Ruminant and methanogen vaccines and their uses

Abstract

Disclosed herein are polynucleotides encoding one or more rumen-associated antigens, and polypeptides encoded by the polynucleotides. Also provided herein are compositions comprising the same, as well as methods of making and using the compositions. The present disclosure identifies particular challenges regarding rising global surface temperatures due to increasing levels of the greenhouse gas, methane (CH4). For example, the present disclosure identifies that increasing agricultural methane emissions represent a major source of greenhouse gas emissions. The present disclosure also identifies that reducing methane emissions from animals, such as ruminants, will be important for reducing greenhouse gas emissions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 422,312, filed November 3, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Control of methane emissions from animals such as ruminants is an emerging field. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure identifies particular challenges with rising Earth's surface temperatures due to increasing levels of the greenhouse gas, namely methane (CH4). For example, the present disclosure identifies that increasing agricultural methane represents a major source of greenhouse gas emissions. The present disclosure also identifies that reducing methane emissions from animals, such as ruminants, will be important in reducing greenhouse gas emissions.

[0004] Among other things, the present disclosure provides techniques for reducing methane emissions from animals, e.g., ruminants, by providing compositions for vaccinating animals, e.g., ruminants, against rumen-associated antigens. In some embodiments, a composition comprising a rumen-associated antigen can further comprise one or more chemokines and / or cytokines. Without wishing to be bound by theory, the present disclosure proposes that a composition comprising a rumen-associated antigen (e.g., a rumen antigen and / or a methanogen antigen) can reduce methane emissions and / or reduce the abundance of microorganisms (e.g., methanogens) in the gastrointestinal tract of an animal (e.g., a ruminant). In some embodiments, reducing methane emissions and / or reducing the abundance of methanogens in the gastrointestinal tract of an animal (e.g., a ruminant) can increase the energy efficiency of the animal.

[0005] The present disclosure provides isolated polynucleotides encoding one or more rumen-associated antigens, fragments thereof, variants thereof, or variant fragments thereof.

[0006] In some embodiments, the one or more rumen-associated antigens comprise one or more rumen antigens.

[0007] In some embodiments, the one or more ruminal antigens are derived from a polypeptide involved in adhesion to fermenting bacteria, or a fragment or variant or variant fragment thereof.

[0008] In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 rumen antigens.

[0009] In some embodiments, the one or more rumen-associated antigens comprise one or more methanogen antigens.

[0010] In some embodiments, one or more methanogen antigens are derived from a polypeptide found on the cell surface of a wild-type methanogen, or a fragment or variant or variant fragment thereof.

[0011] In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 methanogen antigens.

[0012] In some embodiments, the polynucleotide comprises about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 20, about 4 to about 20, about 5 to about 20, about 6 to about 20, about 7 to about 20, about 8 to about 20, about 9 to about 20, about 10 to about 20, or about 15 to about 20 methanogen antigens.

[0013] In some embodiments, one or more methanogen antigens are the same, eg, have the same sequence.

[0014] In some embodiments, one or more of the methanogen antigens are different, eg, have different sequences.

[0015] In some embodiments, the one or more methanogen antigens are:

[0016] (i) one or more peptides with at least 80% sequence identity to a methanogen protein;

[0017] (ii) one or more secreted antigens containing a signal peptide;

[0018] (iii) multiple peptides having at least 80% sequence identity with each other;

[0019] (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species;

[0020] (v) one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity;

[0021] (vi) one or more peptides having at least 80% sequence identity to an AglB polypeptide or having substantially similar function to an AglB polypeptide;

[0022] (vii) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having substantially the same function as an Ig-like domain-containing polypeptide; or

[0023] (viii) Any combination thereof.

[0024] In some embodiments, the one or more methanogen antigens are:

[0025] (i) one or more peptides with at least 80% sequence identity to a methanogen protein;

[0026] (ii) one or more secreted antigens containing a signal peptide;

[0027] (iii) multiple peptides having at least 80% sequence identity with each other;

[0028] (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species;

[0029] (v) one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity; or

[0030] (vi) Any combination thereof.

[0031] In some embodiments, the polynucleotide comprises three methanogen antigens. In some embodiments, the three methanogen antigens are associated with at least three different methanogen species.

[0032] In some embodiments, the one or more methanogen antigens are:

[0033] (i) one or more peptides with at least 80% sequence identity to a methanogen protein;

[0034] (ii) one or more secreted antigens containing a signal peptide;

[0035] (iii) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species;

[0036] (iv) one or more peptides having at least 80% sequence identity to an AglB polypeptide or having substantially the same function as an AglB polypeptide; or

[0037] (v) includes any combination thereof.

[0038] In some embodiments, the polynucleotide comprises five methanogen antigens. In some embodiments, the five methanogen antigens are associated with at least five different methanogen species.

[0039] In some embodiments, the one or more methanogen antigens are:

[0040] (i) one or more peptides with at least 80% sequence identity to a methanogen protein;

[0041] (ii) one or more secreted antigens containing a signal peptide;

[0042] (iii) multiple peptides having at least 80% sequence identity with each other;

[0043] (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species;

[0044] (v) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having substantially the same function as an Ig-like domain-containing polypeptide; or

[0045] (vi) Any combination thereof.

[0046] In some embodiments, the polynucleotide comprises eight methanogen antigens. In some embodiments, the eight methanogen antigens are associated with at least three different methanogen species.

[0047] In some embodiments, the one or more methanogen antigens comprise a plurality of peptides associated with at least 2 different, at least 3 different, at least 4 different, at least 5 different, at least 6 different, at least 7 different, at least 8 different, at least 9 different, at least 10 different, at least 11 different, at least 12 different, at least 15 different, or at least 20 different methanogen species.

[0048] In some embodiments, the one or more methanogen antigens comprise a plurality of peptides associated with about 2 different, about 3 different, about 4 different, about 5 different, about 6 different, about 7 different, about 8 different, about 9 different, about 10 different, about 11 different, about 12 different, about 15 different, or about 20 different methanogen species.

[0049] In some embodiments, one or more methanogen antigens are derived from a polypeptide found on the cell surface of a wild-type methanogen, or a fragment or variant or variant fragment thereof.

[0050] In some embodiments, one or more methanogen antigens are secreted.

[0051] In some embodiments, the methanogen antigen comprises a peptide involved in adhesion, attachment, or motility, or a fragment or variant of a peptide involved in adhesion, attachment, or motility.

[0052] In some embodiments, the secreted methanogen antigen comprises a signal peptide. In some embodiments, the signal peptide can be predicted using a prediction algorithm, optionally with a prediction score of at least 0.5.

[0053] In some embodiments, the one or more methanogen antigens comprise one or more peptides having at least 80% sequence identity to a methanogen protein.

[0054] In some embodiments, the polynucleotide comprises multiple methanogen antigens, each of which has at least 80% sequence identity to each other.

[0055] In some embodiments, the polynucleotide comprises a plurality of methanogen antigens, wherein each of the plurality of methanogen antigens is associated with a different methanogen species. In some embodiments, the polynucleotide comprises a plurality of methanogen antigens, wherein the plurality of methanogen antigens is associated with at least two different, at least three different, at least four different, or at least five different methanogen species. In some embodiments, the polynucleotide comprises a plurality of methanogen antigens, wherein the plurality of methanogen antigens is associated with the same methanogen species. In some embodiments, the methanogen species comprises Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanomicrobium mobile, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp. UBA188, or Methanosarcina Soligelidi. In some embodiments, the methanogen species comprises Methanobrevibacter ruminantium, Methanobrevibacter smithii, or Methanobrevibacter oralis. In some embodiments, the methanogen species comprises Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, or Methanosphaera stadtmanae.

[0056] In some embodiments, the polynucleotide comprises one or more methanogen antigens comprising one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity. In some embodiments, the polypeptide having signal peptidase activity (i) is membrane-bound; (ii) has the ability to cleave one or more signal peptides; (iii) plays a role in converting a secreted protein to a mature form (e.g., from a non-secreted form to a secreted form); and / or (iv), or any combination thereof.

[0057] In some embodiments, the polynucleotide comprises one or more methanogen antigens that comprise one or more peptides that have (1) at least 80% sequence identity to an immunoglobulin (Ig)-like polypeptide, or (2) a substantially similar function to an Ig-like domain-containing polypeptide. In some embodiments, the Ig-like domain-containing polypeptide comprises a polypeptide characterized as a cell surface protein that facilitates binding to other cells and / or cell surfaces.

[0058] In some embodiments, the polynucleotide comprises one or more methanogen antigens including an adhesin or a fragment or variant thereof, a pili protein or a fragment or variant thereof, or a flagellin protein or a fragment or variant thereof.

[0059] In some embodiments, the polynucleotide comprises a sequence encoding one or more methanogen antigens, including those listed in Table 1, or a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0060] In some embodiments, the polynucleotide comprises a sequence encoding one or more methanogen antigens comprising an antigen sequence presented in Table 2, or a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the polynucleotide comprises a sequence encoding one or more methanogen antigens comprising a fragment, variant, or variant fragment of an antigen sequence presented in Table 2. Exemplary antigen sequences presented in Table 2 are not shown in bold or italics.

[0061] In some embodiments, the polynucleotide comprises an antigen nucleic acid sequence presented in Table 2, or a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0062] In some embodiments, the polynucleotide comprises a sequence that encodes a polypeptide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide sequence presented in Table 2.

[0063] In some embodiments, the polynucleotide comprises one or more methanogen antigens that comprise (1) 1 to 80% sequence identity to an archaeal oligosaccharyltransferase (AglB) polypeptide, or (2) one or more peptides that have substantially the same function as an AglB polypeptide. In some embodiments, the AglB polypeptide is characterized by having N-glycosylation activity.

[0064] In some embodiments, the methanogen antigen comprises a peptide involved in adhesion, attachment, or motility, or a fragment or variant of a peptide involved in adhesion, attachment, or motility.

[0065] In some embodiments, the methanogen antigen comprises an adhesin or a fragment or variant thereof. In some embodiments, the methanogen antigen comprises an adhesin protein set forth in Table 1 or a sequence at least 85% identical thereto. In some embodiments, the methanogen antigen comprises mru1499, or a fragment or variant thereof.

[0066] In some embodiments, the methanogen antigen comprises an antigenic sequence in Table 2, or a variant or fragment or variant fragment thereof. In some embodiments, the methanogen antigen comprises an antigenic sequence in Table 2, or a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Exemplary antigenic sequences presented in Table 2 are not shown in bold or italics.

[0067] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 2, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0068] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 5, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 6, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0069] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 7, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 8, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0070] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 15, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0071] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 18, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 17, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0072] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 20, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 19, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0073] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 22, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 21, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0074] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 24, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 23, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0075] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 26, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 25, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0076] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 28, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 27, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0077] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 30, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 29, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0078] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 32, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 31, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0079] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 34, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 33, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0080] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 36, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 35, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0081] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 38, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 37, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0082] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 40, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 39, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0083] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 42, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 41, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0084] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 44, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 43, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0085] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 46, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 45, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0086] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 48, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 47, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0087] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 50, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 49, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0088] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 52, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 51, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0089] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 54, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 53, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0090] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 56, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 55, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0091] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 58, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 57, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0092] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 60, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 59, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0093] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 62, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 61, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0094] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 64, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 63, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0095] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 66, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 65, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0096] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 68, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 67, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0097] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 70, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 69, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0098] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 72, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 71, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0099] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 74, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 73, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0100] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 76, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 75, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0101] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 78, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 77, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0102] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 80, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 79, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0103] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 82, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 81, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0104] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 82, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 83, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0105] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 86, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 85, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0106] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 88, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 87, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0107] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 90, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 89, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0108] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 92, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 91, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0109] In some embodiments, the methanogen antigen comprises the antigenic sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 94, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto. In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 93, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0110] In some embodiments, the methanogen antigen comprises a pili protein or a fragment or variant thereof.

[0111] In some embodiments, the methanogen antigen comprises flagellin or a fragment or variant thereof.

[0112] In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 methanogen antigens.

[0113] In some embodiments, the polynucleotide comprises a signal peptide, in some embodiments, the signal peptide has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to an archaeal signal peptide.

[0114] In some embodiments, the polynucleotide comprises at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homology to a bacterial signal peptide.

[0115] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 97, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0116] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 98, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0117] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 99, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0118] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 100, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0119] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 101, or a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0120] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 102, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0121] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 103, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0122] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 104, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0123] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 105, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0124] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 106, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0125] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 107, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0126] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 108, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0127] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 109, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0128] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 110, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0129] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 111, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0130] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 112, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0131] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 113, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0132] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 114, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0133] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 115, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0134] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 116, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0135] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 117, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0136] In some embodiments, the methanogen antigen comprises the antigen sequence set forth in SEQ ID NO: 118, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0137] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 119, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0138] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 120, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0139] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 121, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0140] In some embodiments, the methanogen antigen comprises the antigenic sequence set forth in SEQ ID NO: 122, or a sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical thereto.

[0141] In some embodiments, the signal peptide is or comprises a PPA2 signal peptide, or a fragment or variant thereof.

[0142] In some embodiments, the signal peptide is or comprises an SSP signal peptide, or a fragment or variant thereof.

[0143] In some embodiments, the signal peptide is or comprises the SARS-CoV-2 spike secretion signal, or a fragment or variant thereof.

[0144] In some embodiments, the signal peptide is located N-terminal to the rumen-associated antigen sequence.

[0145] In some embodiments, the polynucleotide comprises (i) a first nucleotide sequence encoding one or more rumen antigens and (ii) a second nucleotide sequence encoding one or more methanogen antigens.

[0146] In some embodiments, the polynucleotide comprises (i) a first nucleotide sequence encoding one or more rumen antigens, (ii) a second nucleotide sequence encoding one or more methanogen antigens, and (iii) a third nucleotide sequence encoding a chemokine and / or cytokine.

[0147] In some embodiments, the polynucleotide comprises (i) a first nucleotide sequence encoding one or more ruminal antigens and (ii) a second nucleotide sequence encoding a chemokine and / or cytokine.

[0148] In some embodiments, the polynucleotide comprises (i) a first nucleotide sequence encoding one or more methanogen antigens and (ii) a second nucleotide sequence encoding a chemokine and / or cytokine.

[0149] In some embodiments, the polynucleotide comprises nucleotides encoding a chemokine and / or cytokine, hi some embodiments, the chemokine and / or cytokine is selected from APRIL, VIP and / or CXCL10.

[0150] In some embodiments, the polynucleotide comprises a sequence encoding a chemokine or cytokine set forth in SEQ ID NO: 10, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0151] In some embodiments, the polynucleotide comprises the nucleotide sequence of a chemokine or cytokine set forth in SEQ ID NO: 9, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0152] In some embodiments, the polynucleotide comprises a sequence encoding the chemokine or cytokine set forth in SEQ ID NO: 12, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0153] In some embodiments, the polynucleotide comprises the nucleotide sequence of a chemokine or cytokine set forth in SEQ ID NO: 11, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0154] In some embodiments, the polynucleotide comprises a sequence encoding the chemokine or cytokine set forth in SEQ ID NO: 14, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0155] In some embodiments, the polynucleotide comprises the nucleotide sequence of a chemokine or cytokine set forth in SEQ ID NO: 13, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0156] In some embodiments, the nucleotide sequence encoding one or more ruminal antigens comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ruminal antigens.

[0157] In some embodiments, the nucleotide sequence encoding one or more methanogen antigens includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 methanogen antigens.

[0158] In some embodiments, the first nucleotide sequence, the second nucleotide sequence, and / or the third nucleotide sequence are located on a single polynucleotide.

[0159] In some embodiments, the first nucleotide sequence, the second nucleotide sequence, and / or the third nucleotide sequence are located on different polynucleotides.

[0160] In some embodiments, the one or more rumen antigens and / or the one or more methanogen antigens are each located on a separate nucleotide sequence.

[0161] In some embodiments, the polynucleotide comprises at least two methanogen antigens, wherein the at least two methanogen antigens are located on separate nucleotide sequences.

[0162] In some embodiments, the polynucleotide comprises at least three methanogen antigens, wherein the at least three methanogen antigens are located on separate nucleotide sequences.

[0163] In some embodiments, the polynucleotide comprises at least four methanogen antigens, wherein the at least four methanogen antigens are located on separate nucleotide sequences.

[0164] In some embodiments, the polynucleotide comprises at least five methanogen antigens, wherein the at least five methanogen antigens are located on separate nucleotide sequences.

[0165] In some embodiments, the polynucleotide comprises at least six methanogen antigens, wherein the at least six methanogen antigens are located on separate nucleotide sequences.

[0166] In some embodiments, the polynucleotide comprises at least seven methanogen antigens, wherein the at least seven methanogen antigens are located on separate nucleotide sequences.

[0167] In some embodiments, the polynucleotide comprises at least eight methanogen antigens, wherein the at least eight methanogen antigens are located on separate nucleotide sequences.

[0168] In some embodiments, the polynucleotide comprises at least nine methanogen antigens, wherein the at least nine methanogen antigens are located on separate nucleotide sequences.

[0169] In some embodiments, the polynucleotide comprises at least 10 methanogen antigens, wherein the at least 10 methanogen antigens are located on separate nucleotide sequences.

[0170] In some embodiments, one or more rumen antigens and / or one or more methanogen antigens are located on the same nucleotide sequence.

[0171] In some embodiments, the polynucleotide comprises a transmembrane domain sequence.

[0172] In some embodiments, the polynucleotide further comprises a complement C3d-binding polypeptide derived from Staphylococcus aureus immunoglobulin-binding protein (Sbi), hi some embodiments, the complement C3d-binding polypeptide is or comprises one or both of domain III and domain IV of Staphylococcus aureus Sbi, or a functional fragment or variant thereof.

[0173] In some embodiments, the polynucleotide is or comprises DNA.

[0174] In some embodiments, the polynucleotide is or comprises RNA. In some embodiments, the RNA comprises a 5' cap. In some embodiments, the RNA comprises a polyA tail.

[0175] In some embodiments, the polynucleotide sequence comprises one or more ribonucleotides comprising a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has the following structure, where R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate: [ka]

[0176] In some embodiments, the polyribonucleotide further comprises one or more modified ribonucleotides other than N4-acetylcytidine, optionally wherein the nucleosides are selected from adenosine, inosine, guanosine, cytidine, or uridine, or any combination thereof.

[0177] In some embodiments, the nucleoside of one or more modified ribonucleotides is 5-hydroxymethyluridine, and the modified ribonucleotide has the following structure, where R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate: [ka]

[0178] In some embodiments, the polynucleotide sequence comprises one or more ribonucleotides comprising a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine, and the modified ribonucleotide has the following structure, where R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate: [ka]

[0179] In some embodiments, the polyribonucleotide further comprises one or more modified ribonucleotides other than 5-hydroxymethyluridine, wherein the one or more modified ribonucleotides comprise a nucleoside selected from adenosine, inosine, guanosine, cytidine, or uridine, or any combination thereof.

[0180] In some embodiments, the nucleoside of one or more modified ribonucleotides is N4-acetylcytidine, and the modified ribonucleotide has the following structure, where R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate: [ka]

[0181] Also provided herein are polypeptides encoded by the polynucleotides disclosed herein.

[0182] The present disclosure further provides compositions comprising one or more polyribonucleotides disclosed herein or polypeptides disclosed herein.

[0183] In some embodiments, the composition comprises a plurality of polyribonucleotides disclosed herein. In some embodiments, each of the plurality of polyribonucleotides comprises one or more methanogen antigens. In some embodiments, each of the plurality of polyribonucleotides encodes a different methanogen antigen. In some embodiments, each of the plurality of polyribonucleotides encodes the same methanogen antigen.

[0184] In some embodiments of a composition comprising a plurality of polyribonucleotides, the plurality of polyribonucleotides comprises a mixture of polyribonucleotides, for example, a mixture of portions encoding the same methanogen antigen and portions encoding different methanogen antigens.

[0185] In some embodiments, a composition disclosed herein comprises one or more polynucleotides comprising one or more rumen-associated antigens, e.g., methanogen antigens. In some embodiments, a composition (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises multiple polynucleotides (e.g., multiple RNAs), each comprising an antigen. In some embodiments, a composition (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises multiple polynucleotides (e.g., multiple RNAs), each comprising one or more antigens (e.g., the same or different antigens).

[0186] In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 1% of the first polynucleotide and about 99% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 5% of the first polynucleotide and about 95% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 10% of the first polynucleotide and about 90% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 20% of the first polynucleotide and about 80% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 30% of the first polynucleotide and about 70% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 40% of the first polynucleotide and about 60% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 50% of the first polynucleotide and about 50% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 60% of the first polynucleotide and about 40% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 70% of the first polynucleotide and about 30% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 80% of the first polynucleotide and about 20% of one or more additional polynucleotides.In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 90% of the first polynucleotide and about 10% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 99% of the first polynucleotide and about 1% of the one or more additional polynucleotides.

[0187] In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multi-component composition, e.g., a multi-component vaccine composition) comprises about 33% first polynucleotide, about 33% second polynucleotide, and about 33% third polynucleotide.

[0188] In some embodiments, the composition is a pharmaceutical composition.

[0189] In some embodiments, the composition is an immunogenic composition.

[0190] In some embodiments, the composition is a vaccine composition.

[0191] In some embodiments, the composition is formulated for delivery by a carrier, hi some embodiments, the carrier is a lipid nanoparticle, a cationic lipid, or a polymer particle.

[0192] In some embodiments, the composition is formulated for delivery without a carrier.

[0193] The present disclosure provides methods that include administering a composition disclosed herein to a cell, tissue, or animal, such as a ruminant.

[0194] In some embodiments, the method is a method of vaccination.

[0195] In some embodiments, the animal is a ruminant, such as a cow, sheep, goat, buffalo, elk, antelope, caribou, or deer.

[0196] In some embodiments, the animal is a livestock animal.

[0197] In some embodiments, the vaccine reduces methane emissions from an animal compared to animals that are not administered the vaccine or that are administered a different vaccine.

[0198] In some embodiments, the composition is characterized in that administration of the composition to an animal reduces methane emissions from the animal compared to an otherwise similar animal not administered the composition or administered a different composition, hi some embodiments, the reduction in methane emissions compared to an otherwise similar animal not administered the composition or administered a different composition is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0199] In some embodiments, the composition is characterized in that administration of the composition to an animal reduces the population of microorganisms in the animal compared to an otherwise identical animal not administered the composition or administered a different composition. In some embodiments, the microorganism is a methanogen. In some embodiments, the methanogen comprises a methanogen from one or more of the following phylogenetic groups: Methanobrevibacter, Methanosphaera, Methanobacterium, Methanosarcinales, Methanomicrobiales, Methanothermobacter, Candidatus, Methanomethylophilus, Thermoplasmatales.

[0200] In some embodiments, the methanogen is Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp.UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans or any combination thereof.

[0201] In some embodiments, the composition is characterized in that administration of the composition to an animal increases the growth rate of the animal compared to the growth rate of an otherwise similar animal not administered the composition or administered a different composition, in some embodiments, the increase in growth rate comprises daily weight gain of the animal, optionally wherein the daily weight gain of the animal is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% increase in body weight compared to an otherwise similar animal not administered the composition or administered a different composition.

[0202] In some embodiments, the methods disclosed herein comprise administering a single dose of a vaccine composition to an animal.

[0203] In some embodiments, the methods disclosed herein comprise administering multiple doses of a vaccine composition to an animal.

[0204] In some embodiments, the animal is administered a first dose of the composition, followed by one or more subsequent doses of the composition.

[0205] In some embodiments, the first dose and one or more subsequent doses of the composition comprise the same methanogen antigen and / or rumen antigen.

[0206] In some embodiments, the first dose and one or more subsequent doses of the composition comprise different methanogen antigens and / or rumen antigens.

[0207] In some embodiments, the composition is administered to the animal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 times.

[0208] In some embodiments, the composition is administered once every week, every two weeks, three weeks, one month, two months, three months, four months, five months, or six months.

[0209] In some embodiments, the composition is administered in combination with one or more additional agents.

[0210] In some embodiments, the one or more additional agents include chemical additives, biological feed additives.

[0211] In some embodiments, the composition is administered in combination with one or more additional compositions.

[0212] In some embodiments, the additional composition immunizes the animal against a disease, eg, an infectious disease.

[0213] Also disclosed herein are compositions comprising the isolated polynucleotides disclosed herein, or pharmaceutical compositions disclosed herein, for use in administration to an animal (e.g., vaccination).

[0214] The present disclosure also provides the use of a composition comprising an isolated polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein, in the preparation of a medicament for administration (e.g., vaccination) to an animal.

[0215] In some embodiments of any of the compositions for use or uses disclosed herein, the isolated polynucleotide or pharmaceutical composition is administered to an animal.

[0216] In some embodiments of any of the compositions for use or uses disclosed herein, administration of the isolated polynucleotide or pharmaceutical composition results in: a reduction in the risk of rhesus macular degeneration compared to an otherwise identical animal not administered the composition, or administered a different composition

[0217] (i) reduction in methane emissions;

[0218] (ii) a reduction in the abundance of one or more microorganisms (e.g., methanogens) in the animal's gastrointestinal tract; and / or

[0219] (iii) resulting in an increase in the growth rate of animals.

[0220]

[0221] In some embodiments of any of the compositions for use or uses disclosed herein, the animal is a ruminant or livestock.

[0222] In some embodiments of any of the compositions for use or uses disclosed herein, the methanogen is selected from the group consisting of Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp. UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, and Methanocorpusculum. labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans or any combination thereof. [Brief explanation of the drawings]

[0223] [Figure 1A] Antigen-specific IgG titers from serum were shown for an exemplary multicomponent RNA vaccine formulation. Experimental groups consisted of calves receiving one dose (day 20, n=5), two doses (days 20 and 34, n=5), or no treatment / untreated (UTD, n=5) of an exemplary RNA vaccine formulation [50% OVA_HNadj (85% HNmod1, where HNmod1 corresponds to Ac4C) and 50% mru1499_HNadj (85% HNmod1) totaling 0.5 mg]. Serum was collected on days 20, 34, and 90. ELISA was performed for each antigen (OVA, mru1499) of the multicomponent vaccine, respectively. Geometric mean reciprocal titers are provided for the one-dose and two-dose groups, for each time point, and for each time point for measuring OVA-IgG (Figure 1A) and mru1499-IgG (Figure 1B) responses to exemplary RNA vaccines in cattle. [Figure 1B] Antigen-specific IgG titers from serum were shown for an exemplary multicomponent RNA vaccine formulation. Experimental groups consisted of calves receiving one dose (day 20, n=5), two doses (days 20 and 34, n=5), or no treatment / untreated (UTD, n=5) of an exemplary RNA vaccine formulation [50% OVA_HNadj (85% HNmod1, where HNmod1 corresponds to Ac4C) and 50% mru1499_HNadj (85% HNmod1) totaling 0.5 mg]. Serum was collected on days 20, 34, and 90. ELISA was performed for each antigen (OVA, mru1499) of the multicomponent vaccine, respectively. Geometric mean reciprocal titers are provided for the one-dose and two-dose groups, for each time point, and for each time point for measuring OVA-IgG (Figure 1A) and mru1499-IgG (Figure 1B) responses to exemplary RNA vaccines in cattle.

[0224] [Figure 2]1 shows the normalized relative abundance of methanogen species in ruminal fluid for exemplary multi-component RNA vaccine formulations. Experimental groups consisted of calves receiving one dose (day 20, n=5), two doses (days 20 and 34, n=5), or no treatment / untreated (UTD, n=5) of an exemplary RNA vaccine formulation [0.5 mg total of 50% OVA_HNadj (85% HNmod1) and 50% mru1499_HNadj (85% HNmod1)]. Ruminal fluid was collected on days 0, 20, 34, and 90. Samples were sequenced to obtain relative abundance measurements of the methanogen Methanobrevibacter (Mbb.) ruminantium M1 in the one-dose and two-dose groups for each time point in response to the exemplary RNA vaccine in cattle. The value for each time point is the difference from the day 0 value for that group. The y-axis is the average pre-trial M1 / Archaeum for the cohort, normalized to 1.0.

[0225] [Figure 3A] Figure 3 shows the normalized relative abundance of methanogen and archaeal species in ruminal fluid for exemplary RNA vaccine formulations targeting methanogen proteins and containing various secretion signals. Experimental groups consisted of groups of calves (n=4) that received a prime / boost dose of a particular RNA vaccine formulation (0.5 mg, 100% HNmod1). Ruminal fluid was collected on days 0, 20, 34, and 90 and processed into pellets. Samples were sequenced to obtain relative abundance measurements of the methanogen Methanobrevibacter (Mbb.) ruminantium M1 (Figure 3A) and total archaea (Figure 3B) for each formulation group in response to the exemplary RNA vaccine in cattle. Values ​​for each time point are the difference from the day 0 value for that group. The y-axis is the average pre-trial M1 / archaea for the cohort, normalized to 1.0. [Figure 3B]Figure 3 shows the normalized relative abundance of methanogen and archaeal species in ruminal fluid for exemplary RNA vaccine formulations targeting methanogen proteins and containing various secretion signals. Experimental groups consisted of groups of calves (n=4) that received a prime / boost dose of a particular RNA vaccine formulation (0.5 mg, 100% HNmod1). Ruminal fluid was collected on days 0, 20, 34, and 90 and processed into pellets. Samples were sequenced to obtain relative abundance measurements of the methanogen Methanobrevibacter (Mbb.) ruminantium M1 (Figure 3A) and total archaea (Figure 3B) for each formulation group in response to the exemplary RNA vaccine in cattle. Values ​​for each time point are the difference from the day 0 value for that group. The y-axis is the average pre-trial M1 / archaea for the cohort, normalized to 1.0.

[0226] [Figure 4] Figure 1 shows the normalized relative abundance of methanogen and archaeal species in ruminal fluid for an exemplary RNA vaccine formulation containing cytokines and dual chemical modifications (HNmod1 / HNmod2, where HNMod1 corresponds to Ac4C and HNMOD2 corresponds to 5hmU). Experimental groups consisted of groups of calves (n=4) receiving a prime / boost dose of a particular RNA vaccine formulation (0.5 mg, each composition noted in the figure). The experimental setup is similar to Figure 3, except the RNA vaccine formulation contains cytokines and dual chemical modifications (HNmod1 / HNmod2) characteristic. Note that n=1 for F8.

[0227] [Figure 5] Figure 1 shows methane output per feed intake for exemplary RNA vaccine formulations containing various secretory signals targeting methanogenic proteins. Experimental groups consisted of groups of calves (n=4) receiving a prime / boost dose of a particular RNA vaccine formulation (0.5 mg, 100% HNmod1; respective compositions noted in the figure). Enteric methane output was measured for each animal as average daily CH4 (g / d) per total daily feed intake (lbs / d) and then averaged over the 8-day measurement time frame.

[0228] [Figure 6] Figure 1 shows methane emissions per feed intake for exemplary RNA vaccine formulations containing cytokines and a single (HNmod1) or dual chemical modification (HNmod1, HNmod2). Experimental groups consisted of groups of calves (n=4) receiving a prime / boost dose of a particular RNA vaccine formulation (0.5 mg; respective composition noted in the figure). The experimental setup is similar to Figure 5, except the RNA vaccine formulation includes a cytokine signature. Note that n=3 for group F5 and n=2 for group F7 due to calf non-adherence to the GreenFeed Systems.

[0229] [Figure 7A]Figure 1 shows the normalized relative abundance of all archaeal species in ruminal fluid for an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins. Experimental groups consisted of groups of calves (n=10) receiving three doses of a particular RNA vaccine formulation (0.55 mg, 100% HNmod1; respective compositions listed in Table 3). Ruminal fluid was collected and processed into pellets on days 90, 109, and 122, with day 90 representing a pre-injection (before the third injection) sample ("PRE," 14 days before the booster), day 109 representing a 5-day post-injection (after the third injection) sample ("+5d POST"), and day 122 representing an 18-day post-injection (after the third injection) sample ("+18d POST"). In this study, gastric fluid was not collected after the first and second injections to minimize disturbance of the ruminal biome. Samples were sequenced to obtain relative abundance measurements (e.g., % of the total sequence population) for all bacterial and archaeal species that responded to the RNA vaccine formulations CNT(OVA) and F3. The bacterial and archaeal species examined included M. wolinii, M. stadtmanae, M. oralis, M. smithii, and M. ruminantium. Figure 7A shows the relative abundance levels of all archaeal species identified at time points PRE, +5-d POST, and +18-d POST. The "other" category includes species with relative abundances below 0.01% and includes M. mazei, M. soligelidi, M. arboriphilus, Thermoplasmatales sp., M. formicicum, M. boviskoreani, M. mobile, Methanothermobacter sp., Candidatus, Methanomethylophilus alvus, and Nitrososphaera sp. Figure 7B shows the percent change of each archaeal species relative to the PRE time point, normalized to CNT (OVA). [Figure 7B]Figure 1 shows the normalized relative abundance of all archaeal species in ruminal fluid for an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins. Experimental groups consisted of groups of calves (n=10) receiving three doses of a particular RNA vaccine formulation (0.55 mg, 100% HNmod1; respective compositions listed in Table 3). Ruminal fluid was collected and processed into pellets on days 90, 109, and 122, with day 90 representing a pre-injection (before the third injection) sample ("PRE," 14 days before the booster), day 109 representing a 5-day post-injection (after the third injection) sample ("+5d POST"), and day 122 representing an 18-day post-injection (after the third injection) sample ("+18d POST"). In this study, gastric fluid was not collected after the first and second injections to minimize disturbance of the ruminal biome. Samples were sequenced to obtain relative abundance measurements (e.g., % of the total sequence population) for all bacterial and archaeal species that responded to the RNA vaccine formulations CNT(OVA) and F3. The bacterial and archaeal species examined included M. wolinii, M. stadtmanae, M. oralis, M. smithii, and M. ruminantium. Figure 7A shows the relative abundance levels of all archaeal species identified at time points PRE, +5-d POST, and +18-d POST. The "other" category includes species with relative abundances below 0.01% and includes M. mazei, M. soligelidi, M. arboriphilus, Thermoplasmatales sp., M. formicicum, M. boviskoreani, M. mobile, Methanothermobacter sp., Candidatus, Methanomethylophilus alvus, and Nitrososphaera sp. Figure 7B shows the percent change of each archaeal species relative to the PRE time point, normalized to CNT (OVA).

[0230] [Figure 8A]Figure 8 shows raw methane output and methane output per feed intake for an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins. Experimental groups consisted of groups of calves (n = 10) receiving a prime / boost dose of a specific RNA vaccine formulation (0.55 mg, 100% HNmod1; respective compositions listed in Table 3). Enteric methane output was measured for each animal and averaged as mean daily CH4 (g / d) per total daily feed intake (lbs / d) over a 14-day time window (T1) after the prime injection (D0-D14) and a baseline (T0) before injection (D-8-D-2). The differential methane percentage (Figure 8A) and methane / intake (Figure 8B) were determined by dividing the raw methane or methane / intake value for the later time window (T1) by the baseline (T0) value, and then normalized by measuring the difference in this percentage for each treatment group relative to that of the control group (CNT-OVA). Error bars reflect standard error. [Figure 8B] Figure 8 shows raw methane output and methane output per feed intake for an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins. Experimental groups consisted of groups of calves (n = 10) receiving a prime / boost dose of a specific RNA vaccine formulation (0.55 mg, 100% HNmod1; respective compositions listed in Table 3). Enteric methane output was measured for each animal and averaged as mean daily CH4 (g / d) per total daily feed intake (lbs / d) over a 14-day time window (T1) after the prime injection (D0-D14) and a baseline (T0) before injection (D-8-D-2). The differential methane percentage (Figure 8A) and methane / intake (Figure 8B) were determined by dividing the raw methane or methane / intake value for the later time window (T1) by the baseline (T0) value, and then normalized by measuring the difference in this percentage for each treatment group relative to that of the control group (CNT-OVA). Error bars reflect standard error.

[0231] [Figure 9]Figure 1 shows growth efficiency measurements for exemplary RNA vaccine formulations targeting methanogen proteins and containing various secretion signals, cytokines, and single (HNmod1) or dual chemical modifications (HNmod1, HNmod2). Experimental groups consisted of groups of calves (n=4) receiving prime / boost doses of a particular RNA vaccine formulation (0.5 mg; compositions correspond to those described in Figures 5 and 6). Average daily weight gain (ADG, lbs / day), which reflects growth efficiency with higher values ​​indicating higher efficiency, was calculated by linear regression between the time periods of weight measurements. Specifically, "pre-injection baseline" is the average daily weight gain from D-32 to D0 (e.g., 32 days before injection), "D0-D20" is the average daily weight gain from D0 to D20 (e.g., 20 days after the primary injection), and "D20-D90" is the average daily weight gain from D20 to D90 (e.g., 70 days after the booster injection). The delta ADG represents the difference between the ADG and the pre-injection baseline for each treatment group over a specific time frame, and is then normalized by finding the difference in this delta to the control group (UTD). Not shown is vaccine condition F8 (n=1), where the change in ADG at D20 was +1.70 lb / day and the change in ADG at D90 was +0.91 lb / day.

[0232] [Figure 10A]Figure 10 shows growth efficiency measurements for an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins. Experimental groups consisted of groups of calves (n=10) receiving a prime / boost dose of a particular RNA vaccine formulation (0.55 mg, 100% HNmod1; respective compositions listed in Table 3). Average daily weight gain (ADG, lb / day), reflecting growth efficiency with higher values ​​indicating higher efficiency, was calculated by linear regression between the time periods of weight measurements (Figure 10A). Specifically, "pre-injection baseline" is the average daily weight gain from D-49 to D0 (e.g., 50 days before injection), "D25" is the average daily weight gain from D0 to D25 (e.g., 25 days after the prime injection), and "D90" is the average daily weight gain from D-25 to D90 (e.g., 65 days after the boost injection). Figure 10B shows differential ADG in pounds per day (lb / d), representing the difference between ADG and pre-infusion baseline for a specific time frame for each treatment group, normalized by taking the difference between this and the control group (CNT-OVA). The right panel shows differential ADG in percentage (%), representing the ratio of the later time to the earlier time, normalized by taking the difference between this and the control group (CNT-OVA). [Figure 10B]Figure 10 shows growth efficiency measurements for an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins. Experimental groups consisted of groups of calves (n=10) receiving a prime / boost dose of a particular RNA vaccine formulation (0.55 mg, 100% HNmod1; respective compositions listed in Table 3). Average daily weight gain (ADG, lb / day), reflecting growth efficiency with higher values ​​indicating higher efficiency, was calculated by linear regression between the time periods of weight measurements (Figure 10A). Specifically, "pre-injection baseline" is the average daily weight gain from D-49 to D0 (e.g., 50 days before injection), "D25" is the average daily weight gain from D0 to D25 (e.g., 25 days after the prime injection), and "D90" is the average daily weight gain from D-25 to D90 (e.g., 65 days after the boost injection). Figure 10B shows differential ADG in pounds per day (lb / d), representing the difference between ADG and pre-infusion baseline for a specific time frame for each treatment group, normalized by taking the difference between this and the control group (CNT-OVA). The right panel shows differential ADG in percentage (%), representing the ratio of the later time to the earlier time, normalized by taking the difference between this and the control group (CNT-OVA). DETAILED DESCRIPTION OF THE INVENTION

[0233] Specific Definitions About or Approximately: As used herein, the terms "about" and "approximately," when used herein with respect to a value, refer to a similar value in the context of a reference value. Generally, a person of ordinary skill in the art familiar with the context will understand the reasonable degree of variation encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" or "approximately" may encompass a range of values ​​that is 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0234] Administering: As used herein, the terms "administering" or "administration" generally refer to administering a composition to an animal to achieve delivery of an agent that is or is contained in the composition. Those of skill in the art will recognize various routes that may be utilized for administration to animals, e.g., ruminants, in appropriate circumstances. In some embodiments, administration may comprise only a single dose. In some embodiments, administration may comprise the application of a number of doses. In some embodiments, administration may comprise administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) administration. In some embodiments, administration may comprise continuous administration (e.g., perfusion) for at least a selected period of time.

[0235] Antigen: As used herein, the term "antigen" refers to a chemical substance that elicits an immune response and / or (ii) binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., involving the specific interaction of a T cell receptor with the antigen). In some embodiments, an antigen comprises at least one epitope of a target protein. In some embodiments, the epitope may be a linear epitope. In some embodiments, the epitope may be a conformational epitope. In some embodiments, an antigen binds to an antibody and may or may not elicit a specific physiological response in an organism. Generally, an antigen may be or include any chemical substance, such as a small molecule, nucleic acid, polypeptide, carbohydrate, lipid, polymer (in some embodiments, other than a biological polymer [e.g., other than a nucleic acid or amino acid polymer]), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, the antigen is or comprises a glycan. Those skilled in the art will generally understand that antigens can be provided in isolated or pure form, or alternatively in crude form (e.g., together with other materials, such as cell extracts or other relatively crude preparations of antigen-containing sources). In some embodiments, the antigens utilized in accordance with the present invention are provided in crude form. In some embodiments, the antigen is a recombinant antigen.

[0236] Delivery / Contacting: As used interchangeably herein, the terms "delivery," "delivering," or "contacting" refer to the introduction of a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell. The target cell may be cultured in vitro or ex vivo, or may reside in an animal (in vivo). The method of introducing a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell may vary depending on the in vitro, ex vivo, or in vivo application. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) may be introduced into a target cell in cell culture by in vitro transfection. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) may be introduced into a target cell via a delivery vehicle (e.g., complexed with a nanoparticle, a liposome, and / or a cell-penetrating agent). In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in an animal by administering the fusion polynucleotide (e.g., as described herein) or the fusion polypeptide (e.g., as described herein) to the animal.

[0237] Functional: As used herein, the term "functional" is used to refer to forms or fragments of an entity that exhibit particular properties and / or activities.

[0238] Fragment: A "fragment" of a material or entity described herein comprises a discrete portion of the whole, but has a structure that lacks one or more portions found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, a fragment comprises a polynucleotide fragment. In some embodiments, a fragment comprises a polypeptide fragment. In some embodiments, a polynucleotide or polypeptide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) found throughout the entire polynucleotide or polypeptide. In some embodiments, a polynucleotide or polypeptide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomer units (e.g., residues) found in the entire polynucleotide or polypeptide. The entire polypeptide or polynucleotide may, in some embodiments, be referred to as the "parent" of the polynucleotide or polypeptide fragment.

[0239] Methanogen: As used herein, a "methanogen" is a microorganism that produces methane. In some embodiments, methanogens can inhabit the digestive tract of animals and participate in fermentation processes. In some embodiments, methanogens are organisms that conserve energy for ATP synthesis by producing methane gas. In some embodiments, methanogens can inhabit the digestive tract of ruminants. In some embodiments, methanogens can inhabit the digestive tract of non-ruminants. Exemplary methanogens are provided in Buan NR (2018), Emerg. Top life Sci. 2(4): pp. 629-646, the entire contents of which are incorporated herein by reference. In some embodiments, the methanogens comprise one or more methanogens from one or more of the following phylogenetic groups: Methanobrevibacter, Methanosphaera, Methanobacterium, Methanosarcinales, Methanomicrobiales, Methanothermobacter, Candidatus, Methanomethylophilus, Thermoplasmatales.In some embodiments, the methanogen is Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp.UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans or any combination thereof.

[0240] Nucleic Acid / Oligonucleotide / Polynucleotide: As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a polymer of three or more nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single-stranded. In some embodiments, a nucleic acid is double-stranded. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, e.g., "peptide nucleic acids." In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) relative to that of the natural residue.In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template in vivo or in vitro, replication in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid has at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 350 0, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500 , 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length. When the number of nucleotides is used, for example, as an indicator of the size of a fusion polynucleotide, a particular number of nucleotides refers, for example, to the number of nucleotides on one strand of the fusion polynucleotide.

[0241] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three or more amino acids. Those of skill in the art will understand that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides representing functional, biologically active, or characteristic fragments, portions, or domains of such complete polypeptides (e.g., fragments, portions, or domains that retain at least one activity). Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known to those of skill in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, polypeptides may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0242] Polyribonucleotide: As used herein, the term "polyribonucleotide" refers to a polymer of three or more ribonucleotides. In some embodiments, a polyribonucleotide is single-stranded. In some embodiments, a polyribonucleotide is double-stranded. In some embodiments, a polyribonucleotide includes both single-stranded and double-stranded portions. In some embodiments, a polyribonucleotide can include a backbone structure described above in the definition of "nucleic acid / oligonucleotide." A polyribonucleotide can be a regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA) oligonucleotide. In some embodiments, a polyribonucleotide typically includes a poly(A) region at its 3' end. In some embodiments, a polyribonucleotide typically includes an art-recognized cap structure at its 5' end, e.g., for RNA recognition and ribosome binding to initiate translation. In some embodiments, a polyribonucleotide includes an RNA oligonucleotide. When the number of ribonucleotides is used, for example, as an indicator of the size of a polynucleotide, the specified number of nucleotides refers to the number of ribonucleotides on a single strand.

[0243] Ruminant-associated antigen: As used herein, a "rumen-associated antigen" is an antigen expressed in a ruminant. In some embodiments, the rumen-associated antigen is a "rumen antigen," e.g., an antigen encoded by a nucleotide sequence naturally occurring in the genome of a ruminant. In some embodiments, the rumen-associated antigen is not naturally occurring in the genome of a ruminant, e.g., is encoded by a nucleotide sequence that has been introduced into the genome of a ruminant, or is part of a microorganism that can inhabit the digestive tract, e.g., the rumen of a ruminant. In some embodiments, the rumen-associated antigen comprises a methanogen antigen, an antigen derived from a ciliated symbiont of ruminal methanogens; an antigen derived from a hydrogen-producing organism; an antigen derived from a taxon associated with low feed efficiency; or any combination thereof. In some embodiments, the rumen-associated antigen is or comprises a methanogen.

[0244] Variant: As used herein, the term "variant" refers to an entity that exhibits sufficient structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties relative to the reference entity. In many embodiments, a variant also differs functionally from the reference entity. Generally, whether a particular entity is properly considered a "variant" of a reference entity is based on the degree of structural identity with the reference entity. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a reference polypeptide possesses one or more biological activities. In some embodiments, a variant polypeptide shares one or more biological activities of a reference polypeptide. In some embodiments, a variant polypeptide lacks one or more biological activities of a reference polypeptide. In some embodiments, a variant polypeptide exhibits a reduced level of one or more biological activities relative to a reference polypeptide.

[0245] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., RNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The above-described techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and described throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose.

[0246] This disclosure identifies that increased agricultural methane represents a major source of greenhouse gas emissions. This disclosure also confirms that reducing methane emissions from animals, e.g., ruminants, will be important for reducing greenhouse gas emissions.

[0247] Methane is produced in the rumen of ruminants by methanogens, a subgroup of the Archaea. Because methane production by methanogens can account for 2-12% of ingested energy, methanogens can reduce the efficiency of nutrients and / or energy available to ruminants (see Leahy SC et al., (2010) PlosOne; volume 5, issue 1; e8926, the entire contents of which are incorporated by reference). Thus, the present disclosure identifies that reducing methane emissions from ruminants can be beneficial by increasing the energy efficiency of ruminants and reducing methane emissions to the atmosphere.

[0248] Among other things, the present disclosure provides techniques for reducing methane emissions from animals, e.g., ruminants, by providing compositions for administering a rumen-associated antigen to an animal, e.g., a ruminant, (e.g., vaccinating an animal (e.g., a ruminant) against a rumen-associated antigen). The present disclosure also provides techniques for increasing the energy efficiency of an animal, e.g., a ruminant, by administering a rumen-associated antigen to an animal, e.g., a ruminant, (e.g., vaccinating an animal (e.g., a ruminant) against a rumen-associated antigen), thereby reducing the abundance of one or more methanogens in the rumen of the animal, e.g., the ruminant.

[0249] Without wishing to be bound by theory, the present disclosure proposes that compositions comprising rumen-associated antigens (e.g., rumen antigens and / or methanogen antigens) can reduce methane emissions and / or reduce the abundance of microorganisms (e.g., methanogens) in the digestive tract of an animal. In some embodiments, reducing methane emissions and / or reducing the abundance of methanogens in the digestive tract of an animal (e.g., a ruminant) can increase the energy efficiency of the animal. As a result, the animal (e.g., a ruminant) may require less food, experience increased muscle mass, and / or improve overall health. These benefits may translate into lower costs for raising the animal and / or increased profits from the sale of the animal or meat derived from such animals.

[0250] rumen-associated antigens Ruminant-associated antigens described herein include rumen antigens and / or methanogen antigens. In some embodiments, the rumen-associated antigen is endogenous to the ruminant, e.g., encoded by a nucleotide sequence within the ruminant genome. In some embodiments, the rumen-associated antigen is not endogenous to the ruminant, e.g., encoded by a nucleotide sequence introduced into the ruminant, or encoded by a nucleotide sequence derived from a microorganism present in or introduced into the ruminant.

[0251] In some embodiments, the ruminant is selected from cattle, sheep, goats, buffalo, elk, antelope, caribou, or deer, or combinations thereof.

[0252] Exemplary rumen-associated antigens include peptides involved in adhesion to bacteria, eg, fermenting bacteria, and fragments or variants thereof.

[0253] In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 rumen antigens.

[0254] In some embodiments, the polynucleotides are about 2 to 100, about 3 to 100, about 4 to 100, about 5 to 100, about 6 to 100, about 7 to 100, about 8 to 100, about 9 to 100, about 10 to 100, about 20 to 100, about 30 to 100, about 40 to 100, about 50 to 100, about 60 to 100, about 70 to 100, about 80 to 100, The antigens include about 90-100, about 2-90, about 2-80, about 2-70, about 2-60, about 2-50, about 2-40, about 2-30, about 2-20, about 2-10, about 2-15, about 2-14, about 2-13, about 2-12, about 2-11, about 2-10, about 2-9, about 2-8, about 2-7, about 2-6, about 2-5, about 2-4, and about 2-3 rumen antigens.

[0255] Methanogens and methanogen antigens Methanogens are anaerobic archaea characterized by their ability to conserve energy for ATP synthesis by producing methane gas, as described in Buan NR (2018). Methanogens can also be present in the digestive tracts of animals, such as ruminants and humans. Because methane production by methanogens can account for 2–12% of ingested energy, methanogens can reduce the efficiency of nutrients and / or energy available to ruminants (see Leahy SC et al., (2010) PlosOne; volume 5, issue 1; e8926, the entire contents of which are incorporated by reference). Therefore, reducing methane emissions from ruminants can be beneficial by increasing the energy efficiency of ruminants and / or reducing methane emissions to the atmosphere.

[0256] In some embodiments, the methanogens include methanogens from one or more of the following phylogenetic groups: Methanobrevibacter, Methanosphaera, Methanobacterium, Methanosarcinales, Methanomicrobiales, Methanothermobacter, Candidatus, Methanomethylophilus, Thermoplasmatales.

[0257] In some embodiments, the methanogens comprise methanogens from the Methanobrevibacter phylogenetic group. In some embodiments, the methanogens from the Methanobrevibacter phylogenetic group comprise Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter thaueri, Methanobrevibacter gottschalkii, Methanobrevibacter millerae, Methanobrevibacter woesei, Methanobrevibacter arboriphilus, Methanobrevibacter wolinii, Methanobrevibacter olleyae, Methanobrevibacter boviskoreani, or a combination thereof.

[0258] In some embodiments, the methanogens comprise methanogens from the Methanosphaera phylogenetic group. In some embodiments, the methanogens from the Methanosphaera phylogenetic group comprise Methanosphaera stadtmanae.

[0259] In some embodiments, the methanogens comprise methanogens from the Methanobacterium phylogenetic group. In some embodiments, the methanogens from the Methanobacterium phylogenetic group comprise Methanobacterium bryantii or Methanobacterium formicicum.

[0260] In some embodiments, the methanogens comprise methanogens from the Methanosarcinales phylogenetic group. In some embodiments, the methanogens from the Methanosarcinales phylogenetic group comprise Methanosarcina bakeri, Methanosarcina mazei, or Methanosarcina soligelidi.

[0261] In some embodiments, the methanogens comprise methanogens from the Methanomicrobiales phylogenetic group. In some embodiments, the methanogens from the Methanomicrobiales phylogenetic group comprise Methanofollis liminatans, Methanospirillum hungatei, Methanolacinia payteri, Methanoculleus marisngigri, or Methanoculleus sp.

[0262] In some embodiments, the methanogens comprise methanogens from the Methanomicrobium phylogenetic group. In some embodiments, the methanogens from the Methanomicrobium phylogenetic group comprise Methanomicrobium mobile.

[0263] In some embodiments, the methanogen comprises a methanogen from the Thermoplasmatales phylogenetic group. In some embodiments, the methanogen from the Thermoplasmatales phylogenetic group comprises Thermoplasma volcanium or Thermoplasma acidophilum.

[0264] In some embodiments, the methanogen is Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp.UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans or any combination thereof.

[0265] Methanogen antigens include any protein expressed or produced in any methanogen phylogenetic group, for example, as described herein. Exemplary methanogen antigens are provided in Table 1. In some embodiments, the methanogen antigen is an antigen provided in Table 1, or a fragment thereof, or a variant thereof. In some embodiments, the methanogen antigen has a sequence that is at least 85% identical to a methanogen antigen provided in Table 1. In some embodiments, the methanogen antigen is an adhesin, e.g., mru1499.

[0266] In some embodiments, the methanogen antigen is a peptide involved in adhesion, adherence, motility, or any combination thereof, hi some embodiments, the methanogen antigen is an adhesin, a pili protein, a flagellin protein, or any combination thereof.

[0267] In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 methanogen antigens.

[0268] In some embodiments, the polynucleotides are about 2 to 100, about 3 to 100, about 4 to 100, about 5 to 100, about 6 to 100, about 7 to 100, about 8 to 100, about 9 to 100, about 10 to 100, about 20 to 100, about 30 to 100, about 40 to 100, about 50 to 100, about 60 to 100, about 70 to 100, about 80 to 100, about and about 2-90, about 2-80, about 2-70, about 2-60, about 2-50, about 2-40, about 2-30, about 2-20, about 2-10, about 2-15, about 2-14, about 2-13, about 2-12, about 2-11, about 2-10, about 2-9, about 2-8, about 2-7, about 2-6, about 2-5, about 2-4, and about 2-3 methanogen antigens.

[0269] In some embodiments, the polynucleotide comprises one or more methanogen antigens derived from one or more methanogens.

[0270] In some embodiments, the methanogen antigen comprises an antigenic sequence presented in Table 2, or a fragment or variant or variant fragment thereof. In some embodiments, the methanogen antigen comprises an antigenic sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence presented in Table 2. Exemplary antigenic sequences presented in Table 2 are not shown in bold or italics.

[0271] In some embodiments, the methanogen antigen is selected from the group consisting of SEQ ID NOs: 2, 6, 8, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 440, 441, 442, 443, 444, 445, 446, 447, 449, 51 09, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or a sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. Antigen sequences are not shown (not bold or italicized) in SEQ ID NOs: 2, 6, 8, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 98, 99, 100, 101, 102, 103, 14, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122.

[0272] In some embodiments, the methanogen antigen comprises an antigen encoded by an antigen nucleotide sequence set forth in any one of SEQ ID NOs: 1, 5, 7, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, or 94, or a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0273] [Table 1-1] [Table 1-2] [Table 1-3]

[0274] Antigens derived from hydrogen-producing symbionts In some embodiments, the rumen-associated antigen comprises an antigen from a hydrogen-producing symbiont, hi some embodiments, the hydrogen-producing symbiont is a microorganism that produces ruminal hydrogen in a manner that reduces the efficiency of the cow, similar to methanogens.

[0275] In some embodiments, the rumen-associated antigen comprises an antigen from rumen ciliates. In some embodiments, the rumen ciliates are symbiotic with methanogens. In some embodiments, the rumen ciliates comprise Diplodinium dentatum (synonym: Diplodinium denticulatum), Diploplastron affine (synonym: Eudiplodinium affine), Enoploplastron triloricatum (synonym: Ostracodinium triloricatum), Entodinium simplex, Entodinium caudatum, Entodinium longinucleatum, Epidinium ecaudatum, Eremoplastron bovis (synonym: Eudiplodinium neglectum), Eudiplodinium maggii, Ostracodinium obtusum, Polyplastron multivesiculatum, or a combination thereof.

[0276] In some embodiments, the hydrogen-producing symbiont provides nutrients and / or energy (e.g., hydrogen) to the methanogens. In some embodiments, the hydrogen-producing symbiont providing nutrients and / or energy (e.g., hydrogen) to the methanogens comprises Butyrivibrio proteoclasticus, Bacteroides thetaiotaomicron, Ruminococcus flavefaciens, Ruminococcus albus, or a combination thereof.

[0277] In some embodiments, the hydrogen-producing symbiont comprises a taxon associated with low feed efficiency. In some embodiments, the hydrogen-producing symbiont comprises a taxon associated with low feed efficiency. In some embodiments, the hydrogen-producing symbiont comprises a taxon associated with low feed efficiency. The taxon includes Veillonellaceae, Prevotellaceae, Lachnospiraceae, Succinivibrionaceae, Fibrobacteraceae, Anaerovibrio, Clostridiales, Prevotella, and Ruminococcaceae.

[0278] Exemplary Multi-Component Compositions Comprising Ruminant-Associated Antigens Disclosed herein are polynucleotides (e.g., RNA) comprising one or more rumen-associated methanogens, e.g., rumen antigens and / or methanogen antigens. Also disclosed herein are compositions comprising same, as well as methods of making and using same for administration to (e.g., vaccination of) animals, e.g., ruminants. In some embodiments, the polynucleotides disclosed herein comprise multiple rumen-associated methanogens, e.g., multiple rumen antigens and / or multiple methanogen antigens. In some embodiments, compositions comprising such polynucleotides are also referred to as multicomponent vaccine compositions or multiplexed vaccine compositions.

[0279] In some embodiments, the polynucleotide is or comprises RNA. In some embodiments, the RNA comprises a 5' cap, e.g., a 5'-5' triphosphate-linked guanosine. In some embodiments, the RNA comprises a poly-A tail. In some embodiments, the RNA comprises one or more untranslated regions, e.g., a 5' UTR and / or a 3' UTR.

[0280] In some embodiments, the polynucleotide is RNA comprising: (i) a 5' cap; (ii) a 5' UTR region; (iii) a sequence encoding one or more payloads (e.g., one or more rumen-associated antigens); (iv) a polyA tail; and (v) a 3' UTR region. In some embodiments, the polynucleotide further comprises one or more elements, such as one or more sequences encoding additional payloads or one or more sequences capable of forming a secondary structure (e.g., a hairpin or IRES). In some embodiments, the polynucleotide is messenger RNA.

[0281] In some embodiments of the isolated polynucleotides disclosed herein, compositions comprising same, or methods of making or using the polynucleotides disclosed herein, the polynucleotide comprises one or more methanogen antigens. In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 methanogen antigens. In some embodiments, the one or more methanogen antigens are the same, e.g., antigens with the same sequence, structure, and / or function. In some embodiments, the one or more methanogen antigens are different, e.g., antigens with different sequence, structure, and / or function. In some embodiments, the one or more methanogen antigens comprise a mixture of the same and different antigens. In some embodiments, the one or more methanogen antigens are associated with one or more methanogen species. In some embodiments, the one or more methanogen antigens are associated with a single methanogen species.

[0282] In some embodiments, the one or more methanogen antigens include one or more peptides having at least 80% sequence identity to a methanogen protein; (ii) one or more secreted antigens, e.g., each comprising a signal peptide; (iii) a plurality of peptides having at least 80% sequence identity to each other; (iv) a plurality of peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; (v) one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity; (vi) one or more peptides having at least 80% sequence identity to an AglB polypeptide or having a function substantially similar to an AglB polypeptide; (vii) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having a function substantially similar to an Ig-like domain-containing polypeptide; or (viii) one or more, or all, or any combination of, one or more methanogen antigens listed in Table 1 or a sequence at least 85% identical thereto. In some embodiments, the polynucleotide comprises one or more methanogen antigens from each of (i)-(viii).

[0283] In some embodiments, the one or more methanogen antigens comprise one or more peptides having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a methanogen protein, hi some embodiments, the methanogen protein comprises a polypeptide encoded from a methanogen genome, or a variant or fragment thereof.

[0284] In some embodiments, the one or more methanogen antigens include one or more secreted antigens, for example, the one or more methanogen antigens include a signal peptide. In some embodiments, the secreted antigen is or includes an antigen that is a surface-associated protein or a protein secreted from the cell. In some embodiments, the secreted antigen includes a signal peptide. In some embodiments, the signal peptide can be predicted using an algorithm. In some embodiments, the signal peptide has a prediction score of at least 0.5.

[0285] In some embodiments, one or more methanogen antigens comprise multiple peptides that have at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to each other. In some embodiments, one or more methanogen antigens that comprise at least 80% identity to each other are also referred to as cluster homologous.

[0286] In some embodiments, the one or more methanogen antigens comprise a plurality of peptides associated with at least 2 different, at least 3 different, at least 4 different, at least 5 different, at least 6 different, at least 7 different, at least 8 different, at least 9 different, at least 10 different, at least 11 different, at least 12 different, at least 15 different, or at least 20 different methanogen species.

[0287] In some embodiments, the one or more methanogen antigens comprise a plurality of peptides associated with about 2 different, about 3 different, about 4 different, about 5 different, about 6 different, about 7 different, about 8 different, about 9 different, about 10 different, about 11 different, about 12 different, about 15 different, or about 20 different methanogen species.

[0288] In some embodiments, the one or more methanogen antigens comprise a plurality of peptides associated with no more than 2 different, no more than 3 different, no more than 4 different, no more than 5 different, no more than 6 different, no more than 7 different, no more than 8 different, no more than 9 different, no more than 10 different, no more than 11 different, no more than 12 different, no more than 15 different, or no more than 20 different methanogen species.

[0289] In some embodiments, the one or more methanogen antigens comprise a plurality of peptides associated with about 2 to about 20, about 2 to about 15, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 20, about 4 to about 20, about 5 to about 20, about 6 to about 20, about 7 to about 20, about 8 to about 20, about 9 to about 20, about 10 to about 20, about 11 to about 20, about 12 to about 20, or about 15 to about 20 different methanogen species.

[0290] In some embodiments, one or more methanogen antigens associated with a methanogen species refer to antigens that can be found in the methanogen species, e.g., a methanogen antigen associated with a particular methanogen species includes an antigen encoded by a nucleotide sequence that is naturally present in the methanogen species.

[0291] In some embodiments, the one or more methanogen antigens comprise methanogens from one or more or all of the following phylogenetic groups: Methanobrevibacter, Methanosphaera, Methanobacterium, Methanosarcinales, Methanomicrobiales, Methanothermobacter, Candidatus, Methanomethylophilus, Thermoplasmatales.

[0292] In some embodiments, the methanogenic bacterial species is selected from Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp. UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, or Methanosarcina acetivorans.

[0293] In some embodiments, the methanogen is selected from Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp. UBA188, or Methanosarcina soligelidi.

[0294] In some embodiments, the methanogen species is selected from Methanobrevibacter ruminantium, Methanobrevibacter smithii, or Methanobrevibacter oralis.

[0295] In some embodiments, the methanogen species is selected from Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, or Methanosphaera stadtmanae.

[0296] In some embodiments, the one or more methanogen antigens comprise multiple methanogen antigens each associated with the same methanogen species.

[0297] In some embodiments, the one or more methanogen antigens comprise one or more peptides having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a polypeptide having signal peptidase activity. In some embodiments, the polypeptide having signal peptidase activity has one or more (or all) of the following characteristics: (i) being membrane-bound; (ii) having the ability to cleave one or more signal peptides; and / or (iii) playing a role in converting a secreted protein to a mature form (e.g., from a non-secreted form to a secreted form).

[0298] In some embodiments, the one or more methanogen antigens comprise one or more peptides having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to an AglB polypeptide, or having a substantially similar function as an AglB polypeptide. In some embodiments, the AglB polypeptide is characterized by having N-glycosylation activity.

[0299] In some embodiments, the one or more methanogen antigens comprise one or more peptides having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to an Ig-like domain-containing polypeptide, or having a substantially similar function to an Ig-like domain-containing polypeptide. In some embodiments, the Ig-like domain-containing polypeptide comprises a polypeptide characterized as a cell surface protein that facilitates binding to other cells and / or cell surfaces.

[0300] The Ig-like domain-containing polypeptides described herein can include (i) a transglutaminase domain-containing protein or a fragment, mutant, or variant fragment thereof; (ii) a pseudomurein-binding repeat-containing protein or a fragment, mutant, or variant fragment thereof; (iii) a right-handed parallel beta-helical repeat-containing protein or a fragment, mutant, or variant fragment thereof; (iv) a succinylglutamate desuccinylase / aspartoacylase family protein or a fragment, mutant, or variant fragment thereof; or (v) any combination of (i)-(iv).

[0301] In some embodiments, the one or more methanogen antigens comprise one or more antigens set forth in Table 1, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical thereto. In some embodiments, the one or more methanogen antigens comprise an adhesin, or a fragment or variant thereof. In some embodiments, the one or more methanogen antigens comprise a pili protein, or a fragment or variant thereof. In some embodiments, the one or more methanogen antigens comprise a flagellin protein, or a fragment or variant thereof.

[0302] In some embodiments of the isolated polynucleotides disclosed herein, compositions comprising the same, or methods of making or using the polynucleotides disclosed herein, the polynucleotide comprises one or more methanogen antigens. In some embodiments, the polynucleotide comprises one or more methanogen antigens selected from one or more, all, or any combination of: (i) one or more peptides having at least 80% sequence identity to a methanogen protein; (ii) one or more secreted antigens including a signal peptide; (iii) multiple peptides having at least 80% sequence identity to each other; (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; or (v) one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity. In some embodiments, the polynucleotide comprises one or more methanogen antigens from each of (i)-(v). In some embodiments, the polynucleotide comprises three methanogen antigens. In some embodiments, the three methanogen antigens are associated with at least three different methanogen species, e.g., Methanobrevibacter ruminantium, Methanobrevibacter Smithii, Methanobrevibacter oralis. Exemplary multi-component compositions are set forth in Table 3.

[0303] In some embodiments of the isolated polynucleotides disclosed herein, compositions comprising the same, or methods of making or using the polynucleotides disclosed herein, the polynucleotide comprises one or more methanogen antigens. In some embodiments, the polynucleotide comprises one or more methanogen antigens selected from one or more, all, or any combination of: (i) one or more peptides having at least 80% sequence identity to a methanogen protein; (ii) one or more secreted antigens including a signal peptide; (iii) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; or (iv) one or more peptides having at least 80% sequence identity to an AglB polypeptide or having substantially similar function as an AglB polypeptide. In some embodiments, the polynucleotide comprises one or more methanogen antigens from each of (i)-(iv). In some embodiments, the polynucleotide comprises five methanogen antigens. In some embodiments, the five methanogen antigens are associated with at least five different methanogen species, e.g., Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanosphaera stadtmanae. Exemplary multi-component compositions are set forth in Table 3.

[0304] In some embodiments of the isolated polynucleotides disclosed herein, compositions comprising the same, or methods of making or using the polynucleotides disclosed herein, the polynucleotide comprises one or more methanogen antigens. In some embodiments, the polynucleotide comprises one or more methanogen antigens selected from one or more, all, or any combination of: (i) one or more peptides having at least 80% sequence identity to a methanogenic protein; (ii) one or more secreted antigens including a signal peptide; (iii) multiple peptides having at least 80% sequence identity to each other; (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; or (v) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having substantially similar function as an Ig-like domain-containing polypeptide. In some embodiments, the polynucleotide comprises one or more methanogen antigens from each of (i)-(v). In some embodiments, the polynucleotide comprises eight methanogen antigens. In some embodiments, the eight methanogen antigens are associated with at least three different methanogen species, e.g., Methanobrevibacter ruminantium, Methanobrevibacter Smithii, Methanobrevibacter oralis. Exemplary multi-component compositions are set forth in Table 3.

[0305] In some embodiments of the isolated polynucleotides disclosed herein, compositions comprising the same, or methods of making or using the polynucleotides disclosed herein, the polynucleotide comprises one or more methanogen antigens. In some embodiments, the polynucleotide comprises one or more methanogen antigens selected from one or more, all, or any combination of: (i) one or more peptides having at least 80% sequence identity to a methanogenic protein; (ii) one or more secreted antigens comprising a signal peptide; (iii) multiple peptides having at least 80% sequence identity to each other; (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; or (v) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having substantially similar function as an Ig-like domain-containing polypeptide. In some embodiments, the polynucleotide comprises one or more methanogen antigens from each of (i)-(v). In some embodiments, the polynucleotide comprises 34 methanogen antigens. In some embodiments, the one or more methanogen antigens are associated with at least five different methanogen species, e.g., Methanobrevibacter ruminantium, Methanobrevibacter Smithii, Methanobrevibacter oralis, Methanobrevibacter thaueri, Methanobrevibacter sp. UBA 188. Exemplary multi-component compositions are set forth in Table 5.

[0306] In some embodiments, a composition disclosed herein comprises one or more polynucleotides comprising one or more rumen-associated antigens, e.g., methanogen antigens. In some embodiments, a composition (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises multiple polynucleotides (e.g., multiple RNAs), each comprising an antigen. In some embodiments, a composition (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises multiple polynucleotides (e.g., multiple RNAs), each comprising one or more antigens (e.g., the same or different antigens).

[0307] In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 1% of the first polynucleotide and about 99% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 5% of the first polynucleotide and about 95% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 10% of the first polynucleotide and about 90% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 20% of the first polynucleotide and about 80% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 30% of the first polynucleotide and about 70% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 40% of the first polynucleotide and about 60% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 50% of the first polynucleotide and about 50% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 60% of the first polynucleotide and about 40% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 70% of the first polynucleotide and about 30% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 80% of the first polynucleotide and about 20% of one or more additional polynucleotides.In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 90% of the first polynucleotide and about 10% of one or more additional polynucleotides. In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multicomponent composition, e.g., a multicomponent vaccine composition) comprises about 99% of the first polynucleotide and about 1% of the one or more additional polynucleotides.

[0308] In some embodiments, a composition comprising a plurality of polynucleotides (e.g., a multi-component composition, e.g., a multi-component vaccine composition) comprises about 33% first polynucleotide, about 33% second polynucleotide, and about 33% third polynucleotide.

[0309] Exemplary methanogen antigens disclosed herein can be obtained, for example, by sequencing a sample from an animal, e.g., the rumen of a ruminant. Sequencing a sample from an animal, e.g., the rumen of a ruminant, can provide information about specific protein targets and / or specific species, e.g., methanogen species. Furthermore, methanogen antigens can also be obtained by using a list, e.g., a defined list, of target species, e.g., methanogens, that are readily ascertainable by one skilled in the relevant art. Additionally or alternatively, methanogen antigens can be obtained by generating novel proteins or related sequence or structural variants using algorithms or language models, e.g., those known in the art.

[0310] Accordingly, also provided herein are methods for identifying one or more methanogen antigens for use in the polynucleotides disclosed herein or compositions comprising same, e.g., for vaccinating an animal, e.g., a ruminant.

[0311] Acetylated nucleotides In particular, provided herein are polyribonucleotides comprising one or more modified ribonucleotides comprising nucleosides comprising acetyl groups. In some embodiments, the nucleosides of the modified ribonucleotides are N4-acetylcytidine, and the modified ribonucleotides have a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. Further details regarding polyribonucleotides comprising one or more modified ribonucleotides comprising nucleosides comprising acetyl groups are provided in International Patent Application No. PCT / US22 / 27721, filed July 18, 202, the entire contents of which are incorporated herein by reference.

[0312] In some embodiments, the nucleoside of the modified ribonucleotide is N4-acetylcytidine, and the modified ribonucleotide has the following structure, where R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate: [ka]

[0313] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues. In some embodiments, at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, less than 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0314] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0315] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 10% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0316] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 15% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0317] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 20% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0318] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 25% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0319] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 30% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0320] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 35% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0321] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 40% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0322] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 45% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0323] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 50% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0324] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 55% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0325] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0326] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0327] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0328] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0329] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0330] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0331] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0332] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein at least 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0333] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues. In some embodiments, about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% to 10%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% contains N4-acetylcytidine.

[0334] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0335] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0336] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 70% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0337] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0338] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0339] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0340] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0341] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0342] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein greater than about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0343] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 5% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0344] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 10% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0345] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 15% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0346] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 20% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0347] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 25% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0348] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 30% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0349] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 35% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0350] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 40% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0351] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 45% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0352] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 50% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0353] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 55% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0354] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 60% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0355] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 65% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0356] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 75% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0357] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 80% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0358] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 85% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0359] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 90% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0360] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 95% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0361] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein about 99% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0362] In some embodiments, the polyribonucleotides disclosed herein comprise cytidine residues, wherein 100% of the cytidine residues in the polyribonucleotide comprise N4-acetylcytidine.

[0363] In some embodiments, a polyribonucleotide disclosed herein (e.g., a polyribonucleotide comprising cytidine residues in which about 5-100% of the cytidine residues comprise N4-acetylcytidine) comprises one or more additional modified ribonucleotides. In some embodiments, the one or more additional modified ribonucleotides comprise a nucleoside selected from adenosine, inosine, guanosine, cytidine, or uridine, or a combination thereof. In some embodiments, the one or more additional modified ribonucleotides comprise a 5-hydroxymethyl group. In some embodiments, the one or more additional modified ribonucleotides comprise 5-hydroxymethyluridine. In some embodiments, 5% to 100% of the uridine residues in a uridine-containing polyribonucleotide are 5-hydroxymethyluridine.

[0364] In some embodiments, the polyribonucleotide can have a length of at least 5 nucleotides or more. In some embodiments, the polyribonucleotide can have a length of at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides, at least 5000 nucleotides or more.

[0365] In some embodiments, the polyribonucleotide may be from about 5 nucleotides to about 200,000 nucleotides, from about 5 nucleotides to about 150,000 nucleotides, from about 5 nucleotides to about 100,000 nucleotides, from about 5 nucleotides to about 50,000 nucleotides, from about 5 nucleotides to about 10,000 nucleotides, from about 5 nucleotides to about 5000 nucleotides, from about 5 nucleotides to about 1000 nucleotides, from about 5 nucleotides to about 500 nucleotides, from about 5 nucleotides to about 400 nucleotides, from about 5 nucleotides to about 300 nucleotides, from about 5 nucleotides to about 200 nucleotides, from about 5 nucleotides to about 100 nucleotides, from about 5 nucleotides to about 90 nucleotides, or from about 5 nucleotides to about 1000 nucleotides. The length may be from about 5 nucleotides to about 85 nucleotides, from about 5 nucleotides to about 80 nucleotides, from about 5 nucleotides to about 75 nucleotides, from about 5 nucleotides to about 70 nucleotides, from about 5 nucleotides to about 65 nucleotides, from about 5 nucleotides to about 60 nucleotides, from about 5 nucleotides to about 55 nucleotides, from about 5 nucleotides to about 50 nucleotides, from about 5 nucleotides to about 45 nucleotides, from about 5 nucleotides to about 40 nucleotides, from about 5 nucleotides to about 35 nucleotides, from about 5 nucleotides to about 30 nucleotides, from about 5 nucleotides to about 25 nucleotides, from about 5 nucleotides to about 20 nucleotides, from about 5 nucleotides to about 15 nucleotides, or from about 5 nucleotides to about 10 nucleotides.

[0366] In some embodiments, the polyribonucleotide is from about 5 nucleotides to about 200,000 nucleotides, from about 10 nucleotides to about 200,000 nucleotides, from 15 nucleotides to about 200,000 nucleotides, from about 20 nucleotides to about 200,000 nucleotides, from about 30 nucleotides to about 200,000 nucleotides, from about 40 nucleotides to about 200,000 nucleotides, from about 50 nucleotides to about 200,000 nucleotides, from about 100 nucleotides to about 200,000 nucleotides, from about 200 nucleotides to about 200,000 nucleotides, from about 300 nucleotides to about 200,000 nucleotides, from about 400 nucleotides to about 200,000 nucleotides, from about 500 nucleotides to about 200,000 nucleotides, from about 1000 nucleotides to about It can have a length of 200,000 nucleotides, about 2,000 nucleotides to about 200,000 nucleotides, about 3,000 nucleotides to about 200,000 nucleotides, about 4,000 nucleotides to about 200,000 nucleotides, about 5,000 nucleotides to about 200,000 nucleotides, about 10,000 nucleotides to about 200,000 nucleotides, about 20,000 nucleotides to about 200,000 nucleotides, about 30,000 nucleotides to about 200,000 nucleotides, about 40,000 nucleotides to about 200,000 nucleotides, about 50,000 nucleotides to about 200,000 nucleotides, about 100,000 nucleotides to about 200,000 nucleotides, or about 150,000 nucleotides to about 200,000 nucleotides.

[0367] In some embodiments, the polyribonucleotide can have a length of 200,000 nucleotides or less, 150,000 nucleotides or less, 100,000 nucleotides or less, or 50,000 nucleotides or less.

[0368] 5-hydroxymethyl modified nucleotides In particular, provided herein are polyribonucleotides comprising one or more modified ribonucleotides comprising nucleosides comprising 5-hydroxymethyl groups. In some embodiments, the nucleoside of the modified ribonucleotide is 5-hydroxymethyluridine, and the modified ribonucleotide has a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. Further details regarding ribonucleotides comprising nucleosides comprising 5-hydroxymethyl groups are described in International Patent Application PCT / US22 / 27721, filed July 18, 202, the entire contents of which are incorporated herein by reference.

[0369] In some embodiments, the nucleoside of the modified ribonucleotide is 5-hydroxymethyluridine, and the modified ribonucleotide has the following structure, where R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate: [ka]

[0370] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues. In some embodiments, at least 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, less than 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0371] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0372] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 10% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0373] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 15% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0374] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 20% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0375] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 25% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0376] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 30% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0377] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 35% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0378] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 40% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0379] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 45% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0380] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 50% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0381] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 55% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0382] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0383] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0384] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0385] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0386] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0387] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0388] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0389] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0390] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein at least 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0391] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues. In some embodiments, about 5% to 99%, about 5% to 95%, about 5% to 90%, about 5% to 85%, about 5% to 80%, about 5% to 75%, about 5% to 70%, about 5% to 65%, about 5% to 60%, about 5% to 55%, about 5% to 50%, about 5% to 45%, about 5% to 40%, about 5% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, about 5% About 10%, about 10% to 99%, about 15% to 99%, about 20% to 99%, about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99% contains 5-hydroxymethyluridine.

[0392] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0393] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0394] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 70% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0395] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0396] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0397] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0398] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0399] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0400] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein greater than 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0401] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 5% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0402] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 10% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0403] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 15% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0404] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 20% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0405] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 25% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0406] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 30% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0407] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 35% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0408] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 40% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0409] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 45% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0410] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 50% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0411] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 55% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0412] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 60% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0413] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 65% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0414] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 75% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0415] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 80% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0416] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 85% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0417] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 90% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0418] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 95% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0419] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein about 99% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0420] In some embodiments, the polyribonucleotides disclosed herein comprise uridine residues, wherein 100% of the uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0421] In some embodiments, a polyribonucleotide disclosed herein (e.g., a polyribonucleotide comprising uridine residues in which about 5-100% of the uridine residues comprise 5-hydroxymethyluridine) comprises one or more additional modified ribonucleotides other than 5-hydroxymethyluridine. In some embodiments, the one or more additional modified ribonucleotides comprise a nucleoside selected from adenosine, inosine, guanosine, cytidine, or uridine, or a combination thereof. In some embodiments, the one or more additional modified ribonucleotides comprise an acetyl group. In some embodiments, the one or more additional modified ribonucleotides comprise N4-acetylcytidine. In some embodiments, 5% to 100% of the cytidine residues in a cytidine-containing polyribonucleotide are N4-acetylcytidine.

[0422] In some embodiments, the polyribonucleotide can have a length of at least 5 nucleotides or more. In some embodiments, the polyribonucleotide can have a length of at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides, at least 5000 nucleotides or more.

[0423] In some embodiments, the polyribonucleotide may be from about 5 nucleotides to about 200,000 nucleotides, from about 5 nucleotides to about 150,000 nucleotides, from about 5 nucleotides to about 100,000 nucleotides, from about 5 nucleotides to about 50,000 nucleotides, from about 5 nucleotides to about 10,000 nucleotides, from about 5 nucleotides to about 5000 nucleotides, from about 5 nucleotides to about 1000 nucleotides, from about 5 nucleotides to about 500 nucleotides, from about 5 nucleotides to about 400 nucleotides, from about 5 nucleotides to about 300 nucleotides, from about 5 nucleotides to about 200 nucleotides, from about 5 nucleotides to about 100 nucleotides, from about 5 nucleotides to about 90 nucleotides, or from about 5 nucleotides to about 1000 nucleotides. The length may be from about 5 nucleotides to about 85 nucleotides, from about 5 nucleotides to about 80 nucleotides, from about 5 nucleotides to about 75 nucleotides, from about 5 nucleotides to about 70 nucleotides, from about 5 nucleotides to about 65 nucleotides, from about 5 nucleotides to about 60 nucleotides, from about 5 nucleotides to about 55 nucleotides, from about 5 nucleotides to about 50 nucleotides, from about 5 nucleotides to about 45 nucleotides, from about 5 nucleotides to about 40 nucleotides, from about 5 nucleotides to about 35 nucleotides, from about 5 nucleotides to about 30 nucleotides, from about 5 nucleotides to about 25 nucleotides, from about 5 nucleotides to about 20 nucleotides, from about 5 nucleotides to about 15 nucleotides, or from about 5 nucleotides to about 10 nucleotides.

[0424] In some embodiments, the polyribonucleotide is from about 5 nucleotides to about 200,000 nucleotides, from about 10 nucleotides to about 200,000 nucleotides, from 15 nucleotides to about 200,000 nucleotides, from about 20 nucleotides to about 200,000 nucleotides, from about 30 nucleotides to about 200,000 nucleotides, from about 40 nucleotides to about 200,000 nucleotides, from about 50 nucleotides to about 200,000 nucleotides, from about 100 nucleotides to about 200,000 nucleotides, from about 200 nucleotides to about 200,000 nucleotides, from about 300 nucleotides to about 200,000 nucleotides, from about 400 nucleotides to about 200,000 nucleotides, from about 500 nucleotides to about 200,000 nucleotides, or from about 1000 nucleotides. The nucleic acid sequence may have a length of from about 200,000 nucleotides, from about 2000 nucleotides to about 200,000 nucleotides, from about 3000 nucleotides to about 200,000 nucleotides, from about 4000 nucleotides to about 200,000 nucleotides, from about 5000 nucleotides to about 200,000 nucleotides, from about 10,000 nucleotides to about 200,000 nucleotides, from about 20,000 nucleotides to about 200,000 nucleotides, from about 30,000 nucleotides to about 200,000 nucleotides, from about 40,000 nucleotides to about 200,000 nucleotides, from about 50,000 nucleotides to about 200,000 nucleotides, from about 100,000 nucleotides to about 200,000 nucleotides, or from about 150,000 nucleotides to about 200,000 nucleotides.

[0425] In some embodiments, the polyribonucleotide can have a length of 200,000 nucleotides or less, 150,000 nucleotides or less, 100,000 nucleotides or less, or 50,000 nucleotides or less. SBI Adjuvant

[0426] The polynucleotides disclosed herein encoding one or more rumen-associated antigens can further include sequences encoding an adjuvant.

[0427] In some embodiments, the adjuvants disclosed herein can be used to induce and / or modulate an immune response elicited by an antigen (e.g., a fragment antigen or antigen variant) described herein. In some embodiments, the adjuvants disclosed herein comprise a complement-binding polypeptide. In some embodiments, the complement-binding polypeptide comprises a complement C3d-binding polypeptide. An exemplary C3d-binding polypeptide is the immunoglobulin-binding protein (Sbi) of Staphylococcus aureus.

[0428] As disclosed herein, immunoglobulin S. aureus binding agent (Sbi) is an exemplary polypeptide capable of binding complement C3d (as described in Clark et al. (2011), Mol. Immunol. 48(4):452-462, the entire contents of which are incorporated herein by reference). Sbi contains two immunoglobulin-binding domains (domains I and II) and two complement C3d-binding domains (domains III and IV). Sbi domains III and IV can bind C3d (of native C3, iC3b, and C3dg) and can lead to fluid-phase consumption of C3 through activation of the alternative pathway (see Clark et al. 2011). Sbi can also be secreted and has been shown to be involved in immune evasion by S. aureus (Burman et al., 2008, J. Biol. Chem; 283:17579-17593).

[0429] Without wishing to be bound by theory, it is believed that in some embodiments, complement C3d-binding polypeptides derived from S. aureus Sbi can be used as adjuvants to enhance and / or modulate immune responses from the antigens described herein. In some embodiments, the immune response is elicited by a fragment antigen or antigen variant disclosed herein. In some embodiments, the immune response is elicited by a component of S. aureus Sbi.

[0430] Exemplary Sbi adjuvants and compositions containing same are disclosed in International Patent Application No. PCT / US2022 / 018610, filed March 3, 2022, the entire contents of which are incorporated herein by reference.

[0431] composition In particular, the present disclosure provides compositions comprising one or more rumen-associated antigens, such as one or more rumen antigens and / or one or more methanogen antigens. The compositions disclosed herein may also include polynucleotides encoding the same.

[0432] In some embodiments, the compositions disclosed herein comprise a polynucleotide comprising a first nucleotide sequence encoding one or more rumen antigens and a second nucleotide sequence encoding one or more methanogen antigens.

[0433] In some embodiments, the first nucleotide sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 rumen antigens.

[0434] In some embodiments, the first nucleotide sequence is about 2 to 100, about 3 to 100, about 4 to 100, about 5 to 100, about 6 to 100, about 7 to 100, about 8 to 100, about 9 to 100, about 10 to 100, about 20 to 100, about 30 to 100, about 40 to 100, about 50 to 100, about 60 to 100, about 70 to 100, about 80 to 100 The antigens include 0, about 90-100, about 2-90, about 2-80, about 2-70, about 2-60, about 2-50, about 2-40, about 2-30, about 2-20, about 2-10, about 2-15, about 2-14, about 2-13, about 2-12, about 2-11, about 2-10, about 2-9, about 2-8, about 2-7, about 2-6, about 2-5, about 2-4, and about 2-3 rumen antigens.

[0435] In some embodiments, the second nucleotide sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 methanogen antigens.

[0436] In some embodiments, the second nucleotide sequence is about 2 to 100, about 3 to 100, about 4 to 100, about 5 to 100, about 6 to 100, about 7 to 100, about 8 to 100, about 9 to 100, about 10 to 100, about 20 to 100, about 30 to 100, about 40 to 100, about 50 to 100, about 60 to 100, about 70 to 100, about 80 to 100, The methanogen antigens include about 90-100, about 2-90, about 2-80, about 2-70, about 2-60, about 2-50, about 2-40, about 2-30, about 2-20, about 2-10, about 2-15, about 2-14, about 2-13, about 2-12, about 2-11, about 2-10, about 2-9, about 2-8, about 2-7, about 2-6, about 2-5, about 2-4, and about 2-3.

[0437] In some embodiments, the compositions disclosed herein comprise a polynucleotide comprising one or more rumen-associated antigens and a polynucleotide comprising one or more cytokines and / or chemokines. In some embodiments, the cytokines and / or chemokines are expressed in ruminants. In some embodiments, the cytokines and / or chemokines are or comprise VIP, CXCL10, APRIL, or any combination thereof.

[0438] In some embodiments, the cytokine and / or chemokine comprises a sequence provided in any one of SEQ ID NOs: 9, 11, or 13, or a sequence at least 85% identical thereto. The cytokine / chemokine sequence is underlined in SEQ ID NO: 9, 11, or 13.

[0439] In some embodiments, the cytokine and / or chemokine comprises a polypeptide encoded by a cytokine and / or chemokine nucleotide sequence provided in any one of SEQ ID NOs: 8, 10, or 12, or a sequence at least 85% identical thereto. The cytokine and / or chemokine sequences are underlined in SEQ ID NOs: 8, 10, and 12.

[0440] In some embodiments, the compositions disclosed herein are pharmaceutical compositions.

[0441] In some embodiments, the compositions disclosed herein are immunogenic compositions.

[0442] In some embodiments, the compositions disclosed herein are vaccine compositions.

[0443] In some embodiments, the compositions disclosed herein are administered at a dose of about 5 ng to about 1000 ng, about 5 ng to about 900 ng, about 5 ng to about 800 ng, about 5 ng to about 700 ng, about 5 ng to about 600 ng, about 5 ng to about 500 ng, about 5 ng to about 400 ng, about 5 ng to about 300 ng, about 5 ng to about 200 ng, about 5 ng to about 100 ng, about 5 ng to about 90 ng, about 5 ng to about 80 ng, about 5 ng to about 70 ng, about 5 ng to about 60 ng, about 5 ng to about 50 ng, about 5 ng to about 40 ng, about 5 ng to about 30 ng, about 5 ng to about 20 ng, or about 5 ng to about 10 ng. In some embodiments, the compositions disclosed herein are administered at a dose of about 10 ng to about 1000 ng, about 20 ng to about 1000 ng, about 30 ng to about 1000 ng, about 40 ng to about 1000 ng, about 50 ng to about 1000 ng, about 60 ng to about 1000 ng, about 70 ng to about 1000 ng, about 80 ng to about 1000 ng, about 90 ng to about 1000 ng, about 100 ng to about 1000 ng, about 200 ng to about 1000 ng, about 300 ng to about 1000 ng, about 40 ng to about 1000 ng, about 50 ng to about 1000 ng, about 60 ng to about 1000 ng, about 700 ng to about 1000 ng, about 800 ng to about 1000 ng, or about 900 ng to about 1000 ng.

[0444] In some embodiments, the compositions disclosed herein are administered at a dose of about 5 ng, about 10 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 60 ng, about 70 ng, about 80 ng, about 90 ng, about 100 ng, 150 ng, about 200 ng, about 250 ng, about 300 ng, about 350 ng, about 400 ng, about 450 ng, about 500 ng, about 550 ng, about 600 ng, about 650 ng, about 700 ng, about 750 ng, about 800 ng, about 850 ng, about 900 ng, about 950 ng, or about 1000 ng.

[0445] In some embodiments, the compositions disclosed herein are administered at a dose of at least 5 ng, at least 10 ng, at least 20 ng, at least 30 ng, at least 40 ng, at least 50 ng, at least 60 ng, at least 70 ng, at least 80 ng, at least 90 ng, at least 100 ng, at least 150 ng, at least 200 ng, at least 250 ng, at least 300 ng, at least 350 ng, at least 400 ng, at least 450 ng, at least 500 ng, at least 550 ng, at least 600 ng, at least 650 ng, at least 700 ng, at least 750 ng, at least 800 ng, at least 850 ng, at least 900 ng, at least 950 ng, or at least 1000 ng.

[0446] Pharmaceutical Composition In some embodiments, the composition comprising one or more rumen-associated antigens is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The pharmaceutical composition of the present disclosure may comprise a polypeptide disclosed herein, a polynucleotide disclosed herein, or an expression vector comprising a polynucleotide disclosed herein.

[0447] In some embodiments, pharmaceutical compositions can include a pharmaceutically acceptable carrier or excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, glycerol, sugars such as mannitol, sucrose, dextrose, fatty acid esters, and the like, and combinations thereof.

[0448] The pharmaceutical compositions can be mixed with auxiliary substances (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavoring agents, and / or aromatic substances, etc.), as needed, which do not adversely react with the active compound or interfere with its activity. In certain embodiments, water-soluble carriers suitable for intravenous administration are used. In some embodiments, the pharmaceutical compositions can be sterilized.

[0449] Suitable pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The pharmaceutical composition can be a liquid solution, suspension, or emulsion.

[0450] Pharmaceutical compositions can be formulated according to conventional procedures as pharmaceutical compositions suitable for administration to humans. The formulation of the pharmaceutical composition should suit the method of administration. For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity of active agent. When the pharmaceutical composition is to be administered by infusion, it can be dispensed into an infusion bottle containing sterile pharmaceutical-grade water, saline, or dextrose / water. When the pharmaceutical composition is to be administered by infusion, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0451] While the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any type of animal or cell, either in vitro or ex vivo. Modifications of pharmaceutical compositions suitable for administration to humans to provide compositions suitable for administration to a variety of animals or cells, either in vitro or ex vivo, are well understood, and an ordinarily skilled professional, e.g., a veterinary pharmacologist, can design and / or perform such modifications with only routine experimentation, if any.

[0452] Formulations of the pharmaceutical 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 into association the active ingredient with a diluent or other excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0453] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition described herein.

[0454] RNA formulations In particular, provided herein are compositions comprising polyribonucleotides comprising rumen-associated antigens, and formulations thereof. In some embodiments, the compositions comprising polyribonucleotides disclosed herein are formulated into lipid nanoparticle (LNP) formulations.

[0455] In some embodiments, the polyribonucleotides disclosed herein encode polypeptides. In some embodiments, the polyribonucleotides disclosed herein are or comprise messenger RNA. In some embodiments, compositions comprising polyribonucleotides comprising messenger RNA are formulated into lipid nanoparticle (LNP) formulations.

[0456] In some embodiments, the present disclosure provides LNP formulations comprising the polyribonucleotides disclosed herein for use in pharmaceutical compositions, such as immunogenic compositions.

[0457] Methods of using the compositions disclosed herein The present disclosure provides, inter alia, methods of using the polyribonucleotides disclosed herein or compositions comprising same.

[0458] In some embodiments, provided herein are methods of administering a polyribonucleotide disclosed herein or a composition comprising a polyribonucleotide disclosed herein to a cell, tissue, or animal, e.g., a ruminant.

[0459] In some embodiments, provided herein are methods of administration, e.g., vaccination methods, comprising administering a polyribonucleotide disclosed herein or a composition comprising a polyribonucleotide disclosed herein to a cell, tissue, or animal, e.g., a ruminant. In some embodiments, the ruminant comprises a cow, sheep, goat, buffalo, elk, antelope, caribou, or deer, or a combination thereof.

[0460] In some embodiments, the compositions disclosed herein are characterized in that, when administered to an animal, the composition reduces methane emissions from the animal compared to an otherwise similar animal not administered the composition or administered a different composition. In some embodiments, the reduction in methane emissions is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to methane emissions in an otherwise similar animal not administered the composition or administered a different composition. In some embodiments, the reduction in methane emissions is at least 5%.

[0461] In some embodiments, the reduction in methane production is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to methane production in otherwise similar animals not administered the composition or administered a different composition.

[0462] In some embodiments, the reduction in methane emissions is between about 5% and about 95%, between about 5% and about 90%, between about 5% and about 85%, between about 5% and about 80%, between about 5% and about 75%, between about 5% and about 80%, between about 5% and about 70%, between about 5% and about 65%, between about 5% and about 60%, between about 5% and about 55%, between about 5% and about 50%, between about 5% and about 45%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 95%, about 15% to about 95%, about 20% to about 95%, about 25% to about 95%, about 30% to about 95%, about 35% to about 95%, about 40% to about 95%, about 45% to about 95%, about 50% to about 95%, about 55% to about 95%, about 60% to about 95%, about 65% to about 95%, 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, and about 90% to about 95%.

[0463] In some embodiments, the compositions disclosed herein are characterized in that, when administered to an animal, the composition reduces the population of microorganisms in the animal compared to an otherwise identical animal not administered the composition or administered a different composition. In some embodiments, the microorganism comprises a methanogen. In some embodiments, the microorganism is a methanogen from the order Methanobrevibacter, Methanosphaera, Methanobacterium, Methanosarcinales, Methanomicrobiales, Thermoplasmatales, or a combination thereof.In some embodiments, the methanogen is Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp.UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans or any combination thereof.In some embodiments, the abundance of methanogens is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the abundance of methanogens in an otherwise similar animal that is not administered the composition or that is administered a different composition. In some embodiments, the abundance of methanogens is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the abundance of methanogens in an otherwise comparable animal not administered the composition or administered a different composition.

[0464] In some embodiments, a composition disclosed herein is characterized in that, when administered to an animal, the composition reduces the abundance of all or substantially all methanogens (e.g., total methanogen abundance) in the animal compared to the total methanogen abundance in an otherwise comparable animal that is not administered the composition or that is administered a different composition. In some embodiments, the abundance of methanogens, e.g., total methanogen abundance, is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the abundance of methanogens in an otherwise comparable animal that is not administered the composition or that is administered a different composition. In some embodiments, the abundance of methanogens, e.g., the abundance of total methanogens, is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the abundance of methanogens in an otherwise comparable animal that has not been administered the composition or that has been administered a different composition.

[0465] In some embodiments, a composition disclosed herein is characterized in that, when administered to an animal, the composition reduces the abundance of at least one methanogen species in the animal compared to the abundance of the same methanogen species in an otherwise comparable animal that is not administered the composition or that is administered a different composition, hi some embodiments, the abundance of the at least one methanogen species is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the abundance of the same methanogen species in an otherwise comparable animal that is not administered the composition or that is administered a different composition. In some embodiments, at least one methanogen species is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the abundance of the same methanogen species in an otherwise comparable animal not administered the composition or administered a different composition. In some embodiments, the abundance of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 18, at least 19, or at least 20 methanogen species is reduced.

[0466] In some embodiments, the compositions disclosed herein are characterized in that, when administered to an animal, the composition increases the growth rate of the animal compared to the growth rate of an otherwise comparable animal not administered the composition or administered a different composition. In some embodiments, the growth rate is assessed by weighing the animal at one or more time points. In some embodiments, the growth rate is assessed by weighing the animal at one or more time points. In some embodiments, the animal's weight is measured daily. In some embodiments, the growth rate of the animal increases over a period of time. In some embodiments, the period of time includes at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months. In some embodiments, the increase in growth rate includes the daily weight gain of the animal. In some embodiments, the animal's daily weight gain is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% increased compared to the body weight of an otherwise comparable animal not administered the composition or administered a different composition. In some embodiments, the animal's daily weight gain is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% increased compared to the body weight of an otherwise comparable animal not administered the composition or administered a different composition.

[0467] In some embodiments, the compositions disclosed herein are characterized in that, when administered to an animal, the composition increases the energy efficiency of the animal compared to the energy efficiency of an otherwise comparable animal not administered the composition or administered a different composition. In some embodiments, increasing energy efficiency comprises increasing digestive efficiency. In some embodiments, digestive efficiency comprises one or more or all of the following parameters: (i) rate of weight gain, (ii) relationship of weight to intake, or (iii) milk quality. In some embodiments, milk quality may be assessed by a weight gain metric (e.g., average daily gain (ADG) for cattle), an intake metric relative to weight (e.g., food conversion ratio or residual feed intake), and / or a milk composition metric. In some embodiments, increasing digestive efficiency comprises reducing digestion-related disorders in an animal, e.g., a ruminant.

[0468] In some embodiments, the compositions disclosed herein are characterized in that, when administered to an animal, the composition alters the output of cellulose fermentation.

[0469] In some embodiments, the compositions disclosed herein are administered in a single dose. In some embodiments, the compositions are administered in multiple doses. In some embodiments, the composition is administered as a first dose of the composition, followed by one or more subsequent doses of the composition. In some embodiments, the first dose and one or more subsequent doses of the composition comprise the same methanogen antigen and / or rumen antigen. In some embodiments, the first dose and one or more subsequent doses of the composition comprise different methanogen antigens and / or rumen antigens.

[0470] In some embodiments, the one or more methanogen antigens and / or one or more ruminal antigens are specific to an animal. In some embodiments, the one or more methanogen antigens and / or one or more ruminal antigens specific to an animal are obtained by a method comprising: (i) identifying one or more methanogen antigens and / or one or more ruminal antigens expressed in the animal; and (ii) selecting the one or more methanogen antigens and / or one or more ruminal antigens for inclusion in the composition in response to the identification. In some embodiments, the one or more methanogen antigens and / or one or more ruminal antigens specific to an animal are different from one or more methanogen antigens and / or one or more ruminal antigens specific to a different animal. In some embodiments, the different animals include any one or all of: (i) animals of different species; (ii) animals of different sexes; (iii) animals of different ages; (iv) animals located in different geographic locations; or (v) the same animal at different times.

[0471] In some embodiments, the composition is administered to the animal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 times. In some embodiments, the composition is administered once every week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0472] In some embodiments, the composition is administered in combination with one or more additional agents. In some embodiments, the one or more additional agents include chemical additives, biological feed additives. In some embodiments, the composition is administered in combination with one or more additional compositions. In some embodiments, the additional compositions immunize the animal against a disease, e.g., an infectious disease.

[0473] kit Another aspect of the present disclosure further provides pharmaceutical packs or kits. In some embodiments, the kits can include a polyribonucleotide or composition described herein, for example, a polynucleotide including a nucleotide sequence encoding a rumen-associated antigen and / or a nucleotide sequence encoding a chemokine and / or a cytokine. In some embodiments, the kits can be used in any applicable method, for example, a method described herein.

[0474] array [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30 Table 2-31 Table 2-32 Table 2-33 Table 2-34 Table 2-35 Table 2-36 Table 2-37 Table 2-38 Table 2-39 Table 2-40 Table 2-41 Table 2-42 Table 2-43 Table 2-44 Table 2-45 Table 2-46 Table 2-47 Table 2-48 Table 2-49 Table 2-50 Table 2-51 Table 2-52 Table 2-53 Table 2-54 Table 2-55 Table 2-56 Table 2-57 Table 2-58 Table 2-59 Table 2-60 Table 2-61 Table 2-62 Table 2-63 Table 2-64 Table 2-65 Table 2-66 Table 2-67 Table 2-68 Table 2-69 Table 2-70 Table 2-71 Table 2-72 Table 2-73 Table 2-74 Table 2-75 Table 2-76 Table 2-77 Table 2-78 Table 2-79 Table 2-80 Table 2-81 [Table 2-82] [Table 2-83] [Table 2-84] [Table 2-85] [Table 2-86] [Table 2-87] [Table 2-88] [Table 2-89] [Table 2-90]

[0475] For SEQ ID NOs: 1-94, the following key indicates sequence features: nucleotide sequences in lowercase correspond to untranslated regions (UTRs); nucleotide sequences in uppercase correspond to open reading frames. Amino acid sequences in bold correspond to signal peptides, unmarked amino acid sequences correspond to antigens, and italicized amino acid sequences correspond to linkers and Sbi adjuvant fusions. [Example]

[0476] Example 1: Description of methods used in the examples described herein Generation of IVT templates: For experiments testing RNA vaccine candidates in cattle, the constructs defined in this example encoding bovine-optimized versions of Methanogen target antigens or cytokines were amplified from their respective custom-synthesized plasmids using a pUC19 backbone (GenScript). Amplification was performed in 20 μL reactions containing 0.25 μM of each primer, T7-AGG_fwd and 120 pA_rev, 1× Hercules II buffer, 25 mM of each dNTP, 15 ng of plasmid (GeneScript), and 0.4 μL of Hercules II enzyme (Agilent), with an annealing temperature of 50°C. After the PCR program was completed, 20 U of DpnI (NEB) was added to each PCR reaction and incubated at 37°C for 15 minutes to digest the plasmid template. The DpnI-treated PCR products were purified using a DNA Clean & Concentrator-25 kit (Zymo Research) and eluted in 60 μL of nuclease-free water.

[0477] The primer sequences used were as follows:

[0478] T7-AGG_fwd:

[0479] gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc (SEQ ID NO: 95)

[0480] 120pA_rev:

[0481] TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT agaatgtgaa gaaactttct ttttattag (SEQ ID NO: 96)

[0482] In vitro transcription (IVT) of RNA vaccine candidates

[0483] For each vaccine candidate, RNA was synthesized in a 22 μL IVT reaction consisting of 200 ng of each template, 20 mM MgCl2, 7.5 mM each NTP, 7.5 mM CleanCap AG (TriLink), 1× HiScribe transcription buffer, 2 M betaine, and 2 μL HiScribe polymerase mix (NEB) and incubated for 2 hours at 37°C. For RNA conditions using chemically modified nucleotides, UTP was replaced with 5-hydroxymethyluridine triphosphate (TriLink) or cytidine was replaced with N4-acetylCTP (Jena BioScience) at the indicated ratios in the IVT mixture.

[0484] All IVT products were cleaned using the Monarch (500 μg) RNA Clean Up Kit (NEB) and eluted in 83 μL of nuclease-free water. The eluted products were then digested in a 100 μL reaction consisting of 1× DNase I buffer and 10 U of DNase I (RNase-free) (New England Biolabs) at 37°C for 5 minutes to degrade the DNA template and remove residual 5' triphosphate on the RNA. The treated samples were cleaned using the Monarch 500 μg RNA Clean Up Kit (New England Biolabs) and eluted in 100 μL of 1 mM sodium citrate (pH 6.5).

[0485] One-pot in vitro transcription (IVT) of multiplexed RNA vaccine candidates: For multiplexed RNA vaccine candidates, the RNAs can be synthesized in separate reactions, as in the IVT method described above, and normalized and pooled in a final step, or synthesized in a single or "one-pot" reaction. For the latter, multiplexed RNA synthesis was performed by mixing 200 ng of DNA in a 22 μL IVT reaction mixture containing 20 mM MgCl2, 7.5 mM of each NTP, 7.5 mM CleanCap AG (TriLink), 1× HiScribe transcription buffer, 2 M betaine, 2 μL of HiScribe polymerase mix (NEB), and equimolar or predetermined molar ratios of each DNA template, and incubating at 37°C for 2 hours. For RNA conditions using chemically modified nucleotides, UTP was replaced with 5-hydroxymethyluridine triphosphate (TriLink) or cytidine was replaced with N4-acetyl CTP (Jena BioScience) at the indicated ratios in the IVT mixture. All IVT products were cleaned up using the Monarch (500 μg) RNA Clean Up Kit (NEB) and eluted in 83 μL of nuclease-free water. The eluted product was then digested in a 100 μL reaction consisting of 1× DNase I buffer and 10 U of DNase I (RNase-free) (New England Biolabs) for 5 minutes at 37°C to degrade the DNA template and remove residual 5' triphosphate on the RNA. The treated sample was cleaned using the Monarch 500 μg RNA Clean Up Kit (New England Biolabs) and eluted in 100 μL of 1 mM sodium citrate (pH 6.5).

[0486] Preparation for in vivo RNA experiments

[0487] Formulations of RNA in lipid nanoparticles (RNA-LNP) were prepared using a microfluidic mixer (Precision Nanosystems, Vancouver, BC). Briefly, using the manufacturer's recommended formulation parameters, the GenVoy-ILM lipid mixture (Precision Nanosystems NWW0042) was diluted to 12.5 mM with absolute ethanol and combined with an aqueous solution of RNA (0.14 mg / mL) in PNI buffer (Precision Nanosystems NWW0043). The formulation was immediately diluted 30:1 with phosphate-buffered saline (Gibco 10010023), concentrated using an Amicon centrifugal filter (MilliporeSigma UFc901008), and adjusted to the appropriate final volume with PBS. The formulation was stored at 4°C for up to 2 days prior to the primary dose and 25 days prior to the booster dose.

[0488] RNA administration test in cattle

[0489] Animal studies were conducted in accordance with guidelines established by Texas A&M University (College Station, MA, USA) and approved by the TAMU Institutional Animal Care and Use (IACUC) Committee. Cattle, consisting of Angus-crossbred cattle with a Bos indicus composition of 0.25 or less, were raised at the TAMU McGregor Research Facility. Calves were weaned at approximately 205 days of age, acclimated to a GreenFeed Pasture system (C-Lock Inc.) for a minimum of 4 weeks after weaning, and vaccinated at a minimum of 8 weeks later.

[0490] During several RNA vaccine trials, such as M1 and M2, calves (n = 4 or 5 per condition) received two RNA doses: on day 0 (prime) and day 21 (boost). RNA injections were administered intramuscularly in the neck with 2 mL of RNA-LNP formulation (0.5 mg RNA dose per animal) unless otherwise noted. Blood, saliva, and ruminal fluid samples were collected from each animal on days -1, 21, 34, and 90 and shipped on dry ice for analysis, unless otherwise noted.

[0491] In other RNA vaccine trials, such as M3, calves (n=10 per condition) received two RNA doses on days 0 (prime, 1°), 25 (boost, 2°), and 104* (boost, 3°). RNA injections were administered intramuscularly in the neck with 2 mL of RNA-LNP formulation (0.55 mg RNA dose per animal) unless otherwise noted. Blood samples were collected on days -1, 25, 42, 90, 109*, and 122*, and ruminal fluid samples were collected on days -1, 90, 109*, and 122*. Samples were shipped on dry ice for analysis unless otherwise noted. (*Conditions F1 and F3 only.)

[0492] Blood was collected via jugular venipuncture (8-10 mL) and stored on ice for >2 h to allow clotting before centrifugation. Serum was collected and stored at -20 °C until thawed for assay.

[0493] Using a vacuum, saliva was collected into a conical flask. More than 10 mL of saliva was collected per collection and stored at -20°C until thawed for assay.

[0494] Ruminal fluid was collected by withdrawing it using an esophageal tube and pump and either immediately flash-frozen in liquid nitrogen or placed on ice at -4°C until centrifugation. The solid portion (i.e., pellet) of the centrifuged sample was removed, placed in a microtube, flash-frozen in liquid nitrogen, stored at -80°C, and shipped on dry ice to CosmosID Inc. (Germantown, MD) for sequencing. The liquid fraction was stored at -20°C until thawed for assay. In Study M3, separate samples were processed for volatile fatty acid (VFA) analysis. For Studies M1 and M2, animals were randomly assigned to treatment groups by body weight (BW) and caged together.

[0495] Enteric methane (CH4), oxygen (O2), and carbon dioxide (CO2) emissions for each individual calf were measured via GreenFeed Pasture System (C-Lock Inc.) on days -8 to -1 to represent a pre-vaccination baseline and again on days 22 to 29. Feed intake for each individual calf was measured via GrowSafe Feed Inake Systems (GrowSafe Systems, Ltd.) and RFID ear tags on days -32 to 90 during the acclimation and experimental periods.

[0496] For Study M3, animals were monitored for CH4 emissions and dry matter intake (DMI) during a baseline period prior to the study and were allocated to treatment groups based on initial CH4 / DMI and BW and housed separately. Gas emissions per individual calf were continuously measured from D-22 through the end of the study, and pre-vaccination enteric methane baselines were determined after final pen selection and before injection (D-6 to D0). Feed intake per individual calf was measured during the acclimation and experimental periods via GrowSafe Feed Inake Systems (GrowSafe Systems, Ltd.) and RFID ear tags.

[0497] Sequencing and bioinformatics analysis

[0498] For DNA extraction, DNA was isolated from samples using the QIAGEN DNeasy PowerSoidPro kit according to the manufacturer's protocol. Unfractionated samples were thawed at 4°C for up to 16 hours and optionally homogenized and / or centrifuged prior to extraction. Extracted DNA samples were quantified using a Qubit 4 fluorometer and the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific).

[0499] For library preparation and sequencing, DNA libraries were prepared with a total DNA input of 1 ng using the Nextera XT DNA Library Preparation Kit (Illumina) and IDT unique Dual Indexes. Genomic DNA was fragmented using a proportional amount of Illumina Nextera XT Fragmentation Enzyme. Unique dual indexes were added to each sample, followed by 12 cycles of PCR to construct libraries. DNA libraries were purified using AMPure magnetic beads (Beckman Coulter) and eluted with QIAGEN EB buffer. DNA libraries were quantified using a Qubit4 fluorometer and Qubit dsDNA HS Assay Kit. Libraries were then sequenced on an Illumina NextSeq2000 platform (2 × 150 bp).

[0500] For bioinformatics analysis, unfinished sequencing reads were directly analyzed by the CosmosID-HUB Microbiome Platform (CosmosID Inc. Germantown, MD), described elsewhere (Ottensen et al. 2016; Ponnusamy et al. 2016; Hasan et al. 2014; Lax et al. 2014), for multi-kingdom microbiome analysis, antibiotic resistance and virulence gene profiling, and quantification of relative organism abundance. Briefly, this system utilizes a curated genome database and high-performance data mining algorithms to rapidly disambiguate hundreds of millions of metagenomic sequence reads into individual microorganisms encoding specific sequences.

[0501] ELISA

[0502] Antigen-specific IgG and IgA responses to the RNA vaccine formulation were measured using ELISA assays. To coat ELISA plates, antigen proteins were reconstituted from lyophilized state in HO (e.g., OVA protein, 2000 μg / mL) or synthesized via an E. coli-based cell-free protein expression system (Liberum Biotech) with a plasmid template DNA containing the antigen protein sequence (e.g., mru1499), as well as a start / stop codon, an upstream T7 promoter and ribosome binding site, and a downstream T7 terminator. The reaction mixture was incubated at 27°C for 8 hours and then diluted 1:10 in PBS. Nunc ELISA plates (ThermoFisher Scientific) were then coated with the antigen protein solution (50 μL / well) and incubated overnight at 4°C.

[0503] The plates were washed three times with PBS-Tween® 1% (PBST) and blocked with 200 μL of SuperBlock (Thermo Fisher Scientific) for 1 hour at room temperature. The diluent was made from a 1:2 mixture of SuperBlock (Thermo Fisher Scientific) and HO. Serial dilutions of serum samples in diluent (ranging from 1:10 to 1:1010) were added to each well and incubated for 2 hours at room temperature. The plates were washed three times with PBST. For IgG titers, the plates were then incubated with 50 μL / well of HRP-conjugated sheep anti-bovine IgG (ab112618, Abcam) at 1:3,000 in diluent for 1 hour at room temperature. For IgA titers, the plates were incubated with 50 μL / well of HRP-conjugated sheep anti-bovine IgA H&L (ab112755, Abcam) at 1:1,000 in diluent for 1 hour at room temperature. Plates were washed three times with PBST, and 100 μL / well of SigmaFast OPD solution (Sigma Aldrich) was added and incubated for 10 minutes at room temperature. The reaction was stopped by adding 50 μL / well of 2 M HCl. Absorbance was read at 450 nm on a GloMax plate reader (Promega).

[0504] The endpoint titer is defined here as the reciprocal of the highest specimen dilution giving a reading above the cutoff. The cutoff is calculated by adding the standard deviation of the raw (UTD) values ​​to the mean of the raw (UTD) values. The geometric mean titer (GMT) and geometric standard deviation (GSD) are obtained across condition groups.

[0505] Vaccine preparations used in Examples 2 to 5.

[0506] Ssp refers to Secpep1. HNpep refers to PPa2 secretion signal. HNadj refers to Sbi adjuvant. HNmod1 refers to N4-acetylCTP. HNmod2 refers to 5-hydroxymethyluridine triphosphate.

[0507] Vaccine formulation used in Example 5.

[0508] Approaches for antigen cluster generation.

[0509] One approach utilized in Examples 4 and 5 to generate multi-component RNA vaccines involves using bioinformatics techniques to identify groupings of two or more antigens (e.g., DNA or amino acid sequences) that are related to each other (e.g., share a defined level of sequence or structural identity, such as greater than 80%) and that are associated with two or more species of ruminal methanogens or methanogen-associated proteins (e.g., the antigens within a grouping collectively share greater than 80% identity to multiple methanogen genomes).

[0510] In one example, such groupings were generated from a pool of antigens 50 aa in length representing all possible 50 aa frames present in a set of open reading frames (ORFs) generated from ruminal metagenomic data. In some cases, this set of ORFs was restricted to those sharing a defined sequence identity (e.g., greater than 80%) to methanogen species genomes or methanogen-associated proteins. In some cases, "hypothetical proteins" and / or sequences for de novo peptide design that share structural similarity (e.g., structural identity via AlphaFold or LLM predictions) with methanogen-associated proteins were included. Alternatively, in some cases, the pool of 50 aa frames was generated from protein or genome databases and restricted to selected methanogen species (e.g., the most abundant methanogen species in ruminal samples: Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, and Methanosphaera stadtmanae). In some cases, this set of ORFs was limited to those predicted by the algorithm to be surface-associated proteins (e.g., >0.5). In some cases, the generated antigen grouping consisted of 2, 3, 4, or 5 antigens with greater than 80% identity to each other, and the grouping contained antigens associated with 2, 3, 4, or 5 different methanogen species. In some cases, the final antigen grouping was defined as the complete protein sequence from which the 50 aa-frame was derived.

[0511] Approaches for multi-component formulation compositions.

[0512] The multi-component RNA vaccines used in Examples 4 and 5 targeting multiple methanogen proteins and / or proteins associated with multiple species of methanogens / archaea were generated by algorithmically or rationally selecting antigen combinations and groups that encompass multiple species of methane-related microorganisms present in the rumen. The vaccine formulations shown in Table 3 or Table 5 are described as consisting of multiple antigens (e.g., 3, 8, or 5 antigens per formulation), associated with multiple methane-related species (e.g., 3 or 5 per formulation), and combined in specific ratios in terms of molar concentration or mass.

[0513] Specifically, in this example, F2 contains three antigens, C3.1 (SEQ ID NO: 35), C3.2 (SEQ ID NO: 37), and C3.3 (SEQ ID NO: 39), generated via the antigen cluster generation workflow described above. The antigens in the F2 preparation share a defined level of similarity (e.g., sequence identity) and have regions covering at least three methane-related species. The antigens in the F2 preparation are also associated with the name / function of "signal peptidases" (e.g., best BlastP match), thereby characterizing them as membrane-bound enzymes and / or important for converting secreted proteins to their mature forms by cleaving signal peptides from the N-terminus of the protein during or after membrane passage.

[0514] F3 contains two groupings of two antigens each (C7.1 (SEQ ID NO: 55), C7.2 (SEQ ID NO: 57), C7.3 (SEQ ID NO: 59), and C7.4 (SEQ ID NO: 61)) and one grouping of four antigens (C8.1 (SEQ ID NO: 63), C8.2 (SEQ ID NO: 65), C8.3 (SEQ ID NO: 67), and C8.4 (SEQ ID NO: 69)). The former two groupings were generated via an antigen cluster generation workflow, and the latter grouping was selected by rational design. Each of the former two antigen groupings has regions that share a defined level of similarity (e.g., sequence identity) and cover at least two methane-related species. Additionally, antigens in the F2 formulation have regions that are related to the name / function of "adhesin," "adhesin-like," or "immunoglobin (Ig)-like" and / or share a defined level of similarity to adhesin / adhesin-like / Ig-like domain-containing domains (e.g., BlastP match). They are thereby characterized as cell surface components that facilitate binding to other cells or surfaces. Overall, the F3 formulation contains eight antigens covering at least five methane-related species.

[0515] F4 contains a grouping of five rationally designed antigens: C9.1 (SEQ ID NO: 79), C9.2 (SEQ ID NO: 81), C9.3A (SEQ ID NO: 83), C9.4C (SEQ ID NO: 91), and C9.5 (SEQ ID NO: 93). The antigens in the F4 formulation are related in name / function to "archaeal oligosaccharyltransferase" or "AglB" and / or share a defined level of similarity (e.g., BlastP match) with the oligosaccharyltransferase / AglB protein, thereby characterizing them as enzymes important for N-glycosylation (the covalent attachment of oligosaccharides to target proteins). Overall, the F4 formulation contains five antigens covering at least five methane-associated species.

[0516] F1 is a non-methanogen antigen control, ovalbumin (OVA), which is derived from Gallus gallus and is intended to generate an immune response that is not specific to methanogens.

[0517] [Table 3]

[0518] [Table 4-1] [Table 4-2]

[0519] Example 2: Generation of antigen-specific antibodies using multicomponent vaccine formulations This example describes antigen-specific IgG titers generated from vaccination of cattle with exemplary vaccines against OVA and mru1499. The methods used in this example are provided in Example 1 above.

[0520] As shown in Figures 1A-1B, antigen-specific IgG titers against OVA-IgG and mru1499-IgG (adhesin) were detected by ELISA in the serum or saliva of animals that received either one or two doses of the multicomponent RNA vaccine. Furthermore, as shown in Table 4, antigen-specific IgA titers were also detected in the serum or saliva of vaccinated animals.

[0521] [Table 5-1] [Table 5-2]

[0522] This data demonstrates that single- or multi-component RNA vaccine formulations can generate antigen-specific antibody responses in cattle. This data supports the development of single-component or multi-component vaccination approaches (e.g., when using two or more antigens) for vaccinating animals, such as ruminants.

[0523] Example 3: In vivo effects on methanogen abundance and methane emissions in cattle vaccinated against methanogen proteins This example describes the in vivo effects on methanogen abundance and methane emissions in cattle vaccinated with exemplary RNA vaccine formulations targeting methanogen proteins containing various secretion signals. The methods used in this example are provided in Example 1 above.

[0524] As shown in Figure 2, calves vaccinated with an exemplary multicomponent RNA vaccine showed a reduction in the methanogen species Methanobrevibacter (Mbb.) ruminantium M1 in ruminal fluid at 20, 34, and 90 days post-vaccination compared to control animals not treated or administered the vaccine formulation. Similarly, Figures 3A-B show a reduction in the methanogen species Methanobrevibacter (Mbb.) ruminantium M1 in ruminal fluid from calves vaccinated with exemplary RNA vaccines targeting methanogen proteins containing various secretion signals. The normalized relative abundance of archaeal species was also generally reduced in ruminal fluid from calves vaccinated with RNA vaccines targeting methanogen proteins (Figure 3B). Archaeal abundance was increased in animals vaccinated with an RNA vaccine construct with an HNpep secretion signal, the mru1499 antigen, and HNadjuvant.

[0525] Figure 4 shows the abundance of methanogen and archaeal species in ruminal fluid from cattle vaccinated with an exemplary RNA vaccine formulation targeting methanogens and, in some cases, containing cytokines. The RNA used also contained Ac4C and / or 5hmU modifications, as indicated. Animals vaccinated with the RNA vaccine formulation targeting methanogens (mru1499) and animals boosted with cytokines CXCL10 or VIP had fewer methanogen and archaeal species (as evidenced by negative difference values) compared to the respective group's day 0 levels. Furthermore, these groups showed a decrease in total archaeal abundance compared to the untreated group. The RNA vaccine formulation containing both Ac4C and 5hmU modifications decreased the abundance of the methanogen species Mbb. ruminantium M1, but increased total archaeal abundance compared to the untreated group.

[0526] Additionally, methane emissions based on feed intake were measured for calves vaccinated with exemplary RNA vaccine formulations targeting methanogen proteins containing various secretory signals. The data in Figure 5 show that calves vaccinated with an exemplary construct targeting mru1499 and comprising a PPa2 secretory peptide and Sbi adjuvant emitted less methane than pre-vaccination (compare the last two bars on the right). Figure 6 shows reduced methane emissions in animals vaccinated with exemplary RNA vaccine formulations targeting methanogens and / or treated with RNA expressing cytokines (CXCL10, VIP).

[0527] Taken together, the data in this example demonstrate that vaccination with an RNA vaccine formulation targeting methanogens and / or treatment with RNA expressing a cytokine expressed by cattle can reduce the abundance of methanogens and / or reduce methane emissions. The data further support the development of vaccination approaches that include methanogen antigens and / or rumen-associated antigens to reduce methanogens in the gastrointestinal tract of animals and / or reduce methane emissions.

[0528] Example 4: In vivo effects on methanogen abundance and methane emissions in cattle vaccinated with a multicomponent RNA vaccine against multiple methanogen proteins This example describes the in vivo effects on methanogen abundance and methane emissions in cattle vaccinated with an exemplary multi-component RNA vaccine formulation targeting multiple methanogen proteins. The methods used in this example are provided in Example 1 above.

[0529] As shown in Figures 7A-7B, calves vaccinated with an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins showed a reduction in total archaea at +5d and +18d POST compared to abundance at PRE. From PRE to +18d POST, calves vaccinated with the F3 multicomponent RNA vaccine formulation showed a 44% reduction in total archaeal abundance (-32%, normalized to the CNT(OVA) group). Furthermore, calves vaccinated with an exemplary multicomponent RNA vaccine formulation targeting multiple methanogen proteins showed a reduction in individual archaeal species identified at +5d POST compared to abundance at PRE, normalized to the CNT(OVA) group. Note that the CNT(OVA) negative control is a vaccine formulation targeting a non-methanogen protein, ovalbumin, to generate a non-methanogen-specific immune response.

[0530] Additionally, methane emissions per feed intake were measured for calves vaccinated with exemplary multicomponent RNA vaccine formulations targeting multiple methanogens. Figures 8A-8B show that calves vaccinated with the F3 or F4 vaccine formulations (listed in Table 3) emitted less methane (CH4 / day) after injection compared to the control CNT-OVA (Figure 8A, last two right bars). Furthermore, all of the exemplary multicomponent RNA vaccine formulations tested that target multiple methanogen proteins demonstrated lower methane emissions per feed intake (CH4 / day) after injection compared to the control CNT-OVA (Figure 8B).

[0531] Collectively, the data in this example demonstrate that vaccination with a multicomponent RNA vaccine formulation targeting one or more methanogen antigens and / or one or more methanogen species can reduce methanogen abundance and reduce methane emissions. The data further support the development of vaccination approaches that include methanogen antigens or rumen-associated antigens to reduce methanogens in the gastrointestinal tract of animals and to reduce methane emissions.

[0532] Example 5: In vivo effects on growth efficiency of cattle vaccinated against methanogen proteins This example describes the in vivo effects on growth efficiency, as measured by average daily gain (ADG, lbs / day) or average weight gained per day per animal, over a defined feeding period of cattle vaccinated with exemplary RNA vaccine formulations targeting methanogens with various secretory signals, and optionally including a multi-component formulation, cytokines, and Ac4C and / or 5hmU modifications. The methods used in this example are provided in Example 1 above.

[0533] As shown in Figure 9, calves vaccinated with exemplary RNA vaccines targeting methanogen proteins and carrying either the secpep1 or HNpep secretion signal (F3, F4) had significantly increased mean daily gain (ADG) during the "D0-D20" (20-day period following the primary injection) and "D20-D90" (70-day period following the booster injection) periods compared to control animals that remained untreated or were not administered the vaccine formulation. The secpep1 formulation increased mean ADG normalized to untreated (UTD) by more than 0.8 lb / day (a 53% increase from baseline ADG) during the D0-D20 period and more than 1.0 lb / day (a 65% increase from baseline ADG) during the D20-D90 period. Furthermore, the data also showed that exemplary RNA vaccine formulations targeting methanogens and including cytokines and / or 5hmU modifications increased ADG compared to control animals that remained untreated or were not administered the vaccine formulation from D0 to D20 and D20 to D90.

[0534] Additionally, this example describes the in vivo effects on growth efficiency, as measured by average daily gain (ADG, lbs / day), of cattle vaccinated with an exemplary multicomponent RNA vaccine against multiple methanogen proteins. As shown in Figures 10A-10B, calves vaccinated with the multicomponent RNA vaccine against multiple methanogen proteins had increased ADG during the "D0-D25" (25-day period after the primary injection) and "D25-D90" (65-day period after the booster injection) periods compared to animals in the CNT(OVA) group, which had been vaccinated with a formulation targeting the non-methanogen protein ovalbumin to generate a non-methanogen-specific immune response.

[0535] The data in this example demonstrate that vaccination with an RNA vaccine formulation targeting methanogens can increase the growth efficiency of cattle, as measured by the average daily weight gain of cattle. Furthermore, the data demonstrate that vaccination with an RNA vaccine formulation targeting one or more methanogen antigens and / or one or more methanogen species can increase the growth efficiency of cattle compared to non-methanogen-specific vaccines. Without wishing to be bound by any particular theory, in some embodiments, vaccination with a polynucleotide comprising one or more rumen-associated antigens reduces one or more microorganisms (e.g., methanogens) in the digestive tract of an animal (e.g., a ruminant), thereby increasing the growth efficiency of the animal by reducing methane emissions. Furthermore, without wishing to be bound by any particular theory, because methane produced in an animal's digestive tract is energy lost (e.g., not absorbed) from the food consumed by the animal, reducing methane emissions allows the animal to use more energy from the food consumed for growth, thereby increasing the growth efficiency of the animal.

[0536] This data further supports the development of vaccination approaches that include rumen-associated antigens (e.g., rumen antigens and / or methanogen antigens) to enhance growth efficiency in ruminants.

[0537] Numbering Embodiment Embodiment 1. An isolated polynucleotide encoding one or more gastrointestinal-associated antigens, or a fragment or variant thereof.

[0538] Embodiment 2. The isolated polynucleotide of embodiment 1, wherein said one or more rumen-associated antigens comprise one or more rumen antigens and / or one or more methanogen antigens.

[0539] Embodiment 3. The isolated polynucleotide of embodiment 1 or 2, wherein said one or more rumen-associated antigens comprise one or more rumen antigens.

[0540] Embodiment 4. The isolated polynucleotide of any one of the preceding embodiments, wherein said one or more ruminal antigens are derived from a polypeptide involved in adhesion to fermenting bacteria, or a fragment or variant thereof.

[0541] Embodiment 5. The isolated polynucleotide of any one of the preceding embodiments, wherein said polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ruminal antigens.

[0542] Embodiment 6. The isolated polynucleotide of embodiment 1 or 2, wherein the one or more rumen-associated antigens comprise one or more methanogen antigens.

[0543] Embodiment 7. The isolated polynucleotide of embodiment 6, wherein the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 methanogen antigens.

[0544] Embodiment 8. The isolated polynucleotide of embodiment 6, wherein the polynucleotide comprises about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 20, about 4 to about 20, about 5 to about 20, about 6 to about 20, about 7 to about 20, about 8 to about 20, about 9 to about 20, about 10 to about 20, or about 15 to about 20 methanogen antigens.

[0545] Embodiment 9. The isolated polynucleotide of any one of embodiments 6 to 8, wherein the one or more methanogen antigens are the same, e.g., have the same sequence.

[0546] Embodiment 10. The isolated polynucleotide of any one of embodiments 6 to 8, wherein the one or more methanogen antigens are different, e.g., have different sequences.

[0547] Embodiment 11. The isolated polynucleotide of any one of embodiments 1-2 or 6-10, wherein the one or more methanogen antigens are derived from a polypeptide found on the cell surface of a wild-type methanogen, or a fragment or variant thereof.

[0548] Embodiment 12. The isolated polynucleotide of any one of embodiments 1-2 or 6-11, wherein the one or more methanogen antigens are secreted.

[0549] Embodiment 13. The isolated polynucleotide of any one of embodiments 1-2 or 6-12, wherein the one or more methanogen antigens comprise a peptide involved in adhesion, attachment, or motility, or a fragment or variant of a peptide involved in adhesion, attachment, or motility.

[0550] Embodiment 14. The isolated polynucleotide of embodiment 12 or 13, wherein the secreted methanogen antigen comprises a signal peptide.

[0551] Embodiment 15. The isolated polynucleotide of embodiment 14, wherein the signal peptide is predictable using a prediction algorithm, and optionally, the prediction score is at least 0.5.

[0552] Embodiment 16. The isolated polynucleotide of any one of embodiments 1-2 or 6-15, wherein the one or more methanogen antigens comprise one or more peptides having at least 80% sequence identity to a methanogen protein.

[0553] Embodiment 17. The isolated polynucleotide of any one of embodiments 1-2 or 6-16, wherein the polynucleotide comprises multiple methanogen antigens, each of which has at least 80% sequence identity to each other.

[0554] Embodiment 18. The isolated polynucleotide of any one of embodiments 1-2 or 6-17, wherein the polynucleotide comprises a plurality of methanogen antigens, and each methanogen antigen of the plurality is associated with a different methanogen species.

[0555] Embodiment 19. The isolated polynucleotide of any one of embodiments 1-2 or 6-18, wherein the polynucleotide comprises a plurality of methanogen antigens, and the plurality of methanogen antigens are associated with at least two different, at least three different, at least four different, or at least five different methanogen species.

[0556] Embodiment 20. The isolated polynucleotide of any one of embodiments 1-2 or 6-19, wherein the polynucleotide comprises a plurality of methanogen antigens, and each methanogen antigen of the plurality is associated with the same methanogen species.

[0557] Embodiment 21. The methanogenic bacterial species is Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp.UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, or Mathanosarcina acetivorans.

[0558] Embodiment 22. The isolated polynucleotide of any one of embodiments 18 to 21, wherein the methanogen species is or comprises Methanobrevibacter ruminantium, Methanobrevibacter Smithii, or Methanobrevibacter oralis.

[0559] Embodiment 23. The isolated polynucleotide of any one of embodiments 18 to 21, wherein the methanogen species is or comprises Methanobrevibacter ruminantium, Methanobrevibacter Smithii, Methanobrevibacter oralis, Methanomicrobium mobile, or Methanosphaera stadtmanae.

[0560] Embodiment 24. The isolated polynucleotide of any one of embodiments 1-2 or 6-23, wherein the one or more methanogen antigens comprise one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity.

[0561] Embodiment 25. The polypeptide having signal peptidase activity is

[0562] (i) It is membrane-bound;

[0563] (ii) have the ability to cleave one or more signal peptides;

[0564] (iii) plays a role in converting secreted proteins to their mature forms (e.g., from non-secreted to secreted forms); and / or

[0565] (iv) The isolated polynucleotide of embodiment 24, which is a combination thereof.

[0566] Embodiment 26. The isolated polynucleotide of any one of embodiments 1-2 or 6-25, wherein the one or more methanogen antigens (1) have at least 80% sequence identity to an immunoglobulin (Ig)-like peptide, or (2) comprise one or more peptides having substantially similar function as an Ig-like domain-containing polypeptide.

[0567] Embodiment 27. The isolated polynucleotide of embodiment 26, wherein the Ig-like domain-containing polypeptide comprises a polypeptide characterized as a cell surface protein that facilitates binding to other cells and / or cell surfaces.

[0568] Embodiment 28. The isolated polynucleotide of any one of embodiments 1-2 or 6-27, wherein the one or more methanogen antigens comprise an adhesin or a fragment or variant thereof.

[0569] Embodiment 29. The isolated polynucleotide of embodiment 28, wherein said one or more methanogen antigens comprise an adhesin protein listed in Table 1, or a sequence having at least 85% identity thereto.

[0570] Embodiment 30. The isolated polynucleotide of any one of embodiments 1-2 or 6-29, wherein the one or more methanogen antigens comprise mru1499 or a fragment or variant thereof.

[0571] Embodiment 31. The one or more methanogen antigens comprise an antigenic sequence set forth in any one of SEQ ID NOs: 2, 6, or 8, or a sequence that is at least 85% identical thereto, or are encoded by a sequence set forth in any one of SEQ ID NOs: 1, 5, or 7, or a sequence that is at least 85% identical thereto. The isolated polynucleotide of any one of embodiments 1-2 or 6-30.

[0572] Embodiment 32. The isolated polynucleotide of any one of embodiments 1-2 or 6-31, wherein the one or more methanogen antigens comprise a pili protein or a fragment or variant thereof.

[0573] Embodiment 33. The isolated polynucleotide of any one of embodiments 1-2 or 6-32, wherein the methanogen antigen comprises a flagellin protein or a fragment or variant thereof.

[0574] Embodiment 34. The isolated polynucleotide of any one of embodiments 1-2 or 6-33, wherein the one or more methanogen antigens (1) have at least 80% sequence identity to an archaeal oligosaccharyltransferase (AglB) polypeptide, or (2) comprise one or more peptides having substantially similar function as an AglB polypeptide.

[0575] Embodiment 35. The isolated polynucleotide of embodiment 34, wherein the AglB polypeptide has N-glycosylation activity.

[0576] Embodiment 36. The one or more methanogen antigens are:

[0577] (i) one or more peptides with at least 80% sequence identity to a methanogen protein;

[0578] (ii) one or more secreted antigens containing a signal peptide;

[0579] (iii) multiple peptides having at least 80% sequence identity with each other;

[0580] (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species;

[0581] (v) one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity; or

[0582] (vi) The isolated polynucleotide of any one of the preceding embodiments, including combinations thereof.

[0583] Embodiment 37. The isolated polynucleotide of embodiment 36, wherein the polynucleotide comprises three methanogen antigens.

[0584] Embodiment 38. The isolated polynucleotide of embodiment 37, wherein the three methanogen antigens are associated with at least three different methanogen species.

[0585] Embodiment 39. The one or more methanogen antigens are: (i) one or more peptides with at least 80% sequence identity to a methanogen protein; (ii) one or more secreted antigens containing a signal peptide; (iii) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; (iv) one or more peptides having at least 80% sequence identity to an AglB polypeptide or having substantially the same function as an AglB polypeptide; or (vi) The isolated polynucleotide of any one of embodiments 1 to 35, including combinations thereof.

[0586] Embodiment 40. The isolated polynucleotide of embodiment 39, wherein the polynucleotide comprises five methanogen antigens.

[0587] Embodiment 41. The isolated polynucleotide of embodiment 40, wherein said five methanogen antigens are associated with at least five different methanogen species.

[0588] Embodiment 42. The one or more methanogen antigens are:

[0589] (i) one or more peptides with at least 80% sequence identity to a methanogen protein;

[0590] (ii) one or more secreted antigens containing a signal peptide;

[0591] (iii) multiple peptides having at least 80% sequence identity with each other;

[0592] (iv) multiple peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species;

[0593] (v) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having substantially similar function to an Ig-like domain-containing polypeptide; and / or

[0594] (vi) The isolated polynucleotide of any one of embodiments 1 to 35, including combinations thereof.

[0595] Embodiment 43. The isolated polynucleotide of embodiment 42, wherein the polynucleotide comprises eight methanogen antigens.

[0596] Embodiment 44. The isolated polynucleotide of embodiment 43, wherein said eight methanogen antigens are associated with at least three different methanogen species.

[0597] Embodiment 45. The isolated polynucleotide of any one of the preceding embodiments, wherein the one or more rumen antigens and / or the one or more methanogen antigens are each located on a separate nucleotide sequence.

[0598] Embodiment 46. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least two methanogen antigens, and the at least two methanogen antigens are located on separate nucleotide sequences.

[0599] Embodiment 47. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least three methanogen antigens, and the at least three methanogen antigens are located on separate nucleotide sequences.

[0600] Embodiment 48. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least four methanogen antigens, and wherein the at least four methanogen antigens are located on separate nucleotide sequences.

[0601] Embodiment 49. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least five methanogen antigens, and wherein the at least five methanogen antigens are located on separate nucleotide sequences.

[0602] Embodiment 50. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least six methanogen antigens, and wherein the at least six methanogen antigens are located on separate nucleotide sequences.

[0603] Embodiment 51. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least seven methanogen antigens, and wherein the at least seven methanogen antigens are located on separate nucleotide sequences.

[0604] Embodiment 52. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least eight methanogen antigens, and wherein the at least eight methanogen antigens are located on separate nucleotide sequences.

[0605] Embodiment 53. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least nine methanogen antigens, and wherein the at least nine methanogen antigens are located on separate nucleotide sequences.

[0606] Embodiment 54. The isolated polynucleotide of any one of embodiments 1-2 and 6-45, wherein the polynucleotide comprises at least 10 methanogen antigens, and wherein the at least 10 methanogen antigens are located on separate nucleotide sequences.

[0607] Embodiment 55. The isolated polynucleotide of any one of embodiments 1 to 45, wherein the one or more rumen antigens and / or the one or more methanogen antigens are located on the same nucleotide sequence.

[0608] Embodiment 56. The isolated polynucleotide of any one of embodiments 1-2 and 6-55, wherein the one or more methanogen antigens comprise a polypeptide antigen sequence set out in Table 2, or a variant and / or fragment thereof.

[0609] Embodiment 57. The isolated polynucleotide of any one of embodiments 1-2 and 6-55, wherein the one or more methanogen antigens comprise a polypeptide antigen sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an antigen sequence set out in Table 2.

[0610] Embodiment 58. The isolated polynucleotide of any one of the preceding embodiments, wherein the polynucleotide comprises a signal peptide.

[0611] Embodiment 59. The isolated polynucleotide of embodiment 58, wherein the signal peptide has at least 85% homology to an archaeal signal peptide.

[0612] Embodiment 60. The isolated polynucleotide of embodiment 58, wherein the signal peptide has at least 85% homology to a bacterial signal peptide.

[0613] Embodiment 61. The isolated polynucleotide of any one of embodiments 58 to 60, wherein the signal peptide is or comprises a PPA2 signal peptide, or a fragment or variant thereof.

[0614] Embodiment 62. The isolated polynucleotide of any one of embodiments 58 to 60, wherein the signal peptide is or comprises an SSP signal peptide, or a fragment or variant thereof.

[0615] Embodiment 63. The isolated polynucleotide of embodiment 58, wherein the signal peptide is or comprises the SARS-CoV-2 spike secretion signal, or a fragment or variant thereof.

[0616] Embodiment 64. The isolated polynucleotide of any one of embodiments 58 to 63, wherein the signal peptide is located at the N-terminus of the rumen-associated antigen sequence.

[0617] Embodiment 65. The polynucleotide comprises: (i) a first nucleotide sequence encoding one or more rumen antigens; (ii) a second nucleotide sequence encoding one or more methanogen antigens, and / or (iii) the isolated polypeptide of embodiment 2, comprising a third nucleotide sequence encoding a chemokine and / or cytokine.

[0618] Embodiment 66. The isolated polynucleotide of embodiment 65, wherein the first nucleotide sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ruminal antigens.

[0619] Embodiment 67. The isolated polynucleotide of embodiment 65 or 66, wherein the second nucleotide sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 methanogen antigens.

[0620] Embodiment 68. The isolated polynucleotide of any one of embodiments 65 to 67, wherein the first nucleotide sequence, the second nucleotide sequence, and / or the third nucleotide sequence are located on a single polynucleotide.

[0621] Embodiment 69. The isolated polynucleotide of any one of embodiments 65 to 67, wherein the first nucleotide sequence, the second nucleotide sequence, and / or the third nucleotide sequence are located on different polynucleotides.

[0622] Embodiment 70. The isolated polynucleotide of any one of the preceding embodiments, wherein the polynucleotide comprises a transmembrane domain.

[0623] Embodiment 71. The isolated polynucleotide of any one of the preceding embodiments, further comprising a complement C3d-binding polypeptide derived from the immunoglobulin-binding protein (Sbi) of Staphylococcus aureus.

[0624] Embodiment 72. The complement C3d-binding polypeptide is

[0625] (i) domain III of Sbi of Staphylococcus aureus , or a functional fragment or variant thereof;

[0626] (ii) domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof; or

[0627] (iii) The isolated polynucleotide of embodiment 71, which is or comprises both (i) and (ii).

[0628] Embodiment 73. The isolated polynucleotide of any one of the preceding embodiments, wherein the polynucleotide is or comprises DNA.

[0629] Embodiment 74. The isolated polynucleotide of any one of the preceding embodiments, wherein the polynucleotide is or comprises RNA.

[0630] Embodiment 75. The isolated polynucleotide of embodiment 74, wherein the RNA comprises a 5' cap.

[0631] Embodiment 76. The isolated polynucleotide of embodiment 74 or 75, wherein the RNA comprises a poly(A) tail.

[0632] Embodiment 77. The isolated polynucleotide of any one of embodiments 74 to 76, wherein the polynucleotide sequence comprises one or more ribonucleotides comprising a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine, and the modified ribonucleotide has the structure: R is 5' monophosphate, 5' diphosphate, or 5' triphosphate. [ka]

[0633] Embodiment 78. The isolated polynucleotide of embodiment 77, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than N4-acetylcytidine, and optionally the nucleosides are selected from adenosine, inosine, guanosine, cytidine, or uridine, or combinations thereof.

[0634] Embodiment 79. The isolated polynucleotide of embodiment 78, wherein the nucleoside of the one or more modified ribonucleotides is 5-hydroxymethyluridine, and the modified ribonucleotide has the structure: R is 5' monophosphate, 5' diphosphate, or 5' triphosphate. [ka]

[0635] Embodiment 80. The isolated polynucleotide of any one of embodiments 74-76, wherein the polynucleotide sequence comprises one or more ribonucleotides comprising a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and has the structure: R is 5' monophosphate, 5' diphosphate, or 5' triphosphate. [ka]

[0636] Embodiment 81. The isolated polynucleotide of embodiment 80, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than 5-hydroxymethyluridine, and wherein the one or more modified ribonucleotides comprise a nucleoside selected from adenosine, inosine, guanosine, cytidine, or uridine, or a combination thereof.

[0637] Embodiment 82. The isolated polynucleotide of embodiment 80 or 81, wherein the nucleoside of the one or more modified ribonucleotides is N4-acetylcytidine, and the modified ribonucleotide has the structure: R is 5' monophosphate, 5' diphosphate, or 5' triphosphate. [ka]

[0638] Embodiment 83. A polypeptide encoded by a polynucleotide according to any one of the preceding embodiments.

[0639] Embodiment 84. A composition comprising one or more polyribonucleotides according to any one of embodiments 1 to 82, or a polypeptide according to embodiment 83.

[0640] Embodiment 85. The composition of embodiment 84, wherein the composition is a pharmaceutical composition.

[0641] Embodiment 86. The composition of embodiment 84, wherein the composition is an immunogenic composition.

[0642] Embodiment 87. The composition of embodiment 84, wherein the composition is a vaccine composition.

[0643] Embodiment 88. The composition of any one of embodiments 83 to 87, wherein the composition is formulated for delivery by a carrier.

[0644] Embodiment 89. The composition of claim 88, wherein the carrier is a lipid nanoparticle, a cationic lipid, or a polymer particle.

[0645] Embodiment 90. The composition of any one of embodiments 83 to 87, wherein the composition is formulated for delivery without a carrier.

[0646] Embodiment 91. A method comprising administering to a cell, tissue, or animal a composition described in any one of embodiments 84 to 90.

[0647] Embodiment 92. The method of embodiment 91, wherein the method is a vaccination method.

[0648] Embodiment 93 The method of embodiment 90 or 91, wherein the animal is a ruminant.

[0649] Embodiment 94. The method of any one of embodiments 90-92, wherein the ruminant is a cow, sheep, goat, buffalo, elk, antelope, caribou, or deer.

[0650] Embodiment 95. The method of any one of embodiments 90 to 92, wherein the animal is a livestock animal.

[0651] Embodiment 96. The method of any one of embodiments 90 to 95, wherein the composition reduces methane emissions from the animal upon administration of the composition to the animal compared to an otherwise identical animal not administered the composition or administered a different composition.

[0652] Embodiment 97. The method of embodiment 96, wherein the reduction in methane emissions is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to animals not administered the composition or administered a different composition.

[0653] Embodiment 98. The method of embodiment 97, wherein the reduction in methane emissions is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to an otherwise similar animal not administered the composition or administered a different composition.

[0654] Embodiment 99. The method of any one of embodiments 90 to 98, wherein the composition reduces the population of microorganisms in the animal upon administration of the composition to the animal compared to an animal not administered the composition or administered a different composition.

[0655] Embodiment 100. The method of embodiment 99, wherein the microorganism is a methanogen.

[0656] Embodiment 101. The method of embodiment 100, wherein the methanogens comprise methanogens from one or more of the following phylogenetic groups: Methanobrevibacter, Methanosphaera, Methanobacterium, Methanosarcinales, Methanomicrobiales, Methanothermobacter, Candidatus, Methanomethylophilus, Thermoplasmatales.

[0657] Embodiment 102. The methanogen is Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp.UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methancaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans, or any combination thereof.

[0658] Embodiment 103. The method of any one of embodiments 100 to 102, wherein the abundance of said methanogens is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the abundance of methanogens in an animal not administered the composition or administered a different composition.

[0659] Embodiment 104. The method of any one of embodiments 100 to 102, wherein the abundance of methanogens is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the abundance of methanogens in an otherwise equivalent animal not administered the composition or administered a different composition.

[0660] Embodiment 105. The method of any one of embodiments 100 to 104, wherein the composition reduces the abundance of all or substantially all methanogens (e.g., total methanogen abundance) in the animal by administering the composition to the animal compared to the abundance of methanogens in an otherwise equivalent animal that has not been administered the composition or that has been administered a different composition.

[0661] Embodiment 106. The method of embodiment 105, wherein the abundance of total methanogens is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the abundance of methanogens in an otherwise comparable animal not administered the composition or administered a different composition.

[0662] Embodiment 107. The method of embodiment 105, wherein the abundance of total methanogens is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the abundance of methanogens in an otherwise comparable animal not administered the composition or administered a different composition.

[0663] Embodiment 108. The method of any one of embodiments 90 to 107, wherein the composition increases the growth rate of the animal upon administration of the composition to the animal compared to the growth rate of an otherwise comparable animal not administered the composition or administered a different composition.

[0664] Embodiment 109. The method of embodiment 108, wherein growth rate is assessed by measuring the body weight of the animal at one or more time points.

[0665] Embodiment 110. The method of embodiment 109, wherein the body weight of the animal is measured daily.

[0666] Embodiment 111. The method of any one of embodiments 108 to 110, wherein the growth rate of the animal is increased over a period of time.

[0667] Embodiment 112. The method of claim 11, wherein the period comprises at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0668] Embodiment 113. The method of any one of embodiments 108 to 110, wherein said increase in growth rate comprises daily weight gain of said animal.

[0669] Embodiment 114. The method of embodiment 113, wherein the daily weight gain of the animal is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% increase compared to the daily weight gain of an otherwise comparable animal not administered the composition or administered a different composition.

[0670] Embodiment 115. The method of embodiment 113, wherein the daily weight gain of the animal is increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the daily weight gain of an otherwise comparable animal not administered the composition or administered a different composition.

[0671] Embodiment 116. The method of any one of embodiments 91 to 115, wherein the composition modifies the output of cellulose fermentation.

[0672] Embodiment 117. The method of any one of embodiments 91 to 116, wherein the composition is delivered by a carrier.

[0673] Embodiment 118. The method of any one of embodiments 91 to 117, wherein the composition is delivered without a carrier.

[0674] Embodiment 119. The method of any one of embodiments 91 to 118, comprising administering a single dose of the composition to the animal.

[0675] Embodiment 120. The method of any one of embodiments 91 to 119, comprising administering multiple doses of the composition to the animal.

[0676] Embodiment 121. The method of embodiment 120, wherein the animal is administered a first dose of the composition followed by one or more subsequent doses of the composition.

[0677] Embodiment 122. The method of embodiment 121, wherein the first dose and the one or more subsequent doses of the composition comprise the same methanogen antigen and / or rumen antigen.

[0678] Embodiment 123. The method of embodiment 121, wherein the first dose and the one or more subsequent doses of the composition comprise different methanogen antigens and / or rumen antigens.

[0679] Embodiment 124. The method of any one of embodiments 91 to 123, wherein the one or more methanogen antigens and / or the one or more rumen antigens are specific for the animal.

[0680] Embodiment 125. The one or more methanogen antigens and / or the one or more rumen antigens specific for the animal are: (i) identifying one or more methanogen antigens and / or one or more rumen antigens expressed in the animal; and (ii) in response to said identifying, selecting one or more methanogen antigens and / or said one or more rumen antigens for inclusion in said composition.

[0681] Embodiment 126. The method of embodiment 124 or 125, wherein the one or more methanogen antigens and / or the one or more rumen antigens specific to the animal are different from the one or more methanogen antigens and / or the one or more rumen antigens specific to a different animal.

[0682] Embodiment 127. The different animals are:

[0683] (i) animals of different species;

[0684] (ii) animals of different sexes;

[0685] (iii) animals of different ages; or

[0686] (iv) animals located in different geographic locations;

[0687] (v) the same animal at different time points, and / or

[0688] (vi) The method of embodiment 126, including combinations thereof.

[0689] Embodiment 128. The method of any one of embodiments 120 to 127, wherein the composition is administered to the animal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 times.

[0690] Embodiment 129. The method of any one of embodiments 120 to 128, wherein the composition is administered once every week, two weeks, three weeks, one month, two months, three months, four months, five months, or six months.

[0691] Embodiment 130. The method of any one of embodiments 91 to 129, wherein the composition is administered in combination with one or more additional agents.

[0692] Embodiment 131. The method of embodiment 130, wherein one or more additional agents comprise chemical additives, biological feed additives.

[0693] Embodiment 132. The method of any one of embodiments 91 to 131, wherein the composition is administered in combination with one or more additional compositions.

[0694] Embodiment 133. The method of embodiment 132, wherein the additional composition immunizes the animal against a disease, such as an infection.

[0695] Embodiment 134. A method for producing a composition comprising one or more polyribonucleotides according to any one of embodiments 1 to 82.

[0696] Embodiment 135. The method of embodiment 134, wherein the composition is an RNA composition.

[0697] Embodiment 136.

[0698] (i) the method is an in vitro transcription method, and / or

[0699] (ii) The method of embodiment 134 or 135, wherein said method produces a plurality of polyribonucleotides.

[0700] Embodiment 137. The isolated polynucleotide of any one of embodiments 1 to 5 or 58 to 81, wherein the rumen antigen comprises an antigen expressed in any one or all of: (a) rumen ciliates that live symbiotically with methanogens; (b) hydrogen-producing organisms that feed on methanogens; (c) taxa associated with low feed efficiency.

[0701] Embodiment 138. An isolated polynucleotide according to embodiment 137, wherein the ruminal ciliates symbiotic with the methanogens include one or more or any combination of Diplodinium dentatum; Diploplastron affine, Enoploplastron triloricatum; Entodinium simplex; Entodinium caudatum; Entodinium longinucleatum; Epidinium ecaudatum; Eremoplastron bovis; Eudiplodinium maggii; Ostracodinium obtusum; or Polyplastron multivesiculatum.

[0702] Embodiment 139. The isolated polynucleotide of embodiment 137, wherein the hydrogen-producing organism comprises one or more of Butyrivibrio proteoclasticus; Bacteroides thetaiotaomicron; Ruminococcus flavefaciens; Ruminococcus albus, or any combination thereof.

[0703] Embodiment 140. The isolated polynucleotide of embodiment 137, wherein the taxa associated with low feed efficiency include one or more of Veillonellaceae; Prevotellaceae; Lachnospiraceae; Succinivibrionaceae; Fibrobacteraceae; Anaerovibrio; Clostridiales; Prevotella; Ruminococcaceae.

[0704] Embodiment 141. The method of any one of embodiments 100 to 104, wherein the composition reduces the abundance of at least one methanogen species in the animal by administering the composition to the animal, compared to the abundance of the same methanogen species in an otherwise identical animal that has not been administered the composition or that has been administered a different composition.

[0705] Embodiment 142. The method of embodiment 141, wherein the abundance of at least one methanogen species is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the abundance of the same methanogen species in an otherwise comparable animal not administered the composition or administered a different composition.

[0706] Embodiment 143. The method of embodiment 141, wherein the abundance of at least one methanogen species is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% compared to the abundance of the same methanogen species in an otherwise comparable animal not administered the composition or administered a different composition.

[0707] Embodiment 144. The method of any one of embodiments 91 to 123, wherein the one or more methanogen antigens and / or the one or more rumen antigens are present and / or expressed in the animal and one or more additional animals.

[0708] Embodiment 145. The animal and the one or more additional animals:

[0709] (i) Animals of the same or different species;

[0710] (ii) animals of the same or different sex;

[0711] (iii) an animal of substantially the same age or developmental stage; or

[0712] (iv) Animals located in the same or substantially similar geographic location; or

[0713] (v) The method of embodiment 144, which is or comprises any combination of (i) to (iv).

[0714] Embodiment 146. The method of any one of embodiments 144 to 156, wherein the composition is administered to the animal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 times.

[0715] Embodiment 147. The method of any one of embodiments 144 to 146, wherein the composition is administered once every week, two weeks, three weeks, one month, two months, three months, four months, five months, or six months.

[0716] Embodiment 148. The method of any one of embodiments 144 to 147, wherein the composition is administered in combination with one or more additional agents.

[0717] Embodiment 149. The method of embodiment 148, wherein one or more additional agents comprise chemical additives, biological feed additives.

[0718] Embodiment 150. The method of any one of embodiments 144 to 149, wherein the composition is administered in combination with one or more additional compositions.

[0719] Embodiment 151. The method of embodiment 150, wherein the additional composition immunizes the animal against a disease, such as an infection.

[0720] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. It should be understood that the invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive language, etc. from one or more of the recited claims, introduced into another claim that depends from the same base claim (or any other claim, if relevant), unless otherwise indicated or unless a contradiction or inconsistency would arise apparent to one of ordinary skill in the art. It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from a claim, regardless of whether a specific exclusion is recited in the specification. The scope of the invention is not intended to be limited to the detailed description of the invention set forth above, but rather is as set forth in the following claims.

Claims

1. An isolated polynucleotide encoding one or more rumen-associated antigens, a fragment thereof, a variant thereof, or a variant fragment thereof.

2. 2. The isolated polynucleotide of claim 1, wherein the one or more rumen-associated antigens comprise one or more rumen antigens and / or one or more methanogen antigens.

3. 3. The isolated polynucleotide of claim 1 or 2, wherein the one or more rumen-associated antigens comprise one or more rumen antigens.

4. 10. The isolated polynucleotide of any one of the preceding claims, wherein said one or more ruminal antigens are derived from a polypeptide involved in adhesion to fermenting bacteria, or a fragment or variant or variant fragment thereof.

5. 5. The isolated polynucleotide of claim 3 or 4, wherein the one or more ruminal antigens comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ruminal antigens.

6. 3. The isolated polynucleotide of claim 1 or 2, wherein the one or more rumen-associated antigens comprise one or more methanogen antigens.

7. 7. The isolated polynucleotide of claim 6, wherein the one or more methanogen antigens include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 methanogen antigens.

8. 8. The isolated polynucleotide of claim 6 or 7, wherein the one or more methanogen antigens are the same.

9. 8. The isolated polynucleotide of claim 6 or 7, wherein the one or more methanogen antigens are different.

10. The one or more methanogen antigens are: (i) one or more peptides having at least 80% sequence identity to a methanogen protein; (ii) one or more secretory antigens comprising a signal peptide; (iii) a plurality of peptides having at least 80% sequence identity with each other; (iv) a plurality of peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; (v) one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity; (vi) one or more peptides having at least 80% sequence identity to an AglB polypeptide or having substantially the same function as an AglB polypeptide; (vii) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having substantially the same function as an Ig-like domain-containing polypeptide; or (viii) The isolated polynucleotide according to any one of claims 6 to 9, including combinations thereof.

11. The one or more methanogen antigens are: (i) one or more peptides having at least 80% sequence identity to a methanogen protein; (ii) one or more secretory antigens comprising a signal peptide; (iii) a plurality of peptides having at least 80% sequence identity with each other; (iv) a plurality of peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; (v) one or more peptides having at least 80% sequence identity to a polypeptide having signal peptidase activity; or (vi) The isolated polynucleotide according to any one of claims 6 to 9, including combinations thereof.

12. 12. The isolated polynucleotide of claim 11, wherein the polynucleotide comprises three methanogen antigens, optionally wherein the three methanogen antigens are associated with at least three different methanogen species.

13. The one or more methanogen antigens are: (i) one or more peptides having at least 80% sequence identity to a methanogen protein; (ii) one or more secretory antigens comprising a signal peptide; (iii) a plurality of peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; (iv) one or more peptides having at least 80% sequence identity to an AglB polypeptide or having substantially the same function as an AglB polypeptide; or (vi) The isolated polynucleotide according to any one of claims 6 to 9, including combinations thereof.

14. 14. The isolated polynucleotide of claim 13, wherein the polynucleotide comprises five methanogen antigens, optionally wherein the five methanogen antigens are associated with at least five different methanogen species.

15. The one or more methanogen antigens are: (i) one or more peptides having at least 80% sequence identity to a methanogen protein; (ii) one or more secretory antigens comprising a signal peptide; (iii) a plurality of peptides having at least 80% sequence identity with each other; (iv) a plurality of peptides associated with at least two different, at least three different, at least four different, or at least five different methanogen species; (v) one or more peptides having at least 80% sequence identity to an Ig-like domain-containing polypeptide or having substantially the same function as an Ig-like domain-containing polypeptide; or (vi) The isolated polynucleotide according to any one of claims 6 to 9, including combinations thereof.

16. 16. The isolated polynucleotide of claim 15, wherein the polynucleotide comprises eight methanogen antigens, optionally wherein the eight methanogen antigens are associated with at least three different methanogen species.

17. 17. The isolated polynucleotide of any one of claims 1-2 or 6-16, wherein the one or more methanogen antigens are derived from a polypeptide found on the cell surface of a wild-type methanogen, or a fragment or variant thereof.

18. 20. The isolated polynucleotide of any one of claims 1-2 or 6-17, wherein the methanogen antigen is secreted.

19. 19. The isolated polynucleotide of any one of claims 1-2 or 6-18, wherein the one or more methanogen antigens comprise a peptide involved in adhesion, attachment, or motility, or a fragment or variant of a peptide involved in adhesion, attachment, or motility.

20. 19. The isolated polynucleotide of any one of claims 1-2 or 6-18, wherein the methanogen antigen comprises an adhesin, or a fragment or variant thereof; a pili protein, or a fragment or variant thereof; and / or a flagellin protein, or a fragment or variant thereof.

21. 21. The isolated polynucleotide of claim 20, wherein the methanogen antigen comprises an adhesin protein listed in Table 1, or a sequence having at least 85% identity thereto.

22. 22. The isolated polynucleotide of any one of claims 1-2 or 6-21, wherein the methanogen antigen comprises an antigen presented in Table 2, or a fragment or variant or variant fragment thereof, or a sequence that is at least 85% identical to an antigen sequence presented in Table 2.

23. 10. The isolated polynucleotide of any one of the preceding claims, wherein the one or more rumen antigens and / or the one or more methanogen antigens are located on different polynucleotides.

24. 24. The isolated polynucleotide of any one of claims 1 to 23, wherein said one or more rumen antigens and / or said one or more methanogen antigens are located on one polynucleotide.

25. 10. The isolated polynucleotide of any one of the preceding claims, wherein the polynucleotide comprises a signal peptide or a variant or fragment thereof.

26. 26. The isolated polynucleotide of claim 25, wherein the signal peptide has at least 85% homology to an archaeal or bacterial signal peptide.

27. The signal peptide (i) the PPA2 signal peptide, or a fragment or variant thereof; (ii) an SSP signal peptide, or a fragment or variant thereof; (iii) the SARS-CoV-2 spike secretory signal, or a fragment or variant thereof; or (iv) The isolated polynucleotide of claim 25 or 26, which is or comprises any combination of (i) to (iii).

28. 28. The isolated polynucleotide of any one of claims 25 to 27, wherein the signal peptide is located N-terminal to the rumen-associated antigen sequence.

29. the polynucleotide is (i) a first nucleotide sequence encoding one or more rumen antigens; (ii) a second nucleotide sequence encoding one or more methanogen antigens; and / or (iii) an isolated polypeptide according to any one of the preceding claims, comprising a third nucleotide sequence encoding a chemokine and / or a cytokine.

30. the first nucleotide sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 rumen antigens; and 30. The isolated polynucleotide of claim 29, wherein the second nucleotide sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 methanogen antigens.

31. 10. The isolated polynucleotide of any one of the preceding claims, wherein the polynucleotide comprises a transmembrane domain.

32. 10. The isolated polynucleotide of any one of the preceding claims, further comprising a complement C3d-binding polypeptide derived from the immunoglobulin-binding protein (Sbi) of Staphylococcus aureus.

33. the complement C3d-binding polypeptide (i) domain III of Staphylococcus aureus Sbi, or a functional fragment or variant thereof; (ii) domain IV of Staphylococcus aureus Sbi, or a functional fragment or variant thereof; or (iii) The isolated polynucleotide of claim 32, which is or comprises both (i) and (ii).

34. 10. The isolated polynucleotide of any one of the preceding claims, wherein the polynucleotide is or comprises DNA.

35. 35. The isolated polynucleotide of any one of claims 1 to 34, wherein the polynucleotide is or comprises RNA.

36. 36. The isolated polynucleotide of claim 35, wherein the polynucleotide sequence comprises one or more ribonucleotides comprising a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine, and the modified ribonucleotide has the structure: R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. 【Chemistry 1】

37. 37. The isolated polynucleotide of claim 36, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than N4-acetylcytidine, and optionally the nucleosides are selected from adenosine, inosine, guanosine, cytidine, or uridine, or any combination thereof.

38. 37. The isolated polynucleotide of claim 36, wherein the nucleoside of the one or more modified ribonucleotides is 5-hydroxymethyluridine, and the modified ribonucleotide has the structure: R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. 【Chemistry 2】

39. 36. The isolated polynucleotide of any one of claims 33 to 35, wherein the polynucleotide sequence comprises one or more ribonucleotides comprising a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine, and the modified ribonucleotide has the structure: R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. 【Transformation 3】

40. 40. The isolated polynucleotide of claim 39, wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than 5-hydroxymethyluridine, wherein the one or more modified ribonucleotides comprise a nucleoside selected from adenosine, inosine, guanosine, cytidine, or uridine, or any combination thereof.

41. 41. The isolated polynucleotide of claim 39 or 40, wherein the nucleoside of the one or more modified ribonucleotides is N4-acetylcytidine, and the modified ribonucleotide has the structure: R is a 5' monophosphate, a 5' diphosphate, or a 5' triphosphate. 【Chemistry 4】

42. A polypeptide encoded by a polynucleotide according to any one of the preceding claims.

43. A composition comprising one or more polyribonucleotides according to any one of claims 1 to 41, or a polypeptide according to claim 42.

44. 44. The composition of claim 43, wherein the composition is a pharmaceutical composition.

45. 45. The composition of claim 44, wherein the composition is an immunogenic and / or vaccine composition.

46. A method comprising administering to a cell, tissue, or animal the composition of any one of claims 43 to 45.

47. 47. The method of claim 46, wherein said method is a vaccination method and said animal is a ruminant or a livestock animal.

48. 48. The method of claim 46 or 47, wherein the composition reduces methane emissions from the animal upon administration of the composition to the animal compared to an otherwise identical animal not administered the composition or administered a different composition.

49. 49. The method of claim 48, wherein the reduction in methane emissions is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to otherwise identical animals not administered the composition or administered a different composition.

50. 50. The method of any one of claims 46 to 49, wherein the composition reduces the population of microorganisms in the animal upon administration of the composition to the animal compared to an otherwise identical animal not administered the composition or administered a different composition.

51. 51. The method of claim 50, wherein the microorganism is a methanogen, optionally wherein the methanogen comprises a methanogen from one or more of the following phylogenetic groups: Methanobrevibacter, Methanosphaera, Methanobacterium, Methanosarcinales, Methanomicrobiales, Methanothermobacter, Candidatus, Methanomethylophilus, Thermoplasmatales.

52. The methanogenic bacteria species are Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, Methanomicrobium. mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp. UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methanaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete 52. The method of claim 51 , comprising: Azotobacter thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans, or any combination thereof.

53. 53. The method of any one of claims 46 to 52, wherein the composition increases the growth rate of the animal upon administration of the composition to the animal compared to the growth rate of an otherwise comparable animal not administered the composition or administered a different composition.

54. 54. The method of claim 53, wherein the increase in growth rate comprises daily weight gain of the animal, and optionally, the daily weight gain of the animal is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% increase in body weight compared to an otherwise similar animal not administered the composition or administered a different composition.

55. 55. The method of any one of claims 46 to 54, comprising administering a single dose of the composition to the animal.

56. 55. The method of any one of claims 46 to 54, comprising administering multiple doses of the vaccine composition to the animal.

57. 57. The method of claim 56, wherein the animal is administered a first dose of the composition followed by one or more subsequent doses of the composition.

58. 57. The method of claim 56, wherein the first dose and the one or more subsequent doses of the composition comprise the same methanogen antigen and / or rumen antigen.

59. 57. The method of claim 56, wherein the first dose and the one or more subsequent doses of the composition comprise different methanogen antigens and / or rumen antigens.

60. 60. The method of any one of claims 57-59, wherein the composition is administered to the animal at least 1 time, at least 2 times, 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 8 times, 9 times, at least 10 times, at least 20 times, or at least 30 times.

61. 61. The method of any one of claims 46 to 60, wherein the composition is administered in combination with one or more additional agents, optionally wherein the one or more additional agents comprise chemical additives, biological feed additives.

62. 62. The method of any one of claims 46 to 61, wherein the composition is administered in combination with one or more additional compositions, optionally wherein the additional compositions immunize the animal against a disease, such as an infectious disease.

63. 45. A composition comprising an isolated polynucleotide according to any one of claims 1 to 41, or a pharmaceutical composition according to claim 43 or 44, for use in administration to an animal (e.g. vaccination).

64. 45. Use of a composition comprising an isolated polynucleotide according to any one of claims 1 to 41, or a pharmaceutical composition according to claim 43 or 44, in the preparation of a medicament for administration to an animal (e.g. vaccination).

65. 65. The composition of claim 63 or the use of claim 64, wherein the isolated polynucleotide or pharmaceutical composition is administered to the animal.

66. administration of the isolated polynucleotide or pharmaceutical composition compared to an otherwise identical animal not administered the composition or administered a different composition, (i) reduction in methane emissions; (ii) reducing the abundance of one or more microorganisms (e.g., methanogens) in the gastrointestinal tract of the animal; and / or 66. The composition of claim 65 or the use of claim 65, wherein (iii) the composition results in an increased growth rate of the animal.

67. 67. The composition of claim 65 or 66, or the use of claim 65 or 66, wherein the animal is a ruminant or a livestock animal.

68. Methanogenic bacteria include Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter oralis, and Methanomicrobium. mobile, Methanobrevibacter wolinii, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanosphaera stadtmanae, Methanosarcina mazei, Methanobrevibacter thaueri, Methanobrevibacter sp. UBA188, Methanosarcina soligelidi, Methanothermobacter thermautotrophicus, Methanococcus aeolicus, Methanaldoococcus jannaschii, Methanococcus voltae, Methanococcus vannielii, Methanococcus maripaludis, Methanopyrus kandleri, Methanocorpusculum labreanum, Methanococcoides burtonii, Methanosaete 68. The composition of any one of claims 65 to 67, or the use of any one of claims 65 to 67, comprising: Thermophilia, Methanoregula boonei, Methanosphaerula palustris, Methanoculleus marisnigri, Methanospirillim hungatei, Mathanosarcina acetivorans, or any combination thereof.

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