Compositions for reducing methane emissions in ruminants

Abstract

The disclosure provides vaccines, such as polynucleotide vaccines, against methanogens. The disclosure also provides methods of making the vaccines and or administering such vaccines to ruminants (e.g., cattle) to reduce methane emissions.

Description

[0001] COMPOSITIONS FOR REDUCING METHANE EMISSIONS IN RUMINANTS

[0002] REEATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 U.S.C. § 119(e) of United States Provisional Application No. 63 / 670,640 filed July 12, 2024 and United States Provisional Application No. 63 / 751,653 filed January 30, 2025, each of which is incorporated herein in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (P124970000WO00-SEQ-JXV.xml; Size: 294,622 bytes; and Date of Creation: July 8, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Global warming and climate change represent serious challenges to the environment and humans. Both can lead to changes in the environment, leading to rises in sea level, changes in precipitation patterns, increased risks of severe weather, threats to biodiversity, and severe impacts to human health. Greenhouse gas emissions are the largest contributor to global warming. Among the greenhouse gases, methane is responsible for approximately 30% of the current rise in global temperature (IEA, 2024), and cows and other livestock produce approximately 27% of the methane emitted in the US (US EP A).

[0008] Methane emissions represent an ongoing problem with far-reaching current and future implications.

[0009] SUMMARY

[0010] The disclosure, in some aspects, provides a vaccine comprising a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the polynucleotide comprises an open reading frame (ORF) having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 5, 8, 10, 11, 13, and 15-42. In some embodiments, the ORF comprises any one of SEQ ID NOs: 5, 8, 10, 11, 13, and 15-42. In some embodiments, the ORF consists of any one of SEQ ID NOs: 5, 8, 10, 11, 13, and 15-42.

[0011] In some embodiments, the polynucleotide further comprises a 5’UTR, and wherein the 5’

[0012] UTR comprises SEQ ID NO: 1. In some embodiments, the polynucleotide further comprises a 3’ UTR, wherein the 3’UTR comprises SEQ ID NO: 2. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 45, 48, 50, 51, 53, and 55-82. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs: 45, 48, 50, 51, 53, and 55-82. In some embodiments, the polynucleotide consists of any one of SEQ ID NOs: 45, 48, 50, 51, 53, and 55-82.

[0013] In some embodiments, the vaccine further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional polynucleotides, each encoding a different protein or fragment thereof of a methanogen. In some embodiments, the polynucleotide is codon-optimized.

[0014] In some embodiments, the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the RNA is messenger RNA (mRNA) or is selfamplifying RNA.

[0015] In some embodiments, the RNA comprises at least nucleobase comprising at least one chemical modification. In some embodiments, the chemical modification is selected from the group consisting of: Nl-methyl-pseudouridine (mly), Nl-ethyl-pseudouridine (ely), 5- methoxy-uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in mRNAs comprise 5- methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, and any combination thereof.

[0016] In some embodiments, the vaccine further comprises a lipid nanoparticle, a synthetic nanocarrier, a liposome, an archaeosome, an extracellular vesicle, or a hydrogel. In some embodiments, the lipid nanoparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol and a PEG-modified lipid. In some embodiments, the synthetic nanocarriers comprises one or more polymers.

[0017] In some embodiments, the vaccine further comprises an adjuvant.

[0018] The disclosure, in some aspects or embodiments, provides a method of inducing an immune response against at least one methanogen in a subject, the method comprising administering any one of the vaccines described herein.

[0019] In some embodiments, the subject is a ruminant. In some embodiments, the ruminant is selected from the group consisting of cow, heifer, bull calf, bull, steer, ox, sheep, or goat.

[0020] In some embodiments, the vaccine is administered to the ruminant via oral administration, intranasal administration, intramuscular administration, intradermal administration, or subcutaneous administration.

[0021] In some embodiments, the ruminant is administered an initial dose (e.g., prime dose) of the vaccine. In some embodiments, the ruminant is administered an additional dose (e.g., boost dose) of the vaccine. In some embodiments, the ruminant is administered at least one additional dose of the vaccine 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 9 months, 1 year, or 2 years after administration of the initial dose. In some embodiments, the initial dose and the additional dose(s) are identical. In some embodiments, the initial dose and the additional dose(s) are different; for example, they comprise nucleic acids encoding different methanogens, the doses of each vaccine are different, the formulations of each vaccine are different, the administration routes of each vaccine are different, or any combination thereof.

[0022] In some embodiments, the method reduces methane emission from the ruminant by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% relative to the amount of methane emitted by the ruminant before administration of the vaccine.

[0023] A vaccine comprising: (a) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 89 encapsulated in a lipid nanoparticle; (b) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 96 encapsulated in a lipid nanoparticle; (c) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 99 encapsulated in a lipid nanoparticle; (d) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 100 encapsulated in a lipid nanoparticle; (e) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 101 encapsulated in a lipid nanoparticle; and (f) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 113 encapsulated in a lipid nanoparticle is also disclosed.

[0024] In some embodiments, each polynucleotide comprises an open reading frame (ORF) and wherein the ORF of the polynucleotide of (a) comprises SEQ ID NO: 8; the ORF of the polynucleotide of (b) comprises SEQ ID NO: 15; the ORF of the polynucleotide of (c) comprises SEQ ID NO: 18; the ORF of the polynucleotide of (d) comprises SEQ ID NO: 19; the ORF of the polynucleotide of (e) comprises SEQ ID NO: 20; and the ORF of the polynucleotide of (f) comprises SEQ ID NO: 32. In some embodiments, each polynucleotide comprises an open reading frame (ORF) and wherein the ORF of the polynucleotide of (a) consists of SEQ ID NO: 8; the ORF of the polynucleotide of (b) consists of SEQ ID NO: 15; the ORF of the polynucleotide of (c) consists of SEQ ID NO: 18; the ORF of the polynucleotide of (d) consists of SEQ ID NO: 19; the ORF of the polynucleotide of (e) consists of SEQ ID NO: 20; and the ORF of the polynucleotide of (f) consists of SEQ ID NO: 32.

[0025] In some embodiments, each polynucleotide further comprises a 5’UTR, and wherein the 5’ UTR comprises SEQ ID NO: 1. In some embodiments, each polynucleotide further comprises a 3’ UTR, wherein the 3’UTR comprises SEQ ID NO: 2.

[0026] In some embodiments, the polynucleotide of (a) comprises SEQ ID NO: 48; the polynucleotide of (b) comprises SEQ ID NO: 55; the polynucleotide of (c) comprises SEQ ID NO: 58; the polynucleotide of (d) comprises SEQ ID NO: 59; the polynucleotide of (e) comprises SEQ ID NO: 60; and the polynucleotide of (f) comprises SEQ ID NO: 72.

[0027] In some embodiments, each polynucleotide comprises RNA and wherein the RNA comprises at least nucleobase comprising at least one chemical modification. In some embodiments, the chemical modification is selected from the group consisting of: N1 -methylpseudouridine (mly), Nl-ethyl-pseudouridine (ely), 5-methoxy-uridine (mo5U), 5-methyl- uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in mRNAs comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1- methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, and any combination thereof.

[0028] In some embodiments, each lipid nanoparticle comprises an ionizable amino lipid, a noncationic lipid, a sterol and a PEG-modified lipid.

[0029] In some embodiments, a method of inducing an immune response against at least one methanogen in a subject is provided, the method comprising administering any vaccine described herein to the subject.

[0030] In some embodiments, the subject is a ruminant. In some embodiments, the ruminant is selected from the group consisting of: cow, heifer, bull calf, bull, steer, ox, sheep, or goat.

[0031] In some embodiments, the subject is administered an initial dose of the vaccine, optionally wherein the subject is administered an additional dose of the vaccine.

[0032] BRIEF DESCRIPTION OF DRAWINGS

[0033] FIGs. 1A-1C show transfection efficacy of exemplary mRNA backbones in Madin- Darby bovine kidney (MDBK) cells. FIG. 1A shows mean fluorescence intensity of eGFP expression in MDBK cells. FIG. IB shows the percentage of eGFP-expressing MDBK cells. FIG. 1C shows transfection efficiency (TE) of exemplary mRNA backbone constructs in MDBK cells. In FIGs. 1A-1C, GS = eGFP (positive control); G1 = eGFP + bovine beta hemoglobin UTRs, 100 amino acid polyA tail; G2 = eGFP + bovine beta hemoglobin UTRs, 120 amino acid polyA tail with linker; G3 = eGFP + bovine beta hemoglobin UTRs, 120 amino acid polyA tail; G4 = eGFP + human beta hemoglobin UTRs, 100 amino acid polyA tail; G5 = eGFP + human beta hemoglobin UTRs, 120 amino acid polyA tail with linker; G6 = eGFP + human beta hemoglobin UTRs, 120 amino acid polyA tail; G7 = eGFP + UTRs derived from the human hemoglobin a-globin (HBA1) gene; CO = MDBK cell only (negative control); TransIT = MDBK cell + transfection reagent (negative control).

[0034] FIGs. 2A-2C show transfection efficacy of exemplary mRNA backbones in Madin- Darby bovine kidney (MDBK) cells. FIG. 2A shows mean fluorescence intensity of eGFP expression in MDBK cells. FIG. 2B shows the percentage of eGFP-expressing MDBK cells. FIG. 2C shows transfection efficiency (TE) of exemplary mRNA backbone constructions in MDBK cells. In FIGs. 2A-2C, GS = eGFP (positive control); G1 = eGFP + bovine beta hemoglobin UTRs, 100 amino acid polyA tail; G3 = eGFP + bovine beta hemoglobin UTRs, 120 amino acid polyA tail; G6 = eGFP + human beta hemoglobin UTRs, 120 amino acid polyA tail; CO = MDBK cell only (negative control); TransIT = MDBK cell + transfection reagent (negative control).

[0035] FIGs. 3A-3C show transfection efficacy of exemplary mRNA backbones in bovine dendritic cells (DCs). FIG. 3A shows mean fluorescence intensity of eGFP expression in bovine DCs. FIG. 3B shows the percentage of eGFP-expression bovine DCs. FIG. 3C shows transfection efficiency of exemplary mRNA backbone constructs in bovine DCs. In FIGs. 3A- 3C, GS = eGFP (positive control); G1 = eGFP + bovine beta hemoglobin UTRs, 100 amino acid polyA tail; G3 = eGFP + bovine beta hemoglobin UTRs, 120 amino acid polyA tail; G6 = eGFP + human beta hemoglobin UTRs, 120 amino acid polyA tail; CO = MDBK cell only (negative control); TransIT = MDBK cell + transfection reagent (negative control).

[0036] FIG. 4 shows bovine leukocyte antigen II expression in bovine DCs following transfection with different constructs. In FIG. 4, GS = eGFP (positive control); G1 = eGFP + bovine beta hemoglobin UTRs, 100 amino acid polyA tail; G3 = eGFP + bovine beta hemoglobin UTRs, 120 amino acid polyA tail; G6 = eGFP + human beta hemoglobin UTRs, 120 amino acid polyA tail; CO = MDBK cell only (negative control); or TransIT = MDBK cell + transfection reagent (negative control).

[0037] FIGs. 5A-5D show the methane and weight gain results in animals during different periods of the study described in Example 3 (pre-immunization period, prime period, and boost #1 period). FIG. 5A shows the normalized methane emissions; FIG. 5B shows the normalized methane emissions per kg food intake; FIG. 5C shows the normalized average daily weight gain for each period; and FIG. 5D shows the normalized feed efficiency (measured as kg gain per kig feed). FIGs. 6A-6B show ELISA results relating to sera and saliva antibodies levels from Bos taurus subjects during different periods of the study described in Example 3 (prime period and boost#l period). FIG. 6A shows systemic anti-methanogen IgG levels (control group light line, n=18 ; vaccine group dark line, n=18). FIG. 6B shows salivary anti-methanogen IgA levels (control group, light line, n=18 ; vaccine group, dark line, n=18). Logio IgG and IgA titers were analyzed and normalized by dividing the average value of the control group in corresponding time points. Data was analyzed by means of a Mann-Whitney U-test (* p < 0.05, ** p < 0.01).

[0038] DETAILED DESCRIPTION

[0039] Global warming and climate change are increasingly problems facing the world today. The rise in greenhouse gas (GHG) emissions has greatly accelerated both processes. Methane, CH4, is the second most abundant GHG, and its potential global warming effect is 28-fold higher than that of CO2, the most prevalent GHG. Ruminant emissions of methane account for approximately 12-20% of global methane emissions. In this way, ruminant feeding is a significant component of global warming. Without wishing to be bound by theory, it is thought that reducing ruminant methane emissions will decrease the rate of global warming. Attempts to reduce ruminant methane emissions have only had limited success to this point. In particular, many attempted solutions have failed due to low efficacy, toxicity issues, poor selectivity, and / or the accumulation of resistance to various treatments.

[0040] Therefore, the present disclosure provides, in some aspects, vaccines that comprise polynucleotides (e.g., DNA, RNA, mRNA) encoding at least one polypeptide from a methanogen. The vaccines, in some embodiments, are administered to a ruminant and are effective in reducing methane production in the ruminant, for example, by inducing an immune response and / or antibody production against the methanogen. Also provided herein are methods of administering the vaccines, methods of producing the vaccines, compositions comprising the vaccines, and nucleic acids encoding the vaccines.

[0041] Methanogens and. Antigens

[0042] The vaccines described herein comprise polynucleotides encoding a protein (e.g., antigen) derived from a methanogen. “Methanogen,” as used herein, refers to any microorganism that produces methane as a byproduct. Methanogens are anaerobic archaea that produce methane as a byproduct of catabolism. In particular, most methanogens use hydrogen (H2) to reduce carbon dioxide (CO2), resulting in the formation of methane (CH4). There are five orders of methanogens: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, and Methanopyrales . Exemplary non-limiting methanogen species include the following: Methanobacterium bryantii, Methanobacterium formicum, Methanobrevibacter arboriphilicus, Methanobrevibacter gottschalkii, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanococcus chunghsingensis, Methanococcus burtonii, Methanococcus aeolicus, Methanococcus deltae, Methanococcus jannaschii, Methanococcus maripaludis, Methanococcus vannielii, Methanocorpusculum labreanum, Methanoculleus bourgensis (Methanogenium olentangyi and Methanogenium bourgense). Methanoculleus marisnigri, Methanoflorens stordalenmirensis , Methanofollis liminatans, Methanogenium cariaci, Methanogenium frigidum, Methanogenium organophilum, Methanogenium wolfei, Methanomicrobium mobile, Methanopyrus kandleri, Methanoregula boonei, Methanosaeta concilii, Methanosaeta thermophila, Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Methanosphaera stadtmanae, Methanospirillium hungatei, Methanothermobacter defluvii (Methanobacterium defluvii), Methanothermobacter thermautotrophicus (Methanobacterium thermoautotrophicum), Methanothermobacter thermoflexus (Methanobacterium thermoflexum), Methanothermobacter wolfei (Methanobacterium wolfei), Methanothrix soehngenii. In some embodiments, the vaccine encodes a protein or fragment thereof derived from Methanobrevibacter ruminantium, Methanobrevibacter gottschalkii, Methanosphaera stadtmanae, Methanobrevibacter smithii, Methanobacterium sp., Methanobrevibacter sp., or Methanomassiliicoccus luminyensis . In some embodiments, the vaccine encodes a protein or fragment thereof derived from Methanobrevibacter ruminantium. In some embodiments, the vaccine encodes a protein or fragment thereof derived from Methanobrevibacter gottschalkii. In some embodiments, the vaccine encodes a protein or fragment thereof derived from Methanosphaera stadtmanae. In some embodiments, the vaccine encodes a protein or fragment thereof derived from Methanobrevibacter smithii. In some embodiments, the vaccine encodes a protein or fragment thereof derived from a species of Methanobacterium. In some embodiments, the vaccine encodes a protein or fragment thereof derived from a species of Methanobrevibacter. In some embodiments, the vaccine encodes a protein or fragment thereof derived from Methanomassiliicoccus luminyensis.

[0043] The methanogen protein encoded by the polynucleotide of the vaccine is an antigen. Antigens, as used herein, are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens). In some embodiments, the antigen is a naturally occurring antigen (e.g., the methanogen antigenic polynucleotide encodes a naturally occurring antigen). In some embodiments, the methanogen antigenic polypeptide is a non- naturally occurring antigen or an engineered version of the protein. In some embodiments, the vaccines described herein include polynucleotides encoding a surface antigen (e.g., a surface protein) or fragment thereof of a methanogen. In some embodiments, the fragment is an antigenic fragment, a fragment comprising an epitope, or a fragment comprising an extracellular domain of the protein. In some embodiments, the fragment further comprises a transmembrane domain (e.g., a heterologous transmembrane domain). In some embodiments, the fragment does not comprise a transmembrane domain.

[0044] The vaccines described herein include polynucleotide encoding the desired antigen or antigens, which when introduced into the body, i.e., administered to a subject (for example, a ruminant) in vivo, cause the cells of the subject to express the desired antigens.

[0045] Without wishing to be bound by theory, it is thought that, once the polynucleotides are taken up by the cells of the subject, the polynucleotides are translated in the cytosol and the protein is assembled and processed by the host cell machinery. The resulting protein (antigen) is then presented and elicits an adaptive humoral and cellular immune response.

[0046] Exemplary antigens are provided in Table 4. In some embodiments, the antigen encoded by a polynucleotide described herein is selected from the amino acid sequences of SEQ ID NOs: 84-123. In some embodiments, the antigen comprises any one of SEQ ID NOs: 84-123. In some embodiments, the antigen consists of, or consists essentially of, any one of SEQ ID NOs: 84-123. In some embodiments, the antigen is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any one of SEQ ID NOs: 84-123.

[0047] In some embodiments, the antigen encoded by a polynucleotide described herein comprises at least one mutation (addition, deletion, or substitution) relative to its wild-type counterpart (e.g., a reference sequence). The antigen comprising at least one mutation is referred to herein as a “mutant antigen” or an “antigen variant” or a “methanogen variant.”

[0048] In some embodiments, the at least one mutation comprises at least one addition and at least one deletion. In some embodiments, the at least one mutation comprises at least one addition and at least one substitution. In some embodiments, the at least one mutation comprises at least one deletion and at least one substitution. In some embodiments, the at least one mutation comprises at least one addition, at least one deletion, and at least one substitution. In some embodiments, the mutation is the addition of at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid relative to the reference sequence. In some embodiments, the mutation is the deletion of at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid relative to the reference sequence. In some embodiments, the mutation is the substitution of at least 1 amino acid relative to the reference sequence. In some embodiments, the mutation is 1-300 substitutions relative to the reference sequence, for example, 1-50, 51-100, 101-150, 151-200, 201-250, or 251-300 substitutions. In some embodiments, the mutation is 1-300 substitutions relative to the reference sequence, for example, 1-25, 26-50, 51-75, 76-100, 101-125, 126-150, 151-175, 176-200, 201- 225, 226-250, 251-275, or 276-300 substitutions. In some embodiments, the mutation is 1-300, 10-290, 20-280, 30-270, 40-260, 50-250, 60-240, 70-230, 80-220, 90-210, or 100-200 substitutions relative to the reference sequence. In some embodiments, the mutation is 1-300 substitutions relative to the reference sequence, for example, 1, 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 substitutions.

[0049] Multivalent Vaccines

[0050] The compositions, as provided herein, may include a polynucleotide or multiple polynucleotides encoding two or more antigens of the same or different species; that is, the compositions may be multivalent compositions. In some embodiments, the vaccine includes a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding two or more antigens, each from a different methanogen. In some embodiments, a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the vaccine comprises a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding 2-8, 3-7, or 4-6 different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the vaccine comprises a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding 2 different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the vaccine comprises a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding 3 different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the vaccine comprises a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding 4 different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the vaccine comprises a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding 5 different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the vaccine comprises a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding 6 different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the vaccine comprises a polynucleotide (e.g., an RNA) or multiple polynucleotides (e.g., multiple RNAs) encoding 7 different methanogen antigens (e.g., different surface proteins from one methanogen, surface proteins from different methanogens, or a combination of both). In some embodiments, the polynucleotide or polynucleotides are mRNA.

[0051] In some embodiments, two or more different polynucleotides (e.g., RNA polynucleotides) encoding antigens may be formulated in the same formulation. In other embodiments, two or more different polynucleotides (e.g., RNA polynucleotides) encoding antigens may be formulated in separate formulations. The two or more formulations may then be combined and administered as a single vaccine composition (e.g., comprising multiple polynucleotides encoding multiple antigens) or may be administered separately. In some embodiments, the polynucleotide or polynucleotides are mRNA.

[0052] Fusion Proteins

[0053] In some embodiments, a vaccine of the present disclosure includes a polynucleotide encoding an antigenic fusion protein. Thus, the encoded antigen or antigens may include two or more proteins (e.g., protein and / or protein fragment) joined together. Antigenic fusion proteins, in some embodiments, retain the functional property from each original protein.

[0054] In some embodiments, the RNA (e.g., mRNA) further encodes a linker located between at least one or each domain of the fusion protein. In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS (SEQ ID NO: 124) linker. In some embodiments, the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain.

[0055] Signal Peptides

[0056] In some embodiments, a vaccine comprises a polynucleotide comprising an ORF that encodes a signal peptide fused to a methanogen protein. Signal peptides, comprising the N- terminal 15-60 amino acids 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. A signal peptide may also facilitate the targeting of the protein to the cell membrane.

[0057] In some embodiments, the 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, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40- 55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25- 30, 15-25, 20-25, or 15-20 amino acids.

[0058] Polynucleotides

[0059] The compositions of the present disclosure comprise a polynucleotide (e.g., mRNA) having an open reading frame (ORF) encoding a methanogen antigen. Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides. Examples of nucleic acids include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), 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) and / or chimeras and / or combinations thereof. In some embodiments, the polynucleotide comprises RNA, such as selfamplifying RNA, circular RNA, or mRNA.

[0060] In some embodiments, an RNA is a self-amplifying RNA. A self-amplifying RNA is an RNA that encodes at least one protein that is able to replicate the self-amplifying RNA. In some embodiments, the protein or proteins encoded by the self- amplifying RNA are non- structural proteins nsPl, nsP2, nsP3, and nsP4, which form an RNA-dependent RNA polymerase (RdRp), or replicase, that is capable of replicating the self-amplifying RNA. By encoding proteins that are able to replicate the RNA, a self-amplifying RNA is capable of self-amplification in a cell, provided that the cell can translate the RNA and produce the encoded protein(s). The viral non- structural proteins facilitate the replication of the nucleotide sequences encoding the desired protein via the subgenomic viral promoters. A “subgenomic viral promoter” refers to a promoter the drives the transcription of subgenomic mRNAs. Some viruses may transcribe subgenomic mRNAs from an RNA replicon that is produced from its genomic RNA. Many positive-sense RNA viruses produce subgenomic mRNAs as one of the common infection techniques used by these viruses and generally transcribe late viral genes. Subgenomic viral promoters range from 20 nucleotide (Sindbis virus) to over 100 nucleotides (Beet necrotic yellow vein virus) and are usually found upstream of the transcription start.

[0061] In some embodiments, the vaccine comprises a messenger RNA (mRNA) encoding a methanogen protein. Messenger RNA (mRNA) is any RNA that encodes a (at least one) protein and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. mRNA comprises an open reading frame (ORF) encoding the protein or fragment thereof. In some embodiments, the mRNA further comprises a 5' untranslated region (UTR), 3' UTR, a polyA tail, and / or a 5' cap analog. In some embodiments, the mRNA may encode a single protein or fragment or the mRNA may encode more than one protein or fragment separately within the same mRNA molecule (i.e., the mRNA is polycistronic). Additionally or alternatively, the disclosed mRNA may encode a fusion protein or fragment thereof.

[0062] In some embodiments, the polynucleotide comprises a 5' UTR, 3' UTR, a poly(A) tail and / or a 5' cap analog. It should also be understood that the vaccines of the present disclosure may include any 5' untranslated region (UTR) and / or any 3' UTR. Exemplary UTR sequences are provided in Table 1 below; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein. The polynucleotides described herein further comprise an open reading frame (ORF). An ORF is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon or codons (e.g., TAA, TAG, TGA, UAA, UAG, or UGA). For clarity: the stop codon itself is not considered a part of the ORF. An ORF typically encodes a protein or fragment thereof (e.g., a methanogen protein). In some embodiments, the polynucleotide comprises a poly(A) tail. A poly(A) tail may contain 10 to 300 adenosine monophosphates. In some embodiments, the poly(A) tail comprises 80 to 140 adenosine monophosphates. In some embodiments, the poly(A) tail comprises 80 adenosine monophosphates. In some embodiments, the poly(A) tail comprises 90 adenosine monophosphates. In some embodiments, the poly(A) tail comprises 100 adenosine monophosphates. In some embodiments, the poly(A) tail comprises 110 adenosine monophosphates. In some embodiments, the poly(A) tail comprises 120 adenosine monophosphates. In some embodiments, the poly(A) tail comprises 130 adenosine monophosphates. In some embodiments, the polynucleotide comprises a 5' end cap or a “5' terminal cap.” A cap analog may be, for example, a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

[0063] In some embodiments, the polynucleotide (e.g., mRNA) may comprise nucleotides that are not chemically modified (i.e., unmodified nucleotides), nucleotides that are modified, or both nucleotides that are not chemically modified and nucleotides that are chemically modified. In mRNA, nucleotides that are not chemically modified are the standard ribonucleotides consisting of adenosine, guanosine, cytidine, and uridine. In some embodiments, the chemically modified nucleotides comprise: Nl-methyl-pseudouridine (mly), Nl-ethyl-pseudouridine (ely), 5- methoxy-uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in mRNAs comprise 5- methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, or any combination thereof. In some embodiments, the polynucleotide (e.g., mRNA) includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

[0064] In some embodiments, polynucleotides (e.g., mRNAs) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a specific modification. For example, a mRNA can be uniformly modified with Nl-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with Nl-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above. In some embodiments, the ORF is uniformly modified for a particular modification, such as Nl-methyl-pseudouridine. In some embodiments, the uniform modification does not include the mRNA cap.

[0065] In some embodiments, an ORF encoding a protein or fragment thereof is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences listed below may be codon optimized. Codon optimization tools, algorithms, and services are known in the art - non-limiting examples include services from GeneArt (Thermo Fisher Scientific), DNA2.0 (ATUM) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms. In some embodiments, a codon optimized sequence shares less than 95%, less than 90%, less than 85%, less than 80%, or less than 75% sequence identity to a naturally occurring or wild-type sequence open reading frame (e.g., a naturally occurring or wild-type mRNA sequence encoding a protein or fragment thereof). In some embodiments, a codon optimized sequence shares between 65% and 85% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type RNA or DNA sequence encoding a protein or fragment thereof).

[0066] The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “U”s in a representative RNA (e.g., mRNA) sequence but where the sequence represents DNA, the “U”s would be substituted for “T”s. Thus, any of the RNAs (e.g., mRNAs) disclosed and identified by a particular sequence identification number herein also disclose the corresponding DNA sequence complementary to the RNA (e.g., mRNA), where each “U” of the RNA sequence is substituted with “T.” Exemplary nucleotide sequences of the disclosure are provided in Tables 1-3.

[0067] Viral Vectors Some aspects of the disclosure relate to viral vectors comprising or encoding a methanogen protein. In some embodiments, the protein is comprised in a viral vector. In some embodiments, a viral vector comprises a nucleic acid encoding the protein.

[0068] Any suitable virus may be used as a viral vector. Non-limiting examples of viruses that may be used as viral vectors include retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus (AAV), vesicular stomatitis virus (VSV), herpesvirus, Rous sarcoma virus, measles virus, poxvirus, gammavirus, alphavirus, murine stem cell virus, Moloney murine leukemia virus, and bovine leukemia virus. In some embodiments, the viral vector is a VSV vector. In some embodiments, the viral vector is a measles virus vector. In some embodiments, the viral vector is an adenovirus vector. These and other viral vectors suitable for expression of heterologous proteins (z.e., proteins not naturally expressed by a virus from which the viral vector is derived) are known in the art.

[0069] Percent Identity

[0070] In some embodiments, the vaccines of the present disclosure include polynucleotides that encode a structurally altered variant of a methanogen protein. Antigenic variants or structurally altered variants refers to molecules that differ in their amino acid sequence from a wild-type (naturally occurring), native, or reference protein sequence. The antigen / structurally altered variants may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a wild-type, native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a wild-type, native, or reference sequence.

[0071] In some embodiments, a vaccine comprises polynucleotide or a polynucleotide ORF that comprises a nucleotide sequence of any one of the sequences provided herein, or comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of a wild-type (naturally occurring) or variant antigen.

[0072] The term “identity” refers to a relationship between the sequences of two or more polypeptides (e.g. antigens) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among 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 antigens or nucleic acids can be readily calculated by known methods. “Percent (%) 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. It is understood that 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 (e.g., antigen) have at least 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) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman- Wunsch algorithm.

[0073] As such, polynucleotides encoding proteins containing substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure. For example, 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 or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function In addition to structurally altered variants that are identical to the reference protein but are truncated, in some embodiments, a structurally altered variant includes an antigen that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations with respect to a reference antigen.

[0074] In vitro Transcription of Polynucleotides (e.g., RNA)

[0075] Aspects of the present disclosure provide methods of producing (e.g., synthesizing) an RNA transcript (e.g., mRNA transcript) comprising contacting a DNA template (e.g., a first input DNA and a second input DNA) with an RNA polymerase (e.g., a T7 RNA polymerase, a T7 RNA polymerase variant, etc.) under conditions that result in the production of the RNA transcript. This process is referred to as “zh vitro transcription” or “IVT”. IVT conditions typically require a purified linear DNA template containing a promoter, nucleoside triphosphates, a buffer system that includes dithiothreitol (DTT) and magnesium ions, and an RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application. Typical IVT reactions are performed by incubating a DNA template with a RNA polymerase and nucleoside triphosphates, including GTP, ATP, CTP, and UTP (or nucleotide analogs) in a transcription buffer. An RNA transcript having a 5' terminal guanosine triphosphate is produced from this reaction.

[0076] In some embodiments, a wild-type T7 polymerase is used in an IVT reaction. In some embodiments, a modified or mutant T7 polymerase is used in an IVT reaction.

[0077] The input deoxyribonucleic acid (DNA) serves as a nucleic acid template for RNA polymerase. A DNA template may include a polynucleotide encoding a polypeptide of interest (e.g., an antigenic polypeptide). A DNA template, in some embodiments, includes an RNA polymerase promoter (e.g., a T7 RNA polymerase promoter) located 5’ from and operably linked to polynucleotide encoding a polypeptide of interest. A DNA template may also include a nucleotide sequence encoding a poly adenylation (poly A) tail located at the 3’ end of the gene of interest. In some embodiments, an input DNA comprises plasmid DNA (pDNA). As used herein, “plasmid DNA” or “pDNA” refers to an extrachromosomal DNA molecule that is physically separated from chromosomal DNA in a cell and can replicate independently. In some embodiments, plasmid DNA is isolated from a cell (e.g., as a plasmid DNA preparation). In some embodiments, plasmid DNA comprises an origin of replication, which may contain one or more heterologous nucleic acids, for example nucleic acids encoding therapeutic proteins that may serve as a template for RNA polymerase. Plasmid DNA may be circularized or linear (e.g., plasmid DNA that has been linearized by a restriction enzyme digest). In some embodiments, the polynucleotide comprises a 5' end cap or a “5' terminal cap” or cap analog. The 5’ cap structure may be added to the polynucleotide co-transcriptionally, for example, by using a dinucleotide cap analog in the IVT reaction (e.g., using an AMPLICAP™ T7 Kit or a MESSAGEMAX™ T7 ARCA-capped Message Transcription Kit). In some embodiments, co-transcriptional capping is performed with a dinucleotide cap analog, for example, is an anti-reverse cap analog (ARCA).

[0078] In some embodiments, the 5’ cap is added using post-transcriptional capping (e.g., enzymatic capping). As an example, Vaccinia Virus Capping Enzyme (VCE) may be used. VCE comprises the three enzymatic activities necessary to synthesize an m7G cap structure (RNA 5'- triphosphatase, guanylyltransferase, and guanine-7-methyltransferase). Therefore, in some embodiments, in vitro transcripts are capped in the presence of the capping enzyme, reaction buffer, GTP, and the methyl donor S- adenosylmethionine (SAM).

[0079] Kits useful for IVT are known in the art and include, but are not limited to, T7 RIBOMAX™ Express Large Scale RNA Production System (Promega), RIBOMAX™ Large Scale RNA Production Systems — SP6 and T7, MEGASCRIPT™ T7 Transcription Kit (Thermo), IVTPRO™ T7 mRNA synthesis kit (Takara), AMPLISCRIBE™ T7-flash transcription kit (LGC Biosearch), or other conventionally available transcription kits. In some embodiments, the HISCRIBE® T7 mRNA Kit with CLEANCAP® Reagent AG is used to synthesize the mRNA.

[0080] Chemical Synthesis

[0081] Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.

[0082] Liquid Phase Chemical Synthesis. The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.

[0083] Combination of Synthetic Methods. The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone. Ligation of Nucleic Acid Regions or Subregions

[0084] Assembling nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and / or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase.

[0085] Purification

[0086] Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

[0087] A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[0088] In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

[0089] Quantification

[0090] In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or when derived from one or more bodily fluid. Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSL), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchioalveolar lavage fluid, semen, prostatic fluid, Cowper’s fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0091] Assays may be performed using construct specific probes, cytometry, qRT-PCR, realtime PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.

[0092] Vaccine Formulations

[0093] In some embodiments, the polynucleotide (e.g., DNA, RNA, or mRNA) of the disclosure is formulated for delivery to a subject (e.g., a ruminant).

[0094] In some embodiments, the polynucleotide (e.g., DNA, RNA, or mRNA) is formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable amino lipid, noncationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest (e.g., polynucleotide encoding a methanogen protein).

[0095] In some embodiments, the ionizable amino lipid comprises ALC-0315, C12-200, cKK- E12, or DLin-MC3-DMA.

[0096] In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0097] In some embodiments, a PEG modified lipid of the disclosure comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and / or PEG-DPG.

[0098] In some embodiments, a sterol of the disclosure comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alphatocopherol, and mixtures thereof.

[0099] In some embodiments, the polynucleotide is formulated in a synthetic nanocarrier. In some embodiments, the synthetic nanocarrier comprises hydrophobic carrier materials, such as hydrophobic polymers or lipids. Therefore, in some embodiments, the synthetic nanocarriers provided herein comprise one or more lipids. In some embodiments, a synthetic nanocarrier may comprise a lipid bilayer. In some embodiments, a synthetic nanocarrier may comprise a lipid monolayer. In some embodiments, a synthetic nanocarrier may comprise a core comprising a polymeric matrix surrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). Further hydrophobic carrier materials include lipids (synthetic and natural), lipid-polymer conjugates, lipid-protein conjugates, and crosslinkable-oils, waxes, fats, etc. Further examples of lipid materials for use as hydrophobic carrier materials as provided herein can be found, for example, in PCT Publication No. W02000 / 006120 and WO2013 / 056132.

[0100] In some embodiments, the synthetic nanoparticle comprises one or more polymers (i.e., they are polymeric nanoparticles). Examples of polymers include, but are not limited to, polysaccharides (e.g., alginate, dextran, chitosan, agarose, and pullulan), polypeptides (e.g., albumin, gelatin, lectin, legumine, and viciline), polyesters, poly ethers, and polyamides. In some embodiments, the one or more polymers is a water soluble, non-adhesive polymer. In some embodiments, polymer is polyethylene glycol (PEG) or polyethylene oxide (PEG). In some embodiments, the polymer is polyalkylene glycol or polyalkylene oxide. In some embodiments, the one or more polymers is a biodegradable polymer. In some embodiments, the one or more polymers is a biocompatible polymer that is a conjugate of a water soluble, non-adhesive polymer and a biodegradable polymer. In some embodiments, the biodegradable polymer is polylactic acid (PLA), poly(glycolic acid) (PGA), or poly(lactic acid / glycolic acid) (PLGA). In some embodiments, the nanoparticle is composed of PEG-PLGA polymers.

[0101] In some embodiments, polymers can be cationic polymers. In general, cationic polymers are able to condense and / or protect negatively charged strands of nucleic acids (e.g., DNA, RNA, or derivatives thereof). Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7; both of which are incorporated herein by reference), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297; incorporated herein by reference), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372; all of which are incorporated herein by reference) are positively-charged at physiological pH, form ion pairs with nucleic acids, and mediate transfection in a variety of cell lines.

[0102] In some embodiments, the polynucleotide is formulated in a liposome. Liposomes can be produced by standard methods such as those reported by Kim et al. (1983, Biochim. Biophys. Acta 728, 339-348); Liu et al. (1992, Biochim. Biophys. Acta 1104, 95-101); Lee et al. (1992, Biochim. Biophys. Acta. 1103, 185-197), Brey et al. (U.S. Pat. Appl. Pub. 20020041861), Hass et al. (U.S. Pat. Appl. Pub. 20050232984), Kisak et al. (U.S. Pat. Appl. Pub. 20050260260) and Smyth-Templeton et al. (U.S. Pat. Appl. Pub. 20060204566). In some embodiments, the liposome is an archaeal lipid vesicle, or archaeosome. Archaeosomes comprise one or more natural polar ether lipids extracted from archaea or prepared with synthetic archaeal lipids that mimic natural archaeal lipids (e.g., bipolar lipids). In some embodiments, the archaeosome further comprises at least one synthetic phospholipid (Sprott et al., LEMS Microbiology Letters, 154(1): 17-22, 1997).

[0103] In some embodiments, the polynucleotide is formulated in a hydrogel.

[0104] In some embodiments, the polynucleotide is formulated in a protein nanoparticle. Protein nanoparticles are self-assembling, multi- subunit, protein-based polyhedron- shaped structures. Each subunit comprises proteins (e.g., a glycosylated polypeptide), and may further comprise the polynucleotide, other nucleic acids, prosthetic groups, organic and inorganic compounds, or any combination thereof. Exemplary protein nanoparticles include, but are not limited to, ferritin nanoparticles, encapsulin nanoparticles, Sulfur Oxygenase Reductase (SOR) nanoparticles, lumazine synthase nanoparticles, and pyruvate dehydrogenase nanoparticles.

[0105] In some embodiments, the polynucleotide is formulated in a microparticle. Microparticles comprise, in some embodiments, biodegradable polymeric materials. Examples of biodegradable polymeric materials useful including, but not limited to, hydrogels, collagen, alginate, poly(glycolide) (PGA), poly(L-lactide) (PLA), poly(lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), polyesters, poly anhydrides, poly orthoesters, and polyamides. In some embodiments, the microparticle comprises or further comprises non-polymeric biodegradable ceramic materials such as calcium phosphate, hydroxyapatite, tricalcium phosphate, and combinations thereof. In some embodiments, the polynucleotide is formulated in a polymer implant. Polymer implants, in some embodiments, are solids comprising the polynucleotide that may be placed in a tissue and releases the polynucleotide in a controlled (e.g., time-release) manner. The implant comprises at least one polymer, such as a biodegradable polymer. Suitable polymers include, but are not limited to, biocompatible polycaprolactones, poly hydroxybutyrates, polylactides and poly lactaide-co -glycolides, polycarbonates, poly anhydrides, polyurethanes, polyacrylates and poly(ortho esters).

[0106] In some embodiments, the polynucleotide is formulated in a cationic nanoemulsion. Cationic nanoemulsions, in some embodiments, comprise a cationic lipid (e.g., DOTAP (1,2- dioleoyl-sn-glycero-3-phosphocholine)) and emulsion agents (e.g., a hydrophobic surfactant, a hydrophilic surfactant and squalene).

[0107] In some embodiments, the polynucleotide is formulated as a vaccine vector, for example using Salmonella. Therefore, in some embodiments, the polypeptide is cloned into an expression vector and then transformed into Salmonella cells. Salmonella cells, in some embodiments, are engineered to have reduced virulence and / or toxicity.

[0108] In some embodiments, the polynucleotide is formulated with an extracellular vesicle (EV). EVs are nano-sized vesicles, approximately 40 nm to 1000 nm in diameter, comprising a lipid bilayer membrane. EVs include, for example, exosomes, ectosomes, microvesicles, and apoptotic bodies.

[0109] Pharmaceutical Formulations

[0110] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for reducing methane emissions in ruminants. Ruminants, herbivorous grazing or browsing artiodactyls belonging to the suborder Ruminantia that acquire nutrients by fermenting plant-based foods in a specialized stomach before digestion include cattle (Bos laurus), domesticated and wild bovines, sheep, goats, giraffes, deer, gazelles, and antelopes. In some embodiments, the ruminant is cattle (e.g., cow, heifer, bull calf, bull, steer, or ox).

[0111] In some embodiments, the vaccine containing RNA as described herein can be administered to a subject (e.g., a ruminant, such as a cow), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen).

[0112] An “effective amount” of a composition (e.g., comprising RNA) is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and / or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex, and general health of the subject. Typically, an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject. In some embodiments, an effective amount is the amount necessary to reduce methane emissions in the subject based on a single dose of the vaccine or single dose of the vaccine in combination with a booster dose. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation and / or expression 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.

[0113] In some embodiments, an effective amount reduces the amount of methane emitted by the subject by 1%, 2%, 35, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% relative to the amount of methane emitted by the subject before administration of the vaccine or relative to the amount of methane emitted by a second subject that was not administered the vaccine (e.g., a cow that is similarly sized and aged to the cow that received the vaccine). In some embodiments, the amount of methane emitted by the subject is reduced by 10%. In some embodiments, the amount of methane emitted by the subject is reduced by 15%. In some embodiments, the amount of methane emitted by the subject is reduced by 20%. In some embodiments, the amount of methane emitted by the subject is reduced by 25%. In some embodiments, the amount of methane emitted by the subject is reduced by 50%. In some embodiments, the amount of methane emitted by the subject is reduced by 75%. The amount of methane emitted by the subject is reduced by 100%. Methane emissions may be measured using any known method in the art, including a GreenFeed system, respiration chambers, laser methane detectors, open-path lasers, micrometeorological techniques (for a population of ruminants), and with the use of canisters and gas chromatography (e.g., equipped with flame ionization detectors).

[0114] In some embodiments, an effective amount reduces the number of methanogens in the rumen of the subject. For example, the number of methanogens in the rumen of the subject may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% relative to the number of methanogens in the rumen of the subject prior to administration of the vaccine relative to the amount of methane emitted by a second subject that was not administered the vaccine (e.g., a cow that is similarly sized and aged to the cow that received the vaccine).

[0115] In some embodiments, the effective amount maintains or increases the subject’s average daily gain (ADG). In some embodiments, the subject’s ADG after administration of the vaccine is 0.5 lbs, 1 lbs, 1.5 lbs, 2 lbs, 2.5 lbs, 3 lbs, 4 lbs, or 5 lbs (pounds per day). In some embodiments, the effective amount results in an ADG of 1 lb. In some embodiments, the effective amount results in an ADG of 1.5 lb. In some embodiments, the effective amount results in an ADG of 2 lb. In some embodiments, the effective amount results in an ADG of 2.5 lb.

[0116] In some embodiments, the effective amount maintains or decreases the subject’s feed conversion ratio (FCR). The FCR is a measurement of the weight of feed intake divided by the weight gain of the subject; that is, lower FCRs indicate higher efficiencies. In some embodiments, the effective amount results in an FCR of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 in the subject.

[0117] In some embodiments, the effective amount maintains or increases the subject’s daily milk yield. For example, the effective amount, in some embodiments, results in production of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gallons of milk per day in the subject (e.g., cow). In some embodiments, the effective amount results in production of 6 gallons of milk per day in the subject (e.g., cow). In some embodiments, the effective amount results in production of 6.5 gallons of milk per day in the subject (e.g., cow). In some embodiments, the effective amount results in production of 7 gallons of milk per day in the subject (e.g., cow). In some embodiments, the effective amount results in production of 7.5 gallons of milk per day in the subject (e.g., cow). In some embodiments, the effective amount results in production of 8 gallons of milk per day in the subject (e.g., cow).

[0118] In some embodiments, the effective amount maintains or increases the subject’s (e.g., cow’s) lactation yield (that is, the amount of milk produced during a lactation cycle). In some embodiments, the subject’s lactation yield is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to the subject’s lactation yield from a previous lactation cycle or relative to a similar subject that has not been administered the vaccine.

[0119] In some embodiments, the effective amount maintains or increases the milk fat content of the milk produced by the subject (e.g., cow). In some embodiments, the milk fat content of the milk produced by the subject (e.g., cow) is 1%, 2%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, or more. In some embodiments, the effective amount maintains or increases the milk protein content of the milk produced by the subject (e.g., cow). In some embodiments, the milk protein content of the milk produced by the subject (e.g., cow) is 1%, 2%, 3%, 3.5%, 4%, 4.5%, 5%, or more.

[0120] In some embodiments, the effective amount reduces the number of methanogens in the rumen of the subject and / or the amount of methane emitted by the subject for 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, or more. In some embodiments, the effective amount reduces the number of methanogens in the rumen of the subject and / or the amount of methane emitted by the subject for 3 months. In some embodiments, the effective amount reduces the number of methanogens in the rumen of the subject and / or the amount of methane emitted by the subject for 6 months. In some embodiments, the effective amount reduces the number of methanogens in the rumen of the subject and / or the amount of methane emitted by the subject for 9 months. In some embodiments, the effective amount reduces the number of methanogens in the rumen of the subject and / or the amount of methane emitted by the subject for 1 year.

[0121] The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0122] A vaccine, disclosed herein, may be administered to a subject to induce an antigen specific immune response. The vaccine may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” or “booster vaccine” refers to an extra administration of the vaccine composition (e.g., following the primary, or initial, vaccination). In some embodiments, the primary vaccination comprises one dose of the vaccine. In some embodiments, the primary vaccination comprises two doses of the vaccine. In some embodiments, the primary vaccination comprises three or more doses of the vaccine. In some embodiments, the time between each dose in the primary vaccination is about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments, the time between each dose in the primary vaccination is 1 month (four weeks).

[0123] A booster (or booster vaccine) may be given after an earlier administration of the vaccine composition. In some embodiments, the vaccine comprises a booster vaccine (e.g., the combination vaccine is administered annually). The time of administration between the initial administration of the vaccine and the booster may be, but is not limited to, 1 week, 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, 2.5 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer. In some embodiments, the time of administration between the initial (primary) vaccination and the booster is one year. In some embodiments, a booster vaccine is not required. That is, the initial (primary) vaccination is sufficient to reduce methane emissions from the ruminant.

[0124] In some embodiments, a composition may be administered orally, intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art. In some embodiments, the composition is administered orally. In some embodiments, the primary vaccination dose(s) and the booster vaccination dose are administered via the same administration route. In some embodiments, the primary vaccination dose(s) is / are administered via one administration route and the booster vaccination dose is administered via a different administration route.

[0125] Provided herein are pharmaceutical compositions including DNA or RNA and / or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

[0126] The DNA or RNA may be formulated or administered alone or in conjunction with one or more other components. For example, an immunizing composition may comprise other components including, but not limited to, adjuvants.

[0127] In some embodiments, an immunizing composition does not include an adjuvant (they are adjuvant-free).

[0128] An RNA or DNA may be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, vaccine compositions comprise at least one additional active substance, such as, for example, a therapeutically active substance, a prophylactically active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0129] For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the RNA vaccines or DNA vaccines or the polynucleotides contained therein, for example, RNA polynucleotide(s) (e.g., mRNA polynucleotide(s)) or DNA polynucleotide(s) encoding antigens.

[0130] Formulations of the 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 or DNA 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.

[0131] 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 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w / w) active ingredient.

[0132] In some embodiments, an RNA is formulated using one or more excipients to: (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. 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, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

[0133] Dosing

[0134] Provided herein are immunizing compositions (e.g., RNA vaccines, DNA vaccines), methods, kits and reagents for reducing methane emissions in ruminants, such as cattle.

[0135] A subject may be any mammal, including ruminants. Typically, a subject is a cow or steer. In some embodiments, the subject is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years of age or older. In some embodiments, the subject is less than a year old. In some embodiments, the subject is 1-3 years old. In some embodiments, the subject is 3-6 years old. In some embodiments, the subject is less than 10 years old. In some embodiments, the subject is 10-15 years old. In some embodiments, the subject is 15-20 years old.

[0136] In some embodiments, the effective dose is a dose listed in a package insert for the vaccine.

[0137] In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in an unvaccinated subject.

[0138] An immunizing composition (e.g., vaccine) may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to oral, intradermal, intramuscular, intranasal, and / or subcutaneous administration. The present disclosure provides methods comprising administering RNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The RNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0139] The effective amount of the RNA or DNA, as provided herein, may be 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, of subject body weight.

[0140] Vaccine Efficacy

[0141] Some aspects of the present disclosure provide formulations of the immunizing compositions (e.g., RNA vaccines or DNA vaccines), wherein the RNA or DNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to the methanogen). “An effective amount” is a dose of the RNA effective to produce an antigen- specific immune response. Also provided herein are methods of inducing an antigen- specific immune response in a subject.

[0142] As used herein, an immune response to a vaccine of the present disclosure is the development in a subject of a humoral and / or a cellular immune response to a (one or more) methanogen protein(s) present in the vaccine. For purposes of the present disclosure, a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules. When a vaccine is administered, antigen- presenting cells (APCs), such as dendritic cells (DC) process the antigen and present it to T CD4+ cells via MHC class II molecules. These T cells then differentiate into T follicular helper cells (Tfh) to help B cells, enabling them to produce antibodies more efficiently and with higher affinity in secondary lymphoid organs, such as the lymph nodes. Upon binding to the antigen and receiving signals from Tfh cells, B cells become activated and differentiate into memory B cells or plasma cells that produce antibodies. This interaction occurs in the germinal center (lymph nodes), where B cells undergo somatic hypermutation to enhance antibody affinity to the vaccine formulation's antigens, with Tfh cells providing essential signals for B cell maturation. The resulting antibody and immune responses occur systemically and in mucosal tissues, ensuring robust and long-lasting antibody production and maintenance of antibody titers throughout the body.

[0143] As used herein, a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and / or CD8+ T cells (e.g., CTLs) and / or other white blood cells. One important aspect of cellular immunity involves an antigen- specific response by cytolytic T-cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen- specific response by helper T-cells. Helper T-cells act to help stimulate the function and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. Helper T-cells also provide crucial signals to B cells, aiding their differentiation and improving the affinity and specificity of the antibodies produced in response to the target antigen. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and / or other white blood cells including those derived from CD4+ and CD8+ T-cells.

[0144] In some embodiments, the antigen- specific immune response is characterized by measuring an anti-methanogen antigen antibody titer produced in a subject administered an immunizing composition as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example. In some embodiments, anti-methanogen antibody levels are measured from saliva and / or blood samples (to test mucosal and systemic levels, respectively).

[0145] In some embodiments, an antibody titer is used to determine the strength of an immune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by an immunizing composition (e.g., RNA vaccine).

[0146] In some embodiments, an anti-methanogen antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, an anti-methanogen antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control. In some embodiments, the anti-methanogen antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the anti-methanogen antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti- methanogen antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

[0147] In some embodiments, the anti-methanogen antigen antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the anti-methanogen antigen antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control. In some embodiments, the anti-methanogen antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. In some embodiments, the anti-methanogen antigen antibody titer produced in a subject is increased 2-10 times relative to a control. For example, the anti-methanogen antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.

[0148] Detectable Antigen. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce detectable levels of antigen as measured in serum of the subject at 1-72 hours post administration.

[0149] Titer. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-methanogen antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example. EXAMPLES

[0150] Example 1. Efficacy of exemplary mRNA backbones in bovine cells.

[0151] This Example demonstrates the effectiveness of exemplary mRNA backbones into Madin-Darby bovine kidney (MDBK) cells and bovine dendritic cells (DCs). Briefly, MDBK cells and DCs were transfected with eGFP mRNA with or without UTR sequences, a polyA tail (100 or 120 adenosine monophosphates in length), and / or a linker (GCATATGACT, SEQ ID NO: 83) as shown in Table 6 using the TransIT®-mRNA Transfection Kit (Mirus Bio). Untransfected cells (with and without transfection reagent) were used as negative controls.

[0152] Table 6. Experimental groups evaluated in this study.

[0153] In MDBK cells, transfection was found to be highest in groups Gl, G3, and G6 (FIGs. 1A-2C). Based on these results, bovine dendritic cells (DCs) were transfected with groups GS, Gl, G3,G6, CO, or TransIT to evaluate the suitability of different mRNA backbones for intracellular processing and presentation by bovine leukocyte antigen (BoLa) molecules on the cell surface. In dendritic cells, transfection was found to be highest in Gl and G6 samples (FIGs. 3A-3C). Groups Gl and G6 were also found to have the least impact on BoLa II expression in bovine dendritic cells following transfection (FIG. 4). These data show the effectiveness of bovine mRNA backbones for use in vaccine compositions.

[0154] Example 2. Development of an LNP-mRNA to Reduce Methane Production in Ruminants

[0155] Methane emissions from cattle, primarily produced by methanogenic archaea in the rumen, significantly contribute to agricultural greenhouse gas production, necessitating innovative solutions. Using artificial intelligence and machine learning (AI / ML) to streamline antigen selection, several rumen archaeal proteins associated with methanogenesis or critical for the methanogen's survival were selected for vaccine antigens. Antigens were prioritized based on their potential for efficacy, focusing on those predicted to be transmembrane-expressed to enhance immune recognition.

[0156] The selected antigen sequences were transcribed into mRNA via in vitro transcription (IVT) and encapsulated in lipid nanoparticles (LNP-mRNA). Mice were immunized with all selected LNP-mRNA vaccine candidates in a prime-boost regimen, with doses administered 28 days apart. In initial preclinical studies, several vaccine candidates demonstrated strong immunogenicity, including robust and high-affinity anti-methanogen-specific IgG responses, enhanced germinal center B cell and T follicular helper (Tfh) cell activity, and balanced Thl / Th2 antigen- specific T cell responses (Tables 7 and 8). In vitro binding and expression assays using mRNA-transfected Madin-Darby bovine kidney (MDBK) cells confirmed efficient antigen production and high-affinity interactions with target methanogens, supporting the potential of these candidates for further evaluation and their capacity to modulate methanogenesis effectively.

[0157] Table 7. Results

[0158] Table 8. Tfh and T Cell Results

[0159] The results demonstrate the potential for using mRNA vaccines to target methanogenesis in ruminants. Additional work will focus on testing in bovine models to evaluate vaccine efficacy and scalability for real-world applications in livestock. This approach represents a novel and potentially impactful solution to reduce methane emissions and contribute to accelerating the fight against climate change.

[0160] Methods

[0161] Animal Immunizations. C57BL / 6J mice (female, age 6-8 weeks) were separated into 28 groups (27 different antigens, as shown in Table 7, and a saline control group; n=5 / group). The animals were immunized subcutaneously (SQ) in scruff with 5 pg of mRNA-lipid nanoparticle (LNP) formulation (in each experimental group, the mRNA encoded one of the 27 antigens listed in Table 7) on day 0. Four weeks later (day 28), each animal received a booster dose identical to the initial dose the animal received (5 pg of the same mRNA-LNP formulation). Blood was collected weekly for testing methanogen- specific antibodies levels. All mice were euthanized at week 6, at which point, blood, lymph nodes, spleens, and bone marrow were collected for the downstream ex vivo and in vitro immunological assays.

[0162] Enzyme-Linked Immunosorbent Assay (ELISA). ELISA plates were coated with formalin-killed rumen methanogens and blocked with 2% Bovine Serum Albumin (BSA) to prevent non-specific binding. Mice serum samples (containing antibodies) were then added. Plates were then washed and the enzyme (horseradish peroxidase, HRP)-linked secondary antibody was added. The enzyme substrate and stop solution were then added. Absorbance (450nm) was detected using a Biotek Synergy HTX plate reader. Data was analyzed in Microsoft Office Excel and using GraphPad Prism software.

[0163] The binding strength of the antibodies generated by the immune responses induced by mRNA encoding the selected antigens via an avidity ELISA was evaluated in parallel. The same procedure as described above was performed, with one additional step: addition of a chaotropic compound - potassium thiocyanate (KSCN) - to the plates to disrupt weak binding antibodies from binding to their antigen targets after the addition of the serum samples. The control plates (with the same serum samples) received PBS. The absorbance values were obtained as described above. The avidity index was calculated as the antibody titer (log scale) in KSCN-treated plates / antibody titer (log scale) in PBS-treated plates * 100%.

[0164] Enzyme-Linked Immunosorbent Spot (ELISpot). Briefly, bone marrow cells were isolated from collected femurs and tibias and the red blood cells were lysed. The remaining immune cells were added to ELISpot plates previously coated with whole formalin-inactivated methanogen cells. After an overnight incubation (at 37°C, in a 5% CO2 incubator), the cells were washed. The enzyme (alkaline phosphatase, ALP)-linked secondary antibody was added, followed by a substrate that produces spots in the well to evaluate long-lived plasma cells (LLPC) responses, as indicated by the formation of spots on the plate, indicating LLPC activity of methanogen- specific antibodies. Spots were counted by an S6 Lite M2 ELISpot reader.

[0165] Flow Cytometry. Mice cervical lymph nodes cells were collected, processed, and stained with fluorophore-conjugated monoclonal antibodies to evaluate germinal center reaction, switched memory, and IgA-positive and IgG-positive B cells. B cells were phenotyped as CD 19 and B220 (CD45R) positivity. Furthermore, germinal center sizes were characterized by GL7- positive and CD38-low expression. Switched memory B cells were defined as CD38-high expression, and IgM- and IgD-negative. The total memory switched IgA -positive and IgG- positive B populations were identified within the CD38-high cells as IgA-positive and IgG- positive, respectively. T follicular helper cells (Tfh) were phenotyped by staining lymph node cells with the markers CD3 and CD4, followed by the double positivity to CXCR5 (C-X-C chemokine receptor type 5) and PD-1 (Programmed Cell Death- 1). Data acquisition was done using multi-color flow cytometry (Attune NxT Flow Cytometer), followed by FlowJo™ software analyses.

[0166] T Cell Thl / Th2 Response. Bone marrow from control (unimmunized) mice was collected and processed to dissect the bone marrow cells (BM cells). The BM cells were then cultured in sterile conditions for 5 days at 37°C and 5% CO2 in presence of granulocytemacrophage colony- stimulating factor (GM-CSF) and interleukin 4 (IL-4) cytokines to differentiate into dendritic cells (DC). DCs were then transfected with mRNA encoding the methanogen antigens as described above to serve as a source of antigens to restimulate cognate T cells from mice immunized with mRNA encoding methanogen antigens in vitro.

[0167] Spleens from immunized mice were harvested and processed to isolate splenocytes. To assess T cells antigen- specific Thl / Th2 responses, splenocytes (including T cells and other immune cells) were co-cultured with the allogeneic DCs transfected with mRNA encoding methanogen antigens, as described above. The DCs and splenocytes were cultured in sterile conditions overnight at 37°C and 5% CO2. To measure intracellular cytokine production, Golgi Stop reagent was added prior to harvesting the cells for analysis in order to inhibit cytokine secretion. Intracellular cytokines were stained with monoclonal antibodies in both CD3 positive CD4 positive T cells (or helper) and CD3 positive CD8 positive T cells (or cytotoxic T cells) that were membrane -permeabilized. Tumor necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma) represented the Thl cytokines. IL-4 and IL-5 cytokines were used to phenotype Th2 responses. The T cell responses are defined by the combined ratio of Thl cytokines divided by the combination ratio of Th2 cytokines. A result greater than 1 indicates a Thl antigen- specific biased T cell response, while a value between 0 and 1 suggests an antigen- specific Th2 biased T cell response. Data acquisition was done using multi-color flow cytometry (Attune NxT Flow Cytometer), followed by Flow Jo™ software analysis.

[0168] Madin-Darby Bovine Kidney Cells (MDBK) Transfection and Antigen Expression. MDBK cells were transfected with antigen-encoding mRNA using lipofectamine MessengerMax, with untransfected, lipofectamine-only, and isotype- stained samples included as controls. To assess intracellular and secreted versus surface antigen expression, Golgi Stop was added prior to harvest to inhibit secretion. At around 20 hours post-transfection, cells were harvested, washed, and stained with viability dye. After washing, cells were incubated with sera from vaccinated mice. Cells were then stained with secondary antibodies for detection after washing. Following final washes, cells were analyzed by Attune flow cytometry and FlowJo™.

[0169] To assess intracellular and secreted versus surface antigen expression, Golgi Stop (a reagent to trap proteins intracellularly) was added four hours prior to harvesting the cells for flow cytometry analysis in order to inhibit antigen secretion. Surface antigen expression was determined using mean fluorescence intensity (MFI) from outer membrane non-permeabilized and Golgi stop untreated cells stained with mice anti-antigen polyclonal sera. Intracellular antigen levels were determined by staining (as described above above). Membrane- permeabilized cells, Golgi stop-treated cells, and background- subtracted MFI (from mRNA untransfected cells) values were used to quantify internal antigen expression. Secreted antigen levels were inferred by comparing intracellular MFI from permeabilized cells with and without Golgi Stop using following formulas:

[0170] Surface MFI= MFInon-pemieabilized, no Golgi Stop - MFIunstained or isotype control

[0171] Intracellular MFI= MFIpermeabilized + Golgi Stop MHunstained or isotype control

[0172] Combined MFI= Surface MFI + Intracellular MFI

[0173] Secreted MFI — MFIpermeabilized + Golgi Stop M Flpermeabilized, no Golgi Stop

[0174] Background-subtracted MFI = MFItransfected cell - MFIuntransfected cell or lipofectamine only

[0175] Example 3. Immune Response following Vaccination (prime and boost) with an LNP- mRNA in Ruminants

[0176] In this Example, the total immune response generated following vaccination with an mRNA vaccine encoding different methanogens was examined. Administration of the vaccine led to the generation of bovine anti-methanogen antibodies which bind ruminal methanogens and modulate ruminal microbiome function.

[0177] A controlled study to evaluate the performance of the vaccine in cattle was performed with Texas A&M University. Bos taurus heifers (n=36; female, 8-12 months of age) were separated into two groups: a treatment group (n=18) and a control group (n=18) and placed in adjacent pens. Animals were fed the same diet of dry matter rations: corn silage (44%), sorghum Sudan hay (28%), corn (12%), DDG (9%), molasses (5%), and minerals (2%). Methane emissions were evaluated multiple times per day with GREENFEED™ pellet-feeder gas emission detectors, and animal food intake was tracked daily with GROWSAFE™ feed machines.

[0178] The treatment group was immunized subcutaneously (SQ) with mRNA-lipid nanoparticle (LNP) vaccines. The mRNA was encapsulated in the LNP. Each vaccine dose was 1.5 mg and comprised 250 pg of six different mRNA-LNPs , each encoding a single antigen (PBA6 (SEQ ID NO: 89 (protein), SEQ ID NO: 48 (mRNA)), PBA13 (SEQ ID NO: 96 (protein), SEQ ID NO: 55 (mRNA)), PBA16 (SEQ ID NO: 99 (protein), SEQ ID NO: 58 (mRNA)), PBA17 (SEQ ID NO: 100 (protein), SEQ ID NO: 59 (mRNA)), PBA18 (SEQ ID NO: 101 (protein), SEQ ID NO: 60 (mRNA)), and PBA30 (SEQ ID NO: 113 (protein), SEQ ID NO: 72 (mRNA))). The control group received 1.5 mg of saline. Both groups were primed on day 0 and boosted four weeks later (week 4 / day 28) with the same formulation (1.5 mg vaccine or saline). Blood was collected weekly to test methanogen- specific antibody levels.

[0179] The study was divided into four phases. The pre-immunization period was the time in which animals were separated into groups (treatment and control) and methane tracking began, but before first administration of the vaccine (day -14 to day 0). Note that food intake was not recorded for the pre-immunization period. The “prime” period (day 1 to day 28) described the period between the first vaccine injection, the “priming” injection, and the second injection, the “boosting” injection. This period is followed by the “boost #1” period (day 29 to 52), which occurred between the second and third injection. The “boost #2” period took place after the third injection (the second boost dose).

[0180] Methane data was tracked via RFID tag per animal, per feed pellet interaction, and daily methane averages (grams CFU per day) were computed for each animal per GREENFEED™ interaction. When an animal registered more than one GREENFEED™interaction in a day, daily methane estimates for each interaction were combined in an average weighted by duration of GREENFEED™ interaction. Food intake was also tracked once daily per animal. Weight was tracked weekly. A small number of study days lacked methane emission data and / or food intake data for some animals due to a combination of rain, loss of wireless connection, loose cables, or pellets stuck in the feeder machinery. Missing days were filled with estimated measurements via nearest neighbor interpolation. For each missing data point, an estimate was calculated by averaging across a total of six data points, the closest three non-missing data points both before and after the missing day. Missing data points at the beginning or end of the study were back filled or forward filled with the average of the three nearest neighbor points. This estimation was performed for each animal independently.

[0181] Performance was tracked by evaluating two methane metrics and two weight gain metrics. Methane reduction performance was evaluated as both reduction of total daily methane in the treatment versus control groups, and as total daily methane normalized by daily food dry matter intake (DMI). Food intake and subsequent hydrolysis or fermentation supplies the carbon and hydrogen building blocks that methanogens use to create methane. Thus, normalization by food intake is important, as animals may emit less methane simply by eating less, and vice versa, higher intake can result in increased emissions, regardless of vaccine efficiency. Weight gain for any given time period was evaluated by feed efficiency (FE, the total weight gain divided by total food eaten), and average daily gain (ADG, the total weight gain divided by the number of days in the time period).

[0182] The four metrics described above were aggregated into the relevant time periods. Population distributions for the above were calculated, and mean and median values were compared between vaccine and control groups independently within each time period. The median value of the control group within each time period was set to 1 for normalization purposes. Significance p-values were calculated as follows using python stats library. Mean difference p-values were calculated with a 1-sample T-test (stats.ttestlsamp) and median differences were calculated with a two-sided Mann- Whitney test (stats.mannwhitneyu). Data were visualized using box and whisker plots where vaccine group values were compared to control median value within each time period.

[0183] Mid-point results were evaluated in the Boost #1 time period. Methane reduction and methane reduction per kg food intake were both significant, with median values dropping by 8.1% (FIG. 5A) and 7.5% (FIG. 5B), respectively. Average daily gain (FIG. 5C) and feed efficiency (FIG. 5D) showed no significant difference at any time period.

[0184] Additionally, an ELISA was used to examine anti-methanogen antibodies. Briefly, ELISA plates were coated with formalin-killed rumen methanogens and blocked with 4% Bovine Serum Albumin (BSA) to prevent non-specific binding. Mice serum samples (containing antibodies) were then added. Plates were then washed and the enzyme (horseradish peroxidase, HRP)-linked secondary antibody was added. The enzyme substrate and stop solution was added in the last step respectively for the absorbance (450nm) detection using a Biotek Synergy HTX™ plate reader. Data was analyzed in Microsoft Office EXCEL™ and GraphPad PRISIM™ software. Logio IgG and IgA titers were analyzed and normalized by dividing the average value of the control group in corresponding time points. Data was analyzed by means of a Mann- Whitney U-test (* p < 0.05, ** p < 0.01). As is shown in FIG. 6A, the serum (systemic) bovine anti-methanogen IgG titer increased significantly (p < 0.01) at the prime+boostl stage (the “boost #1” time period), as did the salivary bovine anti-methanogen IgA titer (FIG. 6B; (p < 0.05). SEQUENCES OF THE DISCLOSURE

[0185] Table 1. UTR Sequences, Stop Codon

[0186] Table 2. ORF Sequences

[0187] Table 3. Full Length Nucleotide Sequences Table 4. Polypeptide Sequences

[0188] Note: each sequence corresponds to an mRNA sequence above (for example, “PBA1” is the protein encoded by Ml)

[0189] EQUIVALENTS

[0190] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0191] The indefinite articles “a” and “an,” as used herein in the specification and in the embodiments and / or claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0192] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0193] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0194] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value. Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

1. CLAIMSWhat is claimed is:

1. A vaccine comprising a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the polynucleotide comprises an open reading frame (ORF) having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5, 8, 10, 11, 13, and 15-42.

2. The vaccine of claim 1, wherein the ORF comprises any one of SEQ ID NOs: 5, 8, 10, 11, 13, and 15-42.

3. The vaccine of claim 1 or claim 2, wherein the ORF consists of any one of SEQ ID NOs:5, 8, 10, 11, 13, and 15-42.

4. The vaccine of any one of claims 1-3, wherein the polynucleotide further comprises a 5’UTR, and wherein the 5’ UTR comprises SEQ ID NO: 1.

5. The vaccine of any one of claims 1-4, wherein the polynucleotide further comprises a 3’ UTR, wherein the 3’UTR comprises SEQ ID NO: 2.

6. The vaccine of any one of claims 1-5, wherein the polynucleotide comprises a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 45, 48, 50, 51, 53, and 55-82.

7. The vaccine of any one of claims 1-6, wherein the polynucleotide comprises any one of SEQ ID NOs: 45, 48, 50, 51, 53, and 55-82.

8. The vaccine of claim 7, wherein the polynucleotide consists of any one of SEQ ID NOs: 45, 48, 50, 51, 53, and 55-82.

9. The vaccine of any one of claims 1-8, wherein the vaccine further comprises 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 additional polynucleotides, each encoding a different protein or fragment thereof of a methanogen.

10. The vaccine of any one of claims 1-9, wherein the polynucleotide is codon-optimized.

11. The vaccine of any one of claims 1-10, wherein the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

12. The vaccine of claim 11, wherein the RNA is messenger RNA (mRNA) or selfamplifying RNA.

13. The vaccine of claim 11, wherein the RNA comprises at least nucleobase comprising at least one chemical modification.

14. The vaccine of claim 13, wherein the chemical modification is selected from the group consisting of: Nl-methyl-pseudouridine (mly), Nl-ethyl-pseudouridine (ely), 5-methoxy- uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in mRNAs comprise 5 -methoxy methyl uridine, 5- methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, and any combination thereof.

15. The vaccine of any one of claims 1-14, wherein the vaccine further comprises a lipid nanoparticle, a synthetic nanocarrier, a liposome, an archaeosome, an extracellular vesicle, or a hydrogel.

16. The vaccine of claim 15, wherein the lipid nanoparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol and a PEG-modified lipid.

17. The vaccine of claim 16, wherein the synthetic nanocarriers comprises one or more polymers.

18. The vaccine of any one of claims 1-17, further comprising an adjuvant.

19. A method of inducing an immune response against at least one methanogen in a subject, the method comprising administering the vaccine of any one of claims 1-18 to the subject.

20. The method of claim 19, wherein the subject is a ruminant.

21. The method of claim 20, wherein the ruminant is selected from the group consisting of: cow, heifer, bull calf, bull, steer, ox, sheep, or goat.

22. The method of any one of claims 19-21, wherein the vaccine is administered to the ruminant via oral administration, intranasal, intramuscular administration, intradermal administration, or subcutaneous administration.

23. The method of any one of claims 19-22, wherein the ruminant is administered an initial dose of the vaccine.

24. The method of claim 23, wherein the ruminant is administered an additional dose of the vaccine.

25. The method of claim 24, wherein the ruminant is administered at least one additional dose of the vaccine 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 9 months, 1 year, or 2 years after administration of initial dose.

26. The method of any one of claims 19-25, wherein the method reduces methane emission from the ruminant by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or 100% relative to the amount of methane emitted by the ruminant before administration of the vaccine.

27. The method of any one of claims 19-26, wherein the method:(a) induces an anti-methanogen-specific IgG response; and / or(b) enhances germinal center B cell activity; and / or(c) enhances T follicular helper cell activity; and / or(d) induces Thl / Th2 antigen- specific T cell responses in the ruminant.

28. A vaccine comprising:(a) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 89 encapsulated in a lipid nanoparticle;(b) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 96 encapsulated in a lipid nanoparticle;(c) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO:99 encapsulated in a lipid nanoparticle;(d) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO:100 encapsulated in a lipid nanoparticle;(e) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO:101 encapsulated in a lipid nanoparticle; and(f) a polynucleotide encoding a protein or fragment thereof of a methanogen, wherein the protein or fragment thereof of a methanogen comprises the amino acid sequence of SEQ ID NO: 113 encapsulated in a lipid nanoparticle.

29. The vaccine of claim 28, wherein each polynucleotide comprises an open reading frame (ORF) and wherein the ORF of the polynucleotide of (a) comprises SEQ ID NO: 8; the ORF of the polynucleotide of (b) comprises SEQ ID NO: 15; the ORF of the polynucleotide of (c) comprises SEQ ID NO: 18; the ORF of the polynucleotide of (d) comprises SEQ ID NO: 19; the ORF of the polynucleotide of (e) comprises SEQ ID NO: 20; and the ORF of the polynucleotide of (f) comprises SEQ ID NO: 32.

30. The vaccine of claim 28 or 29, wherein each polynucleotide comprises an open reading frame (ORF) and wherein the ORF of the polynucleotide of (a) consists of SEQ ID NO: 8; the ORF of the polynucleotide of (b) consists of SEQ ID NO: 15; the ORF of the polynucleotide of (c) consists of SEQ ID NO: 18; the ORF of the polynucleotide of (d) consists of SEQ ID NO: 19; the ORF of the polynucleotide of (e) consists of SEQ ID NO: 20; and the ORF of the polynucleotide of (f) consists of SEQ ID NO: 32.

31. The vaccine of any one of claims 28-30, wherein each polynucleotide further comprises a 5’UTR, and wherein the 5’ UTR comprises SEQ ID NO: 1.

32. The vaccine of any one of claims 28-31, wherein each polynucleotide further comprises a 3’ UTR, wherein the 3’UTR comprises SEQ ID NO: 2.

33. The vaccine of any one of claims 28-32, wherein the polynucleotide of (a) comprises SEQ ID NO: 48; the polynucleotide of (b) comprises SEQ ID NO: 55; the polynucleotide of (c) comprises SEQ ID NO: 58; the polynucleotide of (d) comprises SEQ ID NO: 59; the polynucleotide of (e) comprises SEQ ID NO: 60; and the polynucleotide of (f) comprises SEQ ID NO: 72.

34. The vaccine of any one of claims 28-33, wherein each polynucleotide comprises RNA and wherein the RNA comprises at least nucleobase comprising at least one chemical modification.

35. The vaccine of claim 34, wherein the chemical modification is selected from the group consisting of: Nl-methyl-pseudouridine (mly), Nl-ethyl-pseudouridine (ely), 5-methoxy- uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in mRNAs comprise 5 -methoxy methyl uridine, 5- methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, and any combination thereof.

36. The vaccine of any one of claims 28-35, wherein each lipid nanoparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol and a PEG-modified lipid.

37. A method of inducing an immune response against at least one methanogen in a subject, the method comprising administering the vaccine of any one of claims 28-36 to the subject.

38. The method of claim 37, wherein the subject is a ruminant.

39. The method of claim 38, wherein the ruminant is selected from the group consisting of: cow, heifer, bull calf, bull, steer, ox, sheep, or goat.

40. The method of any one of claims 37-39, wherein the subject is administered an initial dose of the vaccine, optionally wherein the subject is administered an additional dose of the vaccine.

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