have reduced athletic performance.

[0008] RE is also a pathogen of humans, causing a pneumonia that resembles pulmonary tuberculosis (TB). The majority of human cases manifest as pneumonia and occur in immunocompromised individuals, such as those with impaired cell-mediated immunity due to HIV infection or immunosuppression therapy related to organ transplantation. Most human infections are derived from porcine- or equine-adapted strains, indicating that most human RE infections are zoonotic.

[0009] The current standard method of control of RE pneumonia in horses involves the transfusion of 1 to 2 liters of plasma from donor horses hyper-immunized against RE (RE HIP) to foals shortly afterbirth; however, this procedure poses several challenges: first, the expenses for plasma, catheters and IV drip sets are costly; second, it requires significant labor and time investment; third, plasma transfusion carries risk for foals including transfusion reactions and other adverse outcomes; fourth, handling fractious foals can be risky for handlers and foals alike; and finally, the transfusion of RE HIP only provides partial effectiveness, evidenced by the fact that many foals develop pneumonia despite transfusion.

[0010] To date, a vaccine to prevent RE pneumonia has not been licensed in the United States. A vaccine remains elusive largely because foals appear to be infected very soon after birth when they are highly susceptible to infection, potentially due to their naive and immature immune responses and the interference of maternal antibodies. Despite this, however, neonatal foals are still capable of mounting effective immune responses against intracellular bacteria.

[0011] Lung macrophages serve as the major host cells during foal infection with virulent strains of RE. Upon inhalation, RE survives and replicates within alveolar macrophages in a phagosomal compartment that fails to mature into a lysosome, resulting in the establishment of the EE-containing vacuole. The pathogen carries a plasmid that encodes the virulence- associated protein A (VapA), which is necessary for RE to cause disease in foals. VapA is an immunodominant surface protein; it neutralizes the pH of the endocytic and phagocytic continua of infected macrophages and allows the multiplication of RE within the vacuole. The ability of VapA to neutralize its intracellular environment is likely a key pathogenic feature of RE toward macrophages.

[0012] Nonetheless, due to its surface expression, VapA is also consequently accessible to immune effector cells and antibodies. Foals gavaged with live, virulent RE develop antibody titers to virulent RE and VapA during the first weeks after birth (Bordin et al. Harris et al. Chirino-Trejo et al. Hooper-McGrevy et aL), which correlate with protection against pneumonia following challenge with virulent RE (Chirino-Trejo et al. Hooper-McGrevy et al.). Additionally, passive immunization with anti -VapA antibodies protects foals against EE pneumonia, further indicating a protective role for anti-VapA antibodies (Sanz et al. Martens et al. Madigan et al. Kahn et al.). mRNA vaccines are one approach to stimulate immune responses against foreign antigens and have gained attention for their use against COVID-19 in humans because they can achieve cell-mediated and humoral immune responses that are superior to those from traditional vaccines. Efficacy of mRNA vaccines has been demonstrated in preclinical models of bacterial infection. Furthermore, it was shown that vaccination of neonatal mice against influenza with in vitro transcribed (IVT) mRNA resulted in humoral and CMI responses in the face of maternal antibodies that protected against influenza infection (Willis et al.). Accordingly, the potential applications of mRNA vaccines in human and veterinary medicine are promising. Due to the lack of effective treatment options, and further due to the lack of effective vaccines for equine rhodoccocal pneumonia, the development of a mRNA vaccine for foal immunization against RE would offer immense benefits to foal health, veterinary medicine and the equine breeding industries.

[0013] BRIEF SUMMARY OF THE INVENTION

[0014] The present invention provides a ribonucleic acid (RNA)-based vaccine for immunizing subjects of the Equus genus (hereinafter “equine” subjects), particularly foals, against Rhodococcus equi (RE) pneumonia.

[0015] More specifically, the present invention provides for a mRNA vaccine that contains an open reading frame (ORF) encoding an immunogenic or antigenic bacterial peptide or polypeptide (hereinafter referred to as “antigens” or “antigenic polypeptides”), or an ortholog, homolog, analog, variant or immunogenic fragment thereof, which, when administered to an equine subject, will induce a humoral response and / or a cell-mediated immune (CMI) response against the respiratory pathogen RE that can persist for at least 60 days post-administration, at least 150 days post-administration, or at least 12 months post-administration. In an exemplary embodiment, the mRNA vaccine comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) identity to SEQ ID NO: 1. In some embodiments, the FLAG tag contained within SEQ ID NO: 1 can be omitted.

[0016] In preferred embodiments, the mRNA vaccine encodes the antigen, virulence- associated protein A (VapA), encoded by SEQ ID NO: 2. Other immunogenic or antigenic bacterial peptides or polypeptides may also be utilized, either separately or together with VapA. For example, the mRNA vaccine can encode other antigenic surface-expressed proteins or Vaps, or orthologs, homologs, analogs, variants or immunogenic fragments thereof.

[0017] The immunogenic response to the vaccine can further be enhanced over the native antigen construct through the use of a mRNA polynucleotide having an ORF encoding the antigen and containing at least one modification to the native sequence. Modifications can include, for example, the insertion, deletion, substitution, inversion and / or transposing of nucleotide sequences. Furthermore, the vaccine construct can be, but is not always, codon- optimized for expression in equine subjects.

[0018] In one embodiment, the mRNA sequence is modified to include a membrane-anchoring polynucleotide sequence, which can enhance the immunogenic effect of the vaccine by, e.g., increasing the antigen’s host cell membrane surface attachment and concentration, and / or localizing the antigen to a specific host cellular component that is optimal for initiating an immune response. In an exemplary embodiment, the mRNA vaccine construct can be modified to include an equine-specific glycosylphosphatidylinisotol (GPI) anchoring sequence comprising an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) identity to SEQ ID NO: 3.

[0019] In one embodiment, the mRNA sequence is modified to include a signal peptide fused to either the N-terminus or C-terminus of the antigen, which can increase secretion of the antigen. Signal peptides from heterologous genes are known in the art and can be incorporated into a mRNA sequence of the present disclosure. For example, the mRNA vaccine construct can be modified to include a signal peptide sequence identified by an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) identity to SEQ ID NO: 4.

[0020] Advantageously, the mRNA vaccines of the present invention containing modified sequences over native antigen sequences have superior properties in that they can produce much larger antibody titers and / or CMI response levels, and / or produce immune responses earlier, than vaccines containing only native antigen sequences.

[0021] In certain embodiments, the vaccines of the present invention are formulated within a lipid nanoparticle (LNP). Other formulations, such as polymer-based nanoparticles, can also be employed, as well as formulations containing any number of adjuvants known to the vaccine industry.

[0022] The present invention further provides methods for producing an antigen-specific immune response to RE in an equine subject, which comprises administering to the subject a safe and effective dose of a vaccine composition comprising a mRNA vaccine according to the subject invention, thereby inducing a humoral response and / or a CMI response to the pathogen. The vaccine can be administered via, e.g., oral, intranasal, intradermal, subcutaneous, intranodal, intravenous and / or intramuscular routes. In certain embodiments, the mRNA vaccine is identified by an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) identity to SEQ ID NO: 1. In some embodiments, the FLAG tag contained within SEQ ID NO: 1 can be omitted.

[0023] In some embodiments, the methods comprise administering to an equine subject a 1 pg to 1,000 pg dose, a 5 pg to 750 pg dose, a 10 pg to 600 pg, or a 15 pg to 500 pg dose of a vaccine composition of the present disclosure. The dosage can be adjusted based on, e.g., the age and weight of the animal being treated, which can be determined by a skilled artisan of the veterinary sciences. Furthermore, additional “booster” dosages can be administered as deemed necessary by the skilled veterinarian, for example, after 24 hour of previous administration, after 48 hours, or between 5 days to 30 days, following the previous administration.

[0024] The equine subject can be treated at any age; however, in preferred embodiments, the subject is a foal, ie., an equine animal aged 6 months or less. Even more preferably, the foal is treated with a first dosage of the vaccine at age 3 days or less.

[0025] In some embodiments, administration of the vaccine composition elicits serum and bronchoalveolar lavage fluid (BALF) neutralizing antibody titers against VapA, or another RE- specific antigen.

[0026] In some embodiments, administration of the vaccine composition elicits production of pro-inflammatory cytokines, immune cytokines and / or transcription factors in PBMCs (peripheral blood mononuclear cells) or immune cells (e.g., T cells or B cells) derived from peripheral organs such as thymus, lymph nodes, spleen, intestine, lung and bone marrow.

[0027] In certain embodiments, the methods of the present invention can also include conducting measurements to detect the level of immune response in the subject after administration of the vaccines of this disclosure. Such measurements can include, for example, ELISA and ELISpot assays to measure antigen-specific immune responses and CMI responses, respectively.

[0028] It should be understood that the preferred embodiments of the vaccine compositions of the present disclosure are not naturally-occurring. That is, the mRNA polynucleotides encoding the modified RE antigens, as provided herein, do not occur in nature. It should also be understood that the mRNA polynucleotides described herein are isolated from bacterial proteins and bacterial lipids as they exist in nature. Thus, as provided herein, vaccine composition comprising a mRNA excludes bacteria (z.e., the compositions are not, nor do they contain, bacteria).

[0029] BRIEF DESCRIPTION OF THE SEQUENCES

[0030] SEQ ID NO: 1. Amino acid sequence for a derived antigen from a mRNA vaccine coding for Rhodococcus equi Virulence- Associated Protein A (VapA).

[0031] SEQ ID NO: 2. Amino acid sequence for Rhodococcus equi Virulence-Associated Protein A (VapA).

[0032] SEQ ID NO: 3. Amino acid sequence for glycosylphosphatidylinositol (GPI) anchor signal sequence derived from the horse placental alkaline phosphatase protein.

[0033] SEQ ID NO: 4. Amino acid sequence for human albumin signal peptide. SEQ ID NO: 5. Amino acid sequence for human IgG signal peptide.

[0034] DETAILED DISCLOSURE OF THE INVENTION

[0035] The present invention provides an RNA (e.g., mRNA) vaccine for immunizing subjects of the Equus genus (hereinafter “equine” subjects), particularly foals, against Rhodococcus equi (RE) pneumonia. The RNA (e.g., mRNA) vaccines, in some embodiments, may be used to induce a balanced immune response, comprising both cellular and humoral immunity.

[0036] Nucleic Acids / Polynucleotides

[0037] RE vaccines, as provided herein, comprise at least one (one or more) ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide, or an ortholog, homolog, analog, variant or immunogenic fragment thereof, selected from any of the virulence-associated proteins (Vaps) of RE, e.g., VapA, VapB, VapC, VapD, VapE, VapG, VapH, VapI, and / or VapN. The mRNA vaccine can also encode other antigenic surface-expressed proteins, or orthologs, homologs, analogs, variants or immunogenic fragments thereof.

[0038] As used herein, the term “nucleic acid” includes any compound and / or substance that comprises a polymer of nucleotides (nucleotide monomer). These polymers are referred to as polynucleotides. Thus, the terms “nucleic acid” and “polynucleotide” are used interchangeably.

[0039] Nucleic acids may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino- LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

[0040] In some embodiments, polynucleotides of the present disclosure function as messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “U ”

[0041] The basic components of a mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly- A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and / or structural design features, which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

[0042] Polynucleotides of the present disclosure, in some embodiments, can be codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art — non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0043] In some embodiments, a codon optimized sequence shares less than 95% sequence identity, less than 90% sequence identity, less than 85% sequence identity, less than 80% sequence identity, or less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or antigenic polypeptide)).

[0044] In some embodiments, a codon-optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85%, or between about 67% and about 80%) sequence identity to a naturally-occurring sequence or a wild-type sequence (e.g., a naturally-occurring or wildtype mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, a codon-optimized sequence shares between 65% and 75%, or about 80% sequence identity to a naturally-occurring sequence or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0045] In some embodiments a codon-optimized RNA (e.g., mRNA) may, for instance, be one in which the levels of G / C are enhanced. The G / C-content of nucleic acid molecules may influence the stability of the RNA. RNA having an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 (incorporated herein by reference) discloses a pharmaceutical composition containing a mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA.

[0046] In some embodiments, the vaccine of the present invention elicits a greater immune response when the polynucleotides are not codon optimized.

[0047] Antigens / Antigenic Polypeptides

[0048] The present invention provides for a mRNA vaccine that contains an open reading frame (ORF) encoding an immunogenic or antigenic bacterial peptide or protein (hereinafter referred to as “antigens” or “antigenic polypeptides”), or an ortholog, homolog, analog, variant or immunogenic fragment thereof, which, when administered to an equine subject, will induce a humoral response and / or a cell-mediated immune (CMI) response against the respiratory pathogen RE that can persist for at least 5 days, at least 30 days, at least 50 days, at least 150 days, or at least 12 months post-administration.

[0049] In preferred embodiments, the mRNA vaccine encodes the antigen, virulence- associated protein A (VapA) (SEQ ID NO: 1), or an ortholog, homolog, analog, variant or immunogenic fragment thereof. In some embodiments, the antigen sequence is constructed based on the vapA gene identified by NCBI accession #BAA04768.1. In an exemplary embodiment, the mRNA vaccine comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) identity to SEQ ID NO: 1. In some embodiments, the FLAG tag contained within SEQ ID NO: 1 can be omitted. Other immunogenic or antigenic bacterial peptides or proteins may also be utilized, either separately or together with VapA. For example, the mRNA vaccine can encode other antigenic surface-expressed proteins or Vaps (e.g., VapB, VapC, VapD, VapE, VapG, VapH, VapI, and / or VapN), or orthologs, homologs, analogs, variants or immunogenic fragments thereof.

[0050] Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. Polypeptides may also comprise single chain polypeptides or multichain polypeptides, such as antibodies or insulin, and may be associated or linked to each other. Most commonly, disulfide linkages are found in multichain polypeptides. The term “polypeptide” may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally- occurring amino acid.

[0051] A “polypeptide variant” is a molecule that differs in its amino acid sequence relative to a native sequence or a reference sequence. Amino acid sequence variants may possess substitutions, deletions, insertions, or a combination of any two or three of the foregoing, at certain positions within the amino acid sequence, as compared to a native sequence or a reference sequence. Ordinarily, variants possess at least 50% identity to a native sequence or a reference sequence. In some embodiments, variants share at least 80% identity or at least 90% identity with a native sequence or a reference sequence.

[0052] In some embodiments “variant mimics” are provided. A “variant mimic” contains at least one amino acid that would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and / or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic. For example, phenylalanine may act as an inactivating substitution for tyrosine, or alanine may act as an inactivating substitution for serine.

[0053] “Orthologs” refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is important for reliable prediction of gene function in newly sequenced genomes. “Analogs” is meant to include polypeptide variants that differ by one or more amino acid alterations, for example, substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.

[0054] The present disclosure provides several types of compositions that are polynucleotide or polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is synonymous with the term “variant” and generally refers to a molecule that has been modified and / or changed in any way relative to a reference molecule or a starting molecule.

[0055] As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this disclosure.

[0056] For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal residues or N-terminal residues) alternatively may be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence that is soluble, or linked to a solid support.

[0057] “Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more (e.g., 3, 4 or 5) amino acids have been substituted in the same molecule.

[0058] As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue.

[0059] “Features” when referring to polypeptide or polynucleotide are defined as distinct amino acid sequence-based or nucleotide-based components of a molecule respectively. Features of the polypeptides encoded by the polynucleotides include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini and any combination(s) thereof.

[0060] As used herein, when referring to polypeptides, the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

[0061] As used herein when referring to polypeptides the terms “site” as it pertains to amino acid based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.” As used herein when referring to polynucleotides the terms “site” as it pertains to nucleotide based embodiments is used synonymously with “nucleotide.” A site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide-based or polynucleotide-based molecules.

[0062] As used herein the terms “termini” or “terminus” when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[0063] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or longer than 100 amino acids. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 (contiguous) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided herein or referenced herein. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than 80%, 75%, 70%, 65% to 60% identical to any of the sequences described herein can be utilized in accordance with the disclosure.

[0064] Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term “identity,” as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between two sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al. (1997). Gapped BLAST and PSLBLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147: 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453). More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0065] As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are termed homologous. Homology is a qualitative term that describes a relationship between molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.

[0066] Homology implies that the compared sequences diverged in evolution from a common origin. The term “homolog” refers to a first amino acid sequence or nucleic acid sequence (e.g., gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ancestral sequence. The term “homolog” may apply to the relationship between genes and / or proteins separated by the event of speciation or to the relationship between genes and / or proteins separated by the event of genetic duplication. “Orthologs” are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function in the course of evolution. “Paralogs” are genes (or proteins) related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.

[0067] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same 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, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm described in the art.

[0068] In certain embodiments, the immunogenic response to the vaccine of the present invention can further be enhanced over the native antigen construct through the use of a modified mRNA sequence. In one embodiment, the mRNA sequence is modified to include a membrane-anchoring amino acid sequence. A “membrane-anchoring” amino acid sequence is, for the purposes of the present invention, an amino acid sequence that serves to anchor, or attach, an antigen of the invention in the cell membrane of the cell expressing the protein (for example by embedding of amino acids, in the case of such sequences based on transmembrane domains, or through associated fatty acids, in the case of GPI anchors). The orientation will be such that the antigen is found on the extracellular side of the membrane. This arrangement allows the antigen to interact with cells other than the cell in which the antigen has been expressed.

[0069] Non limiting examples of membrane-anchoring amino acid sequences include: a transmembrane domain; a glycosylphosphatidylinositol (GPI) anchor; a sequence motif that undergoes lipid modification thereby allowing membrane anchorage of the antigen; and another antibody, or antigen-binding fragment thereof, the binding of which anchors the antigen of the present invention to the membrane. Examples of membraneanchoring sequences that go beyond those referred to above will also be known to those skilled in the art, and these too may be used in the vaccines of the invention.

[0070] In the case of transmembrane domains for use in the mRNA sequences of the invention, a suitable domain may, for example, be from a one-pass transmembrane type 1 proteins for example CD4. Alternatively, a suitable domain may be from a one-pass transmembrane type 2 protein, such as CD72. A suitable domain may be from a four-pass transmembrane protein (for example CD37), and may comprise one of more of the four individual transmembrane domains found in such proteins. Alternatively, a suitable domain may be from a multiple transmembrane attachment (type III) protein, such as C5aR, and may comprise one or one of more of the individual transmembrane domains found in proteins of this sort.

[0071] In order to serve their function, membrane-anchoring sequences comprising or based upon transmembrane domains should have sufficient length to allow the sequence to become embedded in the cell membrane.

[0072] Suitable membrane-anchoring sequences that allow lipid modification of the antigens of the invention may facilitate conjugation of a lipid to the antigen ex vivo, or may induce lipid modification in vivo. Suitable membrane-anchoring sequences for that allow lipid modification of the antigens of the invention may allow expression of the antigen at the cell surface.

[0073] By way of example a suitable sequence allowing lipid modification may be one that promotes N-myristoylation of the antigen. N-myristoylation is catalyzed by N-myristoyl transferase which generally recognizes the sequence Met-Gly-X-X-X-Ser / Thr at the amino terminus. Other suitable sequences allowing lipid modification may include those that promote attachment of lipids selected from the group consisting of: fatty acids, isoprenoids, sterols, phospholipids, and glycosylphosphatidyl inositol (GPI). Examples of such sequences are well known to those skilled in the art.

[0074] It will also be appreciated that antigens of the invention may comprise sequences that allow modification by more than one type of lipid. Merely by way of example, antigens of the invention may comprise a sequence that promotes modification by myristate and a sequence that promotes modification by palmitate; a sequence that promotes modification by palmitate and a sequence that promotes modification by cholesterol; or a sequence that promotes modification by farnesyl and a sequence that promotes modification by palmitate.

[0075] Additional antibodies, or antigen-binding fragments thereof, suitable for use as membrane-anchoring amino acid sequences in the RE antigens of the invention may include a cell surface moiety (for example, a moiety selected from the group consisting of: proteins; peptides; lipids; and polysaccharides associated with the membrane). For example, a suitable RE antigen may comprise an anti-CD37 antibody, or an antigen-binding fragment thereof.

[0076] Further examples of membrane-anchoring amino acid sequences that may be used in vaccines of the invention include suitable integral (intrinsic) or peripheral (extrinsic) proteins that leads to expression of the RE antigen at the cell surface.

[0077] In an exemplary embodiment, the mRNA vaccine construct of the present invention comprises an equine-specific GPI membrane-anchoring sequence derived from horse placental alkaline phosphatase protein comprising an amino acid sequence identified by SEQ ID NO: 3.

[0078] In some embodiments, antigenic polypeptides encoded by the present RE vaccine are modified to include a signal peptide. Signal peptides, located at the N-terminal of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway.

[0079] Signal peptides generally include three regions: an N-terminal region of differing length, which usually comprises positively charged amino acids; a hydrophobic region; and a short carboxy -terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by a ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane. The signal peptide, however, is not responsible for the final destination of the mature protein. Secretory proteins devoid of additional address tags in their sequence are by default secreted to the external environment.

[0080] The vaccines of the present disclosure may comprise, for example, RNA (e.g., mRNA) polynucleotides encoding a signal peptide, either homologous or heterologous, wherein the signal peptide coding sequence is operably linked to and is in frame with the coding sequence of the antigenic polypeptide. Thus, vaccines of the present disclosure, in some embodiments, produce an antigenic polypeptide comprising an antigenic polypeptide (e.g., VapA) fused to a signal peptide. In some embodiments, a signal peptide is fused to the N-terminus of the antigenic polypeptide. In some embodiments, a signal peptide is fused to the C-terminus of the antigenic polypeptide.

[0081] A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

[0082] 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-

[0083] 60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, SO-

[0084] 55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-

[0085] 45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-

[0086] 25, 20-25, or 15-20 amino acids.

[0087] In certain embodiments the signal peptide derives from tissue plasminogen activator (TP A) or albumin, especially human serum albumin (HSA), or CD5 (CD — cluster of differentiation) or HLA-A2 (HLA — human leucocyte antigen) or luciferase, especially Gaussian luciferase, or immunoglobulin (e.g. IgG or IgE heavy chain) or IL-2 (human IL-2) or chymotrypsinogen (human chymotrypsinogen), or a fragment, variant or derivative thereof. In some embodiments, the signal peptide fused to the antigenic polypeptide is an artificial signal peptide.

[0088] Preferably, the signal peptide of the present invention is a human serum albumin (HSA) signal peptide according to SEQ ID NO: 4 (MKWVTFISLLFLFSSAYS) or a human IgG signal peptide according to SEQ ID NO: 5 (MGWSCIILFLVATATGVHS). By inclusion of a signal peptide that is aimed to allow co-translational transport of a Vap into the ER followed by protein secretion, it is possible to increase antigen accessibility to the immune system. The examples disclosed herein are not meant to be limiting and any signal peptide that is known in the art to facilitate targeting of a protein to ER for processing and / or targeting of a protein to the cell membrane may be used in accordance with the present disclosure.

[0089] Advantageously, the mRNA vaccines of the present invention containing modified sequences, e.g., comprising a membrane-anchoring sequence and / or a signal peptide, have superior properties in that they can produce much larger antibody titers and / or CMI response levels, and / or produce immune responses earlier, than vaccines containing only native RE antigen sequences.

[0090] In Vitro Transcription of RNA (e.g., mRNA)

[0091] RE vaccines of the present disclosure comprise at least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, for example, is transcribed in vitro from template DNA, referred to as an “z z vitro transcription template.” In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

[0092] A “5' untranslated region” (5 TR) refers to a region of a mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of a mRNA transcript translated by a ribosome) that does not encode a polypeptide.

[0093] A “3' untranslated region” (3 UTR) refers to a region of a mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of a mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

[0094] An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.

[0095] A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.

[0096] In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides. For example, a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0097] Therapeutic and Prophylactic Compositions

[0098] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention, treatment or diagnosis of Rhodococcus equi (RE) pneumonia in equine subjects, and in some embodiments, in humans and / or other mammals. RE mRNA vaccines can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and / or treat infectious disease.

[0099] In some embodiments, the RE vaccines of the present disclosure are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

[0100] In some embodiments, the RE vaccine containing RNA (e.g., mRNA) polynucleotides as described herein can be administered to a subject and the RNA (e.g., mRNA) polynucleotides are translated in vivo to produce an antigenic polypeptide.

[0101] The present vaccines may be induced for translation of an antigen in a cell, tissue or organism. In some embodiments, such translation occurs in vivo, although such translation may occur ex vivo, in culture or in vitro. In some embodiments, the cell, tissue or organism is contacted with an effective amount of a composition containing the RE vaccine containing a polynucleotide that has at least one a translatable region encoding an antigenic polypeptide.

[0102] The RE vaccines of the present invention may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of the RE vaccine of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

[0103] In certain embodiments, the present invention provides pharmaceutical compositions including RE vaccines and RE vaccine compositions and / or complexes optionally in combination with one or more pharmaceutically-acceptable excipients, adjuvants, additional therapeutically-active or prophylactically-active substances, or other components. The vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free.

[0104] Formulations of the RE vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0105] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.001% and 100%, e.g., between 0.05 and 50%, between 0.1-30%, between 1-80%, or at least 80% (w / w) active ingredient (mRNA polynucleotide).

[0106] The vaccines can be formulated using one or more excipients to, for example: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo, and / or (6) alter the release profile of encoded protein (antigen) in vivo.

[0107] In addition to traditional excipients, such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RE vaccines (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, adjuvants and combinations thereof.

[0108] Vaccine adjuvants traditionally are immunopotentiators that bind to pattern recognition receptors (PRRs) of innate immune cells to increase the magnitude or achieve qualitative alteration of immune responses, finally enhancing the efficacy of vaccines. The adjuvants can include, for example, aluminum salts, emulsions, polymers, saponins and immunostimulating complexes (ISCOMs). In some embodiments, the vaccines do not include an adjuvant (they are adjuvant free). In certain embodiments, the mRNA polynucleotide of the vaccine possess self-adjuvant characteristics through the triggering of innate immune signaling in a host in addition to the immune response achieved by translation of an antigen protein. In certain embodiments, a vaccine delivery system is utilized, such as a lipid nanoparticle, which contain components such as ionizable cationic lipids that can enhancing mRNA delivery and immune response.

[0109] Suitable adjuvants are known in the immunological arts, including those described in US 11,896,666 B2; US 10,265,395 B2; Xie, C., et al. The advances of adjuvants in mRNA vaccines, npj Vaccines 8, 162 (2023). https: / / doi.org / 10.1038 / s41541-023-00760-5; and Carnet, F., et al. An inventory of adjuvants used for vaccination in horses: the past, the present and the future. Vet Res 54, 18 (2023). https: / / doi.org / 10.1186 / sl3567-023-01151-3 (each of which is incorporated by reference herein).

[0110] In certain embodiments, the vaccine contains a modification increasing resistance to in vivo degradation (e.g. degradation by an exo- or endo-nuclease) and / or ex vivo degradation (e.g. by the manufacturing process prior to vaccine administration, e.g. in the course of the preparation of the vaccine solution to be administered). Stabilization of RNA can, e.g., be achieved by providing a 5 '-CAP- Structure, a poly(A) tail, a histone stem-loop or any other UTR-modification. It can also be achieved by chemical modification or modification of the G / C-content of the nucleic acid. Various other methods are known in the art and conceivable in the context of the invention.

[0111] In some embodiments, the RE vaccine may or may not contain a enhancer and / or promoter sequence, which may be modified or unmodified or which may be activated or inactivated.

[0112] In some embodiments the RE vaccine may have one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3'UTR. The AURES may be removed from the RE vaccines. Alternatively the AURES may remain in the vaccine.

[0113] The vaccine compositions of the present invention can be refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 4 °C or lower, such as a temperature between about -150 °C. and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C). For example, the pharmaceutical composition is a solution that is refrigerated for storage and / or shipment at, for example, about -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C.

[0114] Delivery of mRNA vaccines

[0115] The mRNA according to the present invention may be prepared using any method known in the art, including synthetic methods such as e.g. solid phase RNA synthesis, as well as in vitro methods, such as RNA in vitro transcription reactions.

[0116] In certain embodiments, the mRNA compound according to the invention in encapsulated by a delivery carrier, such as a lipid nanoparticle (LNP). Polymers (e.g., polyamines, dendrimers, polysaccharide and copolymers) and / or peptides (e.g., protamine) can also serve as delivery agents. In some embodiments, the mRNA is delivered as free mRNA in solution. In some embodiments, the mRNA is delivered via autologous dendritic cells pretreated ex vivo with the mRNA.

[0117] In certain preferred embodiments, the mRNA is produced The term “lipid nanoparticle,” or “LNP,” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids. In some embodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipients selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a PEGylated lipid).

[0118] In some embodiments, the mRNA, or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response. In some embodiments, the mRNA or a portion thereof is associated with the lipid nanoparticles.

[0119] In the context of the present invention, lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and / or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a LNP.

[0120] In various embodiments, the LNP have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, the mRNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease. As used herein, the mean diameter may be represented by the z-average as determined by dynamic light scattering.

[0121] In certain embodiments, the LNP have a hydrodynamic diameter in the range from about 50 nm to about 300 nm, or from about 60 nm to about 250 nm, from about 60 nm to about 150 nm, or from about 60 nm to about 120 nm, respectively.

[0122] An LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0123] In one embodiment, the mRNA-comprising LNP comprises one or more cationic lipids as defined herein, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.

[0124] In certain embodiments, the cationic lipid is cationisable, i.e., it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. When positively charged, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease. The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0125] In certain embodiments, the LNP may comprise any further cationic or cationisable lipid, i.e. any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3- dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(l,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide (DMRIE).

[0126] Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn- 3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l- (2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), di((Z)-non-2-en-l-yl) 9-((4-

[0127] (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)-N,N-dimethyl-2- nonylhenicosa-12,15-dien-l-amine (L608), and N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl]heptadecan-8-amine (L530).

[0128] In one embodiment, the further cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in W02012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2- dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy- 3 morpholinopropane (DLin-MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy- 3 dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin- TAP.C1), l,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3- (N,Ndilinoleylamino)- 1 ,2-propanediol (DLinAP), 3 -(N,N-di oleylamino)- 1 ,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), and dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA).

[0129] In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.

[0130] Suitable stabilizing lipids include neutral lipids and anionic lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0131] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoylphosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn- glycero-3 phosphocholine (DSPC).

[0132] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.

[0133] In various embodiments, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[0134] In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid to cholesterol ranges from about 5:1 to 1 : 1. The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N— dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0135] In certain embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GMi).

[0136] In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-s-DMG) and the like.

[0137] In certain embodiments, the LNP comprises an additional, stabilizing-lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycolipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2000)carbamyl]-l,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as

[0138] 4-O-(2',3 '-di(tetradecanoyloxy)propyl- 1 -0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-

[0139] 5-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25: 1.

[0140] In some embodiments, the LNP comprises a cationic lipid, a PEG or PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the lipid nanoparticle carrier comprises a molar ratio of about 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol; and 0.5-15% PEG-modified lipid.

[0141] In some embodiments, the lipid nanoparticle has a poly dispersity value of less than 0.4. In some embodiments, the nanoparticle has a net neutral charge at a neutral pH value.

[0142] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a vaccine composition may vary, depending upon the identity, size, and / or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.001% and 99% (w / w) of the active ingredient. By way of example, the composition may comprise between 0.001% and 100%, e.g., between 0.05 and 50%, between 0.1-30%, between 1-80%, or at least 80% (w / w) active ingredient.

[0143] In some embodiments, the ratio of lipid to RNA (e.g., mRNA) in LNPs may be 5: 1 to 20: 1, 10:1 to 25: 1, 15: 1 to 30: 1 (w / w). In one embodiment, the mRNA to total lipid ratio is less than 0.06 vi / vi, or between 0.03 and 0.04 w / w.

[0144] In certain embodiments, the mRNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

[0145] In some embodiments, the RE mRNA vaccine composition may comprise a polynucleotide described herein, formulated in a lipid nanoparticle comprising 45-55 mole percent ionizable cationic lipid (e.g., Clin-MC3-DMA), 5-15 mole percent DSPC, 35-40 mole percent cholesterol, and optionally 1-2 mole percent DMG-PEG. Additional components can include the buffer trisodium citrate, sucrose and water for injection.

[0146] In certain embodiments, the LNP comprises one or more targeting moieties which are capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand which directs the LNP to a receptor found on a cell surface.

[0147] In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains which bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo. Other non-limiting exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20210251898 Al, which is incorporated by reference in its entirety.

[0148] The compositions of the present invention can further comprise one or more pharmaceutically acceptable carriers and / or excipients, and can be formulated into preparations in, for example, solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, gels, lotions, solutions, suppositories, drops, patches, injections, inhalants and aerosols.

[0149] The term “pharmaceutically acceptable” as used herein means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0150] Carriers and / or excipients according the subject invention can include any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for, e.g., IV use, solubilizers (such as, e.g., Tween 80, Polysorbate 80), alcohols (e.g., methanol, ethanol, isopropanol, propanol), colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, aromatizers, thickeners, coatings, preservatives (such as, e.g., Thimerosal, benzyl alcohol), antioxidants (such as, e.g., ascorbic acid, sodium metabisulfite), tonicity controlling agents, absorption delaying agents, adjuvants, bulking agents (such as, e.g., lactose, mannitol) and the like. The use of carriers and / or excipients in the field of drugs and supplements is well known. Except for any conventional media or agent that is incompatible with the target health-promoting substance or with the composition, its use in the present compositions may be contemplated.

[0151] In one embodiment, the vaccine composition can be made into aerosol formulations so that, for example, it can be nebulized or inhaled. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions. Formulations for oral or nasal aerosol or inhalation administration may also be formulated with illustrative carriers, including, for example, saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents or fluorocarbons. Aerosol formulations can be placed into pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Illustratively, delivery may be by use of a single-use delivery device, a mist nebulizer, a breath-activated powder inhaler, an aerosol metered-dose inhaler (MDI) or any other of the numerous nebulizer delivery devices available in the art. Additionally, mist tents or direct administration through endotracheal tubes may also be used.

[0152] In one embodiment, the vaccine composition can be formulated for administration via injection, for example, as a solution or suspension. The solution or suspension can comprise suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3- butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. One illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI). Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion. Water or saline solutions and aqueous dextrose and glycerol solutions may be preferably employed as carriers, particularly for injectable solutions. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.

[0153] Further components can be added to the compositions as are determined by the skilled artisan such as, for example, buffers, carriers, viscosity modifiers, preservatives, flavorings, dyes and other ingredients specific for an intended use. One skilled in this art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulations techniques and pharmaceutically-acceptable excipients and carrier solutions suitable for particular modes of administration are well-known to those skilled in the art.

[0154] Methods of Treatment

[0155] Further provided herein are methods for producing an antigen-specific immune response to RE in an equine subject, which comprises administering to the subject a prophylactically-effective dose of a vaccine composition according to the present invention thereby inducing a humoral and / or a CMI response to an antigenic polypeptide associated with the pathogen. In certain embodiments, the mRNA vaccine identified is by SEQ ID NO: 1. The vaccine can be administered via, e.g., oral, intranasal, intradermal, subcutaneous, intranodal, intravenous and / or intramuscular routes.

[0156] An “effective” or “prophylactically effective” amount or dose of a RE vaccine is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the vaccine, and other determinants. In general, an effective amount of the vaccine composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the native antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA, e.g., mRNA, vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.

[0157] In certain embodiments, the “effective” or “prophylactically effective” amount or dose is a dose that prevents infection with the RE pathogen at a clinically acceptable level.

[0158] As used herein, an “equine subject” includes any animal of the genus Equus, including horses, wild ass, zebra, onager, kiang, and donkeys. Example species include, but are not limited to, E. caballus, E. ferus ferus, E. ferus przewalskii, E. africanus somaliensis, E. africanus africanus, E. kiang, E. hemionus onager, E. hemionus kulan, E. zebra, E. quagga, E. quagga chapmani, E. quagga quagga, and E. greyvi.

[0159] In certain embodiments, the subject of treatment may also be another mammal, including cats, dogs, rhinoceros, tapir, and humans. RE is capable of infecting humans with compromised immune systems, e.g., as a result of AIDS infection or cancer treatment.

[0160] Vaccines can be administered once, twice, three times, four times or more. In certain embodiments, primary immunization is followed by at least one booster dose. It is possible, although less desirable, to administer the vaccine to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

[0161] In some embodiments, the methods comprise administering to an equine subject a 1 pg to 1,000 pg dose, a 5 pg to 750 pg dose, a 10 pg to 600 pg, a 15 pg to 500 pg, a 25 pg to 250 pg dose, a 100 pg to 400 pg dose, a 200 to 500 pg dose, a 300 to 600 pg dose, or a 400 to 800 pg dose of a mRNA vaccine of the present disclosure.

[0162] The dosage can be adjusted based on, e.g., the age and weight of the animal being treated, which can be determined by a skilled artisan of the veterinary sciences.

[0163] The equine subject can be treated at any age; however, in preferred embodiments, the subject is a foal, ie., an equine animal aged one year or less.

[0164] In some embodiments, the subject is about 5 years old or younger when treated with an initial dosage of the mRNA vaccine of the present disclosure. In some embodiments, the subject is about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month). In some embodiments, the subject is between the ages of about 1 year and about 5 years (e.g., about 1, 2, 3, 5 or 5 years), or between the ages of about 6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months), or between the ages of 12 hours and 6 months (e.g., about 1 to 10 days, or 1 day to 30 days, or 30 days to 60 days, or about 60 days to 6 months). In some embodiments, the subject is 5 to 10 years of age, or 5 to 15 years of age, or 5 to 20 years of age, or 5 to 30 years of age or 5 to 50 years of age.

[0165] In a preferred embodiments, the subject is a foal is treated with a first dosage of the vaccine at age 5 days or less, 4 days or less, 3 days or less, 48 hours or less, or 24 hours or less.

[0166] In some embodiments, the subject is a pregnant mare. Thus, the present disclosure provides RNA (e.g., mRNA) vaccines for maternal immunization to improve mother-to-child transmission of protection against the RE pathogen.

[0167] In some embodiments, the subject has been exposed to RE, or the subject is at risk of infection by RE. In some embodiments, the subject is immunocompromised (i.e., has an impaired immune system due to, e.g., age, infection, illness, an immune disorder or an autoimmune disorder).

[0168] The RE vaccines may be administrated with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an adjuvant or a booster.

[0169] As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, or 45 years. In some embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 30 days, 6 months or 1 year.

[0170] In some embodiments, the disclosure provides a method of delivering a therapeutic and / or prophylactic composition to a cell, tissue or organ of a subject (e.g., a lung cell, or the lung / lung tissue). This method includes the step of administering to a subject a mRNA vaccine composition of the present invention such that the composition contacts the cell, tissue and / or organ, whereby the composition is thus delivered to the cell, tissue and / or organ.

[0171] In some embodiments, the disclosure provides a method of producing a polypeptide of interest in a cell (e.g., a mammalian cell). The method includes the step of contacting the cell with a mRNA vaccine composition of the present invention encoding the polypeptide of interest, whereby the mRNA is capable of being translated in the cell to produce the polypeptide.

[0172] In some embodiments, the disclosure provides a method of treating and / or preventing a disease or disorder in subject in need thereof. The method includes the step of administering to the subject a therapeutically effective amount of a mRNA vaccine composition of the present invention. In some embodiments, the disease or disorder is characterized by dysfunctional or aberrant protein or polypeptide activity. For example, the disease or disorder can be selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases. In some embodiments, the disease or disorder is an infection with RE.

[0173] As used herein, the term “treatment” refers to eradicating, reducing, ameliorating, or reversing a sign or symptom of a health condition, disease or disorder to any extent, and includes, but does not require, a complete cure of the condition, disease or disorder. Treating can be curing, improving, or partially ameliorating a disorder. “Treatment” can also include improving or enhancing a condition or characteristic, for example, bringing the function of a particular system in the body to a heightened state of health or homeostasis.

[0174] As used herein, “preventing” a health condition, disease or disorder refers to avoiding, delaying, forestalling, or minimizing the onset of a particular sign or symptom of the condition, disease or disorder. Prevention can, but is not required to be, absolute or complete, meaning the sign or symptom may still develop at a later time. Prevention can include reducing the severity of the onset of such a condition, disease or disorder, and / or inhibiting the progression of the condition, disease or disorder to a more severe (i.e., causing more harm) condition or disorder.

[0175] In some embodiments, the present invention provides methods for eliciting an immune response in a subject against RE, which comprises administering to the subject a mRNA vaccine comprising at least one RNA polynucleotide having an ORF encoding VapA or another RE antigenic polypeptide, wherein anti-antigenic polypeptide antibody titer in the subject is increased following vaccination relative to baseline. In certain embodiments, the mRNA vaccine is identified by SEQ ID NO: 1. In some embodiments the FLAG tag can be omitted from SEQ ID NO: 1.

[0176] In some embodiments, administration of the vaccine composition elicits serum and bronchoalveolar lavage fluid (BALF) neutralizing antibody titers against VapA, or another RE- specific antigen.

[0177] In some embodiments, administration of the vaccine composition elicits production of pro-inflammatory cytokines, immune cytokines and / or transcription factors in PBMCs (peripheral blood mononuclear cells) or immune cells derived from peripheral organs such as thymus, lymph nodes, spleen, intestine, lung and bone marrow.

[0178] In preferred aspects, vaccines of the invention produce prophylactically- and / or therapeutically-efficacious levels, concentrations and / or titers of antigen-specific antibodies, or anti-antigenic polypeptide antibodies, in the blood or serum of a vaccinated subject. As defined herein, the term antibody titer refers to the amount of antigen-specific antibody produces in a subject.

[0179] An “antigen-specific antibody,” or “anti-antigenic polypeptide antibody” is a serum antibody the binds specifically to the antigenic polypeptide.

[0180] In exemplary embodiments, antibody titer is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result. In exemplary embodiments, antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titer is determined or measured by neutralization assay, e.g., by microneutralization assay. In certain aspects, antibody titer measurement is expressed as a ratio, such as 1 :40, 1 : 100, etc.

[0181] Thus, antigen-specific immune responses in a subject may be determined, in some embodiments, by assaying for antibody titer (titer of an antibody that binds to a RE antigenic polypeptide) following administration to the subject of any of the mRNA vaccines of the present disclosure. For example, in some embodiments, activities of anti-VapA antibodies such as IgGi, IgG4 / 7 and / or IgA in serum and BALF can be measured using indirect ELISA.

[0182] In exemplary embodiments of the invention, an efficacious vaccine produces an antibody titer of greater than 1 :40, greater that 1 : 100, greater than 1 :400, greater than 1 : 1000, greater than 1 :2000, greater than 1 :3000, greater than 1 :4000, greater than 1 :500, greater than 1 :6000, greater than 1 :7500, greater than 1 : 10000.

[0183] In exemplary aspects of the invention, antigen-specific antibodies are measured in units of pg / ml or are measured in units of IU / L (International Units per liter) or mIU / ml (milli International Units per ml). In exemplary embodiments of the invention, an efficacious vaccine produces >0.5 pg / ml, >0.1 pg / ml, >0.2 pg / ml, >0.35 pg / ml, >0.5 pg / ml, >1 pg / ml, >2 pg / ml, >5 pg / ml or >10 pg / ml. In exemplary embodiments of the invention, an efficacious vaccine produces >10 mIU / ml, >20 mIU / ml, >50 mIU / ml, >100 mIU / ml, >200 mIU / ml, >500 mIU / ml or >1000 mIU / ml.

[0184] In exemplary embodiments, the antibody level or concentration is produced or reached by 24 hours following vaccination, 48 hours following vaccination, 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In exemplary embodiments, the level or concentration is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose).

[0185] In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti- antigenic polypeptide antibody titer produced in the subject is increased by 1 log to 10 log, 2 log to 9 log, 3 log to 8 log, 4 log to 7 log, or 5 log to 6 log following vaccination relative to a control. In some embodiments, the “control” is an anti-antigenic polypeptide antibody titer produced in a subject who has not been administered a RNA (e.g., mRNA) vaccine of the present disclosure (i.e., baseline). In some embodiments, the control is an anti -antigenic polypeptide antibody titer produced in a subject who has been administered a mRNA vaccine encoding only a native form RE antigen, without an anchoring sequence and / or without a signal peptide. In some embodiments, the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated RE vaccine, or wherein the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified RE protein vaccine.

[0186] In some embodiments, the anti-antigenic polypeptide antibody titer produced in a subject is increased at least 2 times, at least 5 times, at least 10 times, at least 25 times, or at least 50 times relative to a control.

[0187] In some embodiments, the antigen-specific immune response is measured as a geometric mean titer (GMT) of serum neutralizing antibodies to RE Vaps, and wherein the GMT in serum neutralizing antibodies to RE Vaps increases in the subject at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold or greater within 1 day, 5 days, 10 days, or 30 days relative to baseline.

[0188] In some embodiments, the immune response in the subject is induced 2 days earlier, or 3 days earlier, relative to an innate immune response in an unvaccinated subject and / or relative to a subject vaccinated with a mRNA vaccine encoding only a native RE antigenic polypeptide. In some embodiments the immune response in the subject is induced 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a control subject.

[0189] In some embodiments, the subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the vaccine. Seroconversion is the time period during which a specific antibody develops and becomes detectable in the blood. After seroconversion has occurred, antibodies to a pathogen can be detected in blood. During an infection or immunization, the immune system begins to produce antibodies in response to the presence of antigens. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are considered absent or at low levels. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection. In certain embodiments, cell-mediated immune (CMI) responses in a subject treated with the vaccines of the present disclosure can be assessed, by measuring Thl cytokine production from isolated foal PBMCs (peripheral blood mononuclear cell). The Thl-oriented immune response promotes the elimination of infected cells by stimulating cytotoxic cells, especially CD8+ T lymphocytes. Specific populations of CD4+ T lymphocytes are associated to the Thl profiles of the immune response, and can be distinguished by the secretion of specific cytokines such as interferon gamma (IFN-y) and interleukin (IL-) 12. Production of cytokines from isolated foal PBMCs stimulated with VapA can be measured using a commercially available ELISpot kit.

[0190] In some embodiments, the efficacy (or effectiveness) of a RNA (e.g., mRNA) vaccine is greater than 20%, greater than 30%, greater than 40%, greater than 50%, and preferably greater than 60%.

[0191] Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11): 1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:

[0192] Efficacy=(ARU-ARV) / ARUx l00; and

[0193] Efficacy=(l-RR)x l00.

[0194] Likewise, vaccine effectiveness may be assessed using standard analyses (see, e.g., Id.). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination: Effectiveness = (1-OR) x 100.

[0195] In some embodiments, the efficacy (or effectiveness) of a RNA (e.g., mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0196] In some embodiments, the vaccine immunizes the subject against RE, for up to 2 years. In some embodiments, the vaccine immunizes the subject against RE for more than 2 years, more than 3 years, more than 4 years, or for 5-10 years.

[0197] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 20 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0198] By “reduces” is meant a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0199] By “reference” is meant a standard or control condition.

[0200] The transitional term “comprising,” which is synonymous with “including,” or “containing,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention, e.g., the ability to improve the bioavailability of a substance. Use of the term “comprising” contemplates embodiments that “consist” or “consist essentially” of the recited element(s).

[0201] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a,” “an” and “the” are understood to be singular or plural.

[0202] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In instances where time is discussed, the term “about” provides for a variation of up to 20% of the listed timeframe. In this context, a “month” should be considered at containing 30 days and a “week” contains 7 days.

[0203] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0204] MATERIALS AND METHODS

[0205] In certain embodiments, the following materials and methods may be utilized for designing and testing efficacy of mRNA vaccines for equine rhodococcal pneumonia (Legere et al. 2023).

[0206] Design of mRNA vaccine constructs

[0207] Four alternative mRNA constructs encoding versions of the VapA protein were designed based on the vapa gene (NCBI accession, #BAA04768.1). The unmodified sequence (AA1-189) was used to generate the native VapA construct. This sequence was codon optimized (CO) using an Equus caballus CGC codon frequency table in Geneious Prime (2021.1.1, Biomatters Ltd.) to generate the VapA-CO construct. The transmembrane domain (TM) of the VapA sequence was predicted by SignalP-5.0 (https: / / services. healthtech. dtu.dk / services / SignalP-5.0 / ) and Polyphobius

[0208] (https: / / phobius.sbc.su.se / poly.html) software. This TM region (AA 1-37) was deleted from the VapA sequence and replaced with the leader sequence from an equine immunoglobulin light chain variable region (AA 1-21; NCBI accession, #KR190600.1) to generate the mod-VapA construct. This construct then was codon optimized for equine tissues to generate the mod- VapA-CO construct.

[0209] Culture and transfection of equine respiratory tract cells

[0210] Primary cultures were established as previously described. Briefly, the lungs were removed en bloc within 1 hour of euthanasia and transported to the laboratory on ice. The bronchial epithelium was sharply dissected from the underlying submucosa and then minced into approximately 1-mm2segments. Aliquots of minced tissue were digested with 0.25% trypsin - 0.6 mM EDTA solution for 2 hours at 37°C in 5% CO2. The suspension was filtered through a sterile cell strainer (pore size 40 pm) and then centrifuged at 250 * g for 10 minutes at 4°C. The resulting cell pellet was resuspended in warm supplemented airway culture media (Airway Epithelial Cell Growth Basal Media and Growth Medium Supplement Pack, Promocell) and then placed into a tissue-treated T75 cell culture flask and incubated for 30 minutes at 37°C in 5% CO2 environment. The media was gently removed from the T75 flasks, allowing adhered fibroblasts to remain within the flask. Fibroblast growth media (DMEM, supplemented with 10% FBS, penicillin [200 U / mL], streptomycin [200 pg / mL], and amphotericin B [2.5 pg / mL]) was then added back to the flask for expansion of fibroblasts. The cellular suspension containing EBECs that had been removed from the T75 flask was centrifuged at 200 x g for 10 minutes at 4°C. The resulting cell pellet was resuspended in supplemented airway culture media. Cells were counted using a cell counter (CellometerAuto T4, Nexcelom Bioscience) with trypan blue for viability assessment, and then plated at a concentration of 2.5 x io5live cells / mL on a collagen-coated tissue-culture-treated 24-well plate. Media for both cell lines was changed at 24 hours, and then every 2 days afterward. Fibroblasts were expanded and serially passaged every 3-4 days until plated at a concentration of 2.5 x io5live cells / mL on a tissue-culture-treated 24-well plate in fibroblast media. All experiments were performed on first-passage EBECs and EBFs between 2-4 passages.

[0211] Each of the 4 mRNA constructs was formulated at a dose of 1 pg mRNA / well in a 24- well plate, with a commercially available transfection reagent (Lipofectamine MessengerMAX, ThermoFisher Scientific) according to manufacturer’s instructions. Formulated mRNA was delivered to complete cell media in each well of adherent cultured cells, and plates were incubated at 37°C with 5% CO2. Each condition was repeated in duplicate in each experiment, and the experiment was replicated 3 times. After 24 hours, supernatants and cells were harvested to assess secretion and intracellular expression of VapA. Samples were harvested, aliquoted, and stored at -80°C for subsequent analysis.

[0212] Western immunoblotting

[0213] Western immunoblotting was performed using supernatants and lysates of cultured EBECs and EBFs. Quantities of 30 pg protein from cell lysates and volumes of 30 pL supernatants were separated by SDS-PAGE, and then probed with a monoclonal mouse anti- VapA antibody (Santa Cruz Biotechnologies; Cat #sc-390576; 1 :500 dilution) and a peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories; Cat #115-035-062; 1 :2,000 dilution). Recombinant VapA (rVapA) was used as a positive control. For loading control, a separate aliquot of each cell lysate during each immunoblot was probed with a monoclonal mouse anti-human GAPDH antibody (Invitrogen; Cat #43700; 1 :500 dilution) and a peroxidase-conjugated goat anti-mouse secondary antibody (Abeam; Cat #6789; 1 :5,000 dilution). Protein bands were developed using Radiance Plus® Femtogram HRP Substrate (Azure Biosystems) and visualized using the Bio-Rad Chemidoc Touch imaging system.

[0214] Formulation of mRNA vaccine in lipid nanoparticles

[0215] Based on in vitro expression results, the mod-VapA construct was selected as a vaccine candidate and in vitro transcription was repeated in larger scale under identical conditions. Lipid nanoparticles (LNPs) were synthesized using a microfluidic benchtop system. The composition of the LNPs included DLin-MC3-DMA (an ionizable lipid for mRNA conjugation), DSPC (l,2-Distearoyl-sn-glycero-3-phosphorylcholine), cholesterol, and DMG- PEG2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), with molar ratios of 50: 10.5:38: 1.5, respectively. To achieve a total lipid concentration of 10 mM, these components were dissolved in ethanol. VapA mRNA was dissolved in the aqueous phase, which consisted of 50 mM sodium acetate buffer (pH=4). The mass ratio of lipids to mRNA was maintained at 1 :20.

[0216] Prior to synthesis, the organic phase was heated for 3 minutes at 45°C, while the aqueous phase was briefly warmed up to 42°C for less than 60 seconds. The synthesis process utilized a flow ratio (organic phase to aqueous phase) of 1 :3, with a total flow rate of 2 mL / min for dispensing the formulation. Following synthesis, the samples were dialyzed against l x PPBS (overnight, 4°C), followed by sterile filtration before characterization (0.22pm filters). Fresh samples were prepared for each vaccination time point and kept at 4°C until vaccination.

[0217] The RNA concentration in the LNP formulation was determined using the RiboGreen RNA Assay Kit (Cat #R11490, Invitrogen) with l x TE buffer, both with and without Triton X-100 detergent. The encapsulation efficiency of the RNA exceeded 85% for all batches. The average size and poly dispersity index (PDI) of the LNPs were evaluated using dynamic light scattering (DLS) with a Malvern Zetasizer. The VapA LNPs exhibited an average diameter of 86.3 nm (lot #1, prime vaccination) and 77.7 nm (lot #2, boost vaccination) based on DLS analysis. The poly dispersity (PDI) was less than 0.12 for both lots. Bronchoalveolar lavage (BAL)

[0218] BAL was performed at age 2 days in lateral recumbency under general anesthesia, using xylazine for sedation (0.5 mg / kg), followed by ketamine (2.2 mg / kg) and midazolam (0.04 mg / kg) for induction. At ages 22 and 35 days, the BAL was performed with standing sedation using xylazine (0.5 mg / kg) and butorphanol (0.04 mg / kg). Briefly, a BAL catheter (Jorgensen Laboratories) was passed down the trachea and lodged into a bronchus, and the air cuff was inflated. A volume of 50 mL sterile saline solution (0.9% NaCl) was infused through the catheter and immediately aspirated.

[0219] Anti-VapA antibody testing via ELISA

[0220] Indirect ELISA was used to determine anti-VapA IgGl, IgG4 / 7, and IgA antibody activities in serum and BALF. Immunoassay plates (Nunc Maxisorp immunoassay plate, Thermo Fisher Scientific) were coated with rVapA diluted in sensitization buffer (0.04 M PO4, pH 7.2) at a concentration of 1 pg / mL for serum testing and 2 pg / mL for BALF testing, and then incubated overnight at 4°C. Plates were washed with PBS with 0.1% Tween-20 and then blocked with 1% non-fat milk in PBS at 37°C for 1 hour. Serum from a horse hyperimmunized against R equi was added to the plate in duplicate for positive control at dilutions of 1 :640 for IgGl, 1 :40,960 for IgG4 / 7, and 1 : 160 for IgA testing. Foal serum samples were diluted in blocking buffer (1 :20 for IgGl, 1 :80 for IgG4 / 7, and 1 :40 for IgA) and added to the plate in duplicate. BALF samples were added to the plate undiluted in duplicate. Following washes, the subisotype-specific peroxidase-conjugated detection antibody for IgGl (Bethyl Laboratories; Cat #A70-124P; 1 :40,000 dilution), IgG4 / 7 (Bethyl Laboratories; Cat #A70- 123P; 1 :80,000 dilution), or IgA (Bethyl Laboratories; Cat #A70-123P; 1 :2,500 dilution) was added to the plate, then incubated at room temperature for 1 hour.

[0221] For serum IgG subisotype ELISAs, plates were treated with SureBlue Reserve TMB Peroxidase Substrate (SeraCare) and incubated 20 minutes at room temperature in the dark. The reaction was stopped by adding sulfuric acid solution (0.18 M H2SO4) and optical densities.

[0222] (ODs) were determined at 450 nm using a microplate reader. For serum IgA and all BALF ELISAs, plates were treated with QuantaRed Enhanced Chemifluorescent Peroxidase Substrate (Cat #15159, ThermoFisher Scientific) and incubated 15 minutes at room temperature in the dark. The reaction was stopped by adding QuantaRed stop solution and ODs were determined at 570 nm using a microplate reader. An endpoint OD titer was calculated by dividing the experimental OD values with that achieved by the positive control on the same plate.

[0223] ELISpot Testing

[0224] The CMI response to vaccination was assessed by measuring interferon gamma (IFN- y) production from isolated foal PBMCs stimulated with a lysate of the virulent R equi (strain ATCC 33701) using a commercially available ELISpot kit (Cat #3117-2A, Mabtech) performed according to the manufacturer’s directions. A 96-well PVDF filter plate (Cat #S2EM004M99, MilliporeSigma) was treated with the mouse anti-bovine IFN-y capture antibody (diluted to 15 pg / mL in PBS), then incubated overnight at 4°C. PBMCs were isolated using a Ficoll-Paque gradient separation (Histopaque-1077, GE Healthcare) and resuspended in RPMI-1640 media with L-glutamine, 15% FBS, and 1.5% penicillin-streptomycin. The PBMCs were added to the ELISpot plate at 100,000 cells / well in triplicate per condition, with 3 conditions: 1) media only as negative control; 2) the mitogen concanavalin A as positive control (Sigma-Aldrich; Cat #C5278; 2.5 pg / mL), or 3) R. equi lysate (2.5 pg / mL). PBMCs were cultured for 48 hours at 37°C in 5% CO2. Plates were washed to remove unbound cells, treated with a biotinylated mouse anti-bovine IFN-y monoclonal detection antibody (diluted to 0.5 pg / mL in PBS and 5% FBS), and incubated 2 hours at room temperature. Plates were washed and treated with streptavidin-alkaline phosphatase (diluted to 1 : 1,000 in PBS and 5% FBS) for 1 hour at room temperature. The plate was washed, then treated with BCIP / NBT substrate solution and developed for 10 minutes in the dark. Development was stopped by washing the plate thoroughly with DI water, then the plate was dried overnight in the dark. Spots were quantified using an ELISpot plate reader (AID Plate Reader, Advanced Imaging Devices GmbH). For each condition, the number of spot-forming cells was quantified by subtracting the background spots from a foal’s negative control condition (media only) from that foal’s PBMCs stimulated with the virulent R equi lysate (containing VapA).

[0225] Jet Nebulizer

[0226] A jet nebulizer administration can be utilized for aerosol delivery of mRNA vaccine to neonatal foals. A custom-made mask with rubber flange gasket around the muzzle was directly connected to a jet nebulizer cup (AeroEclipse-II Breath Actuated Nebulizer, Monaghan Medical) in front of the nostrils with a commercially available equine jet nebulizer (Elite Equine Nebulizer System, Equi-Resp) at a running pressure of 35 psi. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0227] Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

[0228] EXAMPLE 1 — Derived Antigen from a mRNA Vaccine Candidate Coding for Rhodococcus equi Virulence- Associated Protein A (VapA) This antigen is engineered to be anchored to the plasma membrane, containing the

[0229] VapA amino acid sequence fused to a human albumin signal peptide at its N-terminus and a GPI anchor signal sequence derived from the horse placental alkaline phosphatase protein at its C-terminus. In some embodiments, the FLAG tag and / or the linker sequences can be omitted from the vaccine sequence.

[0230] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated within the scope of the invention without limitation thereto.

[0231] REFERENCES

[0232] Altschul, SF et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Research, 25: 17 (1997), 3389-

[0233] 3402, https: / / doi.org / 10.1093 / nar / 25.17.3389.

[0234] Bordin Al, Pillai S, Brake C, et al. Immunogenicity of an electron beam inactivated Rhodococcus equi vac-cine in neonatal foals. PLoS ONE. 2014;9(8):el05367. doi: 10.1371 / journal. pone.0105367 (“Bordin et al."y

[0235] Carnet, F., et al. An inventory of adjuvants used for vaccination in horses: the past, the present and the future. Vet Res 54, 18 (2023). https: / / doi.org / 10.1186 / sl3567-023-01151-3.

[0236] Chirino-Trejo JM, Prescott JF, Yager JA. Protection of foals against experimental Rhodococcus equi pneumonia by oral immunization. Can J Vet Res. 1987;51(4):444- 447. (“Chirino-Trejo et al.”).

[0237] Harris SP, Hines MT, Mealey RH, Alperin DC, Hines SA. Early development of cytotoxic T lymphocytes in neo-natal foals following oral inoculation with Rhodococcus equi. Vet Immunol Immunopathol. 2011; 141(3— 4): 312-316. doi: 10.1016 / j.vetimm.2011.03.015 (“Harris et al. ”).

[0238] Hooper-McGrevy KE, Wilkie BN, Prescott JF. Virulence-associated protein-specific serum immunoglobulin G-isotype expression in young foals protected against Rhodococcus equi pneumonia by oral immunization with virulent R equi. Vaccine. 2005;23(50):5760-5767. doi: 10.1016 / j.vaccine.2005.07.050. (“Hooper-McGrevy et alN).

[0239] Kahn SK, Cohen ND, Bordin Al, Coleman MC, Heird JC, Welsh TH Jr. Transfusion of hyperimmune plasma for protecting foals against Rhodococcus equi pneumonia. Equine Vet J. 2023;55(3):376-388. (“Kahn et al ”).

[0240] Legere, et al. Intramuscular but not nebulized administration of a mRNA vaccine against Rhodococcus equi stimulated humoral immune responses in neonatal foals. Am. J Vet Res. 2023. Doi.org / 10.2460 / ajvr.23.9.0208.

[0241] Madigan JE, Hietala S, Muller N. Protection against naturally acquired Rhodococcus equi pneumonia in foals by administration of hyperimmune plasma. J Reprod Fertil Suppl. 1991;44:571-578. (“Madigan et airy Martens RJ, Martens JG, Fiske RA, Hietala SK. Rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals. Equine Vet J. 1989;21(4):249-255. doi: 10.1111 / j.2042-3306.1989. tb02161.x (“Martens et al.n

[0242] Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453).

[0243] Sanz M, Loynachan A, Sun L, Oliveira A, Breheny P, Horohov DW. The effect of bacterial dose and foal age at challenge on Rhodococcus equi infection. Vet Microbiol. 2013; 167(3— 4):623— 631. doi: 10.1016 / j.vetmic. 2013.09.018. (“Sanz et al.”).

[0244] Smith TF, Waterman MS. Identification of common molecular subsequences. J Mol Biol. 1981 Mar 25;147(l): 195-7. doi: 10.1016 / 0022-2836(81)90087-5. PMID: 7265238.

[0245] Weinberg GA, Szilagyi PG. Vaccine epidemiology: efficacy, effectiveness, and the translational research roadmap. J Infect Dis. 2010 Jun l;201(l l): 1607-10. doi: 10.1086 / 652404. PMID: 20402594.

[0246] Willis E, Pardi N, Parkhouse K, et al. Nucleoside-modified mRNA vaccination partially overcomes maternal antibody inhibition of de novo immune responses in mice. Sci Transl Med. 2020;12(525):eaav5701. doi: 10.1126 / scitranslmed. aav5701. (“Willis et al.”).

[0247] Xie, C., et al. The advances of adjuvants in mRNA vaccines, npj Vaccines 8, 162 (2023). https: / / doi .org / 10.1038 / s41541 -023 -00760-5.

[0248] Von der Mulbe et al. “Stabilised mRNA with an increased g / c content and optimised codon for use in gene therapy.” WO02 / 098443. Pub. 12 / 12 / 2002.

[0249] Manoharan etal. “Methods and compositions for delivery of active agents.” W02012 / 016184. Pub. 02 / 02 / 2012.

[0250] Miller et al. “Adjuvant compositions and related methods.” US 10,265,395 B2. Pub. 07 / 20 / 2017.

[0251] Baumhof et al. “Lipid nanoparticle mRNA vaccines.” US20210251898A1. Pub. 08 / 19 / 2021.

[0252] Dominowski et al. “Adjuvant compositions.” US 11,896,666 B2. Pub. 11 / 04 / 2021.

Claims

CLAIMSWe claim:

1. A pharmaceutical composition comprising:(a) a prophylactically-effective amount of a mRNA comprising an open reading frame encoding a Rhodococcus equi (RE) antigenic polypeptide, or an ortholog, homolog, analog, variant or immunogenic fragment thereof, and one or both of a membrane-anchoring sequence and a signal peptide; and(b) a pharmaceutically-acceptable carrier, adjuvant and / or delivery agent.

2. The pharmaceutical composition of claim 1, wherein the mRNA of (a) is identified by an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

3. The pharmaceutical composition of claim 1, wherein the antigenic polypeptide is a RE virulence-associated protein (Vap).

4. The pharmaceutical composition of claim 2, wherein the Vap is VapA.

5. The pharmaceutical composition of claim 3, wherein the Vap is VapA, identified by an amino acid sequence having at least 80% identity to SEQ ID NO: 2.

6. The pharmaceutical composition of claim 1, wherein the membrane-anchoring sequence is an equine-specific glycosylphosphatidylinisotol (GPI) anchoring sequence comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 3.

7. The pharmaceutical composition of claim 1, wherein the signal peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or SEQ ID NO:

58. The pharmaceutical composition of claim 1, wherein the pharmaceutically- acceptable delivery agent is a lipid nanoparticle (LNP) comprising a cationic lipid, a noncationic lipid, a sterol and PEG-modified lipid at a molar ratio of about 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol; and 0.5-15% PEG-modified lipid.

9. The pharmaceutical composition of claim 8, wherein the LNP comprises dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); distearoylphosphatidylcholine (DSPC); cholesterol; and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000).

10. A method of inducing an antigen-specific immune response in a subject, the method comprising administering to the subject a pharmaceutical composition comprising:(a) a prophylactically-effective amount of a mRNA comprising an open reading frame encoding a Rhodococcus equi (RE) antigenic polypeptide, or an ortholog, homolog, analog, variant or immunogenic fragment thereof, and one or both of a membrane-anchoring sequence and a signal peptide; and(b) a pharmaceutically-acceptable carrier, adjuvant and / or delivery agent, wherein, relative to baseline, serum and bronchoalveolar lavage fluid (BALF) anti- antigenic polypeptide antibody titer in the subject is increased following vaccination and / or cellular-mediated immune (CMI) response markers are increased following vaccination.

11. The method of claim 10, wherein the mRNA of (a) is identified by an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

12. The method of claim 10, wherein the antigenic polypeptide is a RE virulence- associated protein (Vap).

13. The method of claim 12, wherein the Vap is VapA.

14. The method of claim 13, wherein the Vap is VapA, identified by an amino acid sequence having at least 80% identity to SEQ ID NO: 2.

15. The method of claim 10, wherein the membrane-anchoring sequence is an equine-specific glycosylphosphatidylinisotol (GPI) anchoring sequence comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 3.

16. The method of claim 10, wherein the signal peptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or SEQ ID NO: 5.

17. The method of claim 10, wherein the anti-antigenic polypeptide antibodies are selected from IgGi, IgG4 / 7 and IgA.

18. The method of claim 10, further comprising measuring the anti-antigenic polypeptide antibody titer using an enzyme-linked immunosorbent assay (ELISA).

19. The method of claim 10, wherein the CMI response markers are pro- inflammatory cytokines, immune cytokines and / or transcription factors in the subject’s PBMCs (peripheral blood mononuclear cells) or immune cells derived from the subject’s thymus, lymph nodes, spleen, intestine, lung or bone marrow.

20. The method of claim 19, wherein the cytokines include interferon gamma (IFN- y) and interleukin- 12 (IL- 12).

21. The method of claim 10, further comprising measuring the CMI response using an enzyme-linked immunospot assay (ELISpot).

22. The method of claim 10, wherein the subject is a mammal.

23. The method of claim 22, wherein the subject is a member of the Equus genus or is a human.

24. The method of claim 23, wherein the subject is a horse, wild ass, zebra, onager, kiang, or donkey.

25. The method of claim 23, wherein the subject is a foal aged 5 days or less.

26. The method of claim 23, wherein the subject is a pregnant mare.

27. The method of claim 10, wherein the prophylactically effective amount is a 300 pg dose to 600 pg dose.

28. The method of claim 10, comprising repeating the administration of the composition to the subject after at least 24 hours.

Images