Mpox virus nucleic acid vaccine

Abstract

The present invention relates to the fields of biomedicine and virology, and in particular to a nucleic acid vaccine for preventing and / or treating mpox virus infections. In particular, provided in the present invention are a polypeptide or a polypeptide combination, a nucleic acid or a nucleic acid combination (in particular an mRNA or an mRNA combination), a composition, a pharmaceutical composition, a kit and the use thereof for preventing and / or treating mpox virus infections. Further provided in the present invention are an expression vector containing the nucleic acid or the nucleic acid combination and a host cell containing the nucleic acid or the nucleic acid combination or the expression vector.

Description

Monkeypox virus nucleic acid vaccine Technical Field

[0001] The present invention relates to the fields of biomedicine and virology, and in particular to a nucleic acid vaccine (preferably an mRNA vaccine) for preventing and / or treating monkeypox virus infection. Background Art

[0002] Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV). Monkeypox virus is an enveloped DNA virus with a diameter of approximately 200-250 nm. It is a member of the Poxviridae family and the genus Orthopoxvirus (see, for example, Alakunle E et al., Monkeypox Virus in Nigeria: Infection Biology, Epidemiology, and Evolution. Viruses. 2020 Nov 5; 12(11): 1257. and Cho CT, Wenner HA. Monkeypox virus. Bacteriol Rev. 1973 Mar; 37(1): 1-18.). Although the World Health Organization (WHO) announced on May 12, 2023 that the monkeypox outbreak is no longer a public health emergency of international concern, the number of monkeypox cases worldwide continues to grow.

[0003] Monkeypox virus is transmitted through direct contact and droplet transmission, with an incubation period of 7-14 days, sometimes up to 21 days (see, for example, Moore MJ et al., Mpox (Monkeypox). 2023 May 3. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan.). The main symptoms of monkeypox include high fever, headache, rash, muscle pain, and local lymphadenopathy. These symptoms are usually transient, lasting 2-4 weeks before resolving on their own. However, patients who develop a rash may be left with erythema, hyperpigmentation, and even scarring after the scabs fall off, and these scars can persist for years. Severe monkeypox infection can also be fatal in people with weakened immune systems (such as those with AIDS).

[0004] Monkeypox virus, highly pathogenic variola virus, and cowpox virus all belong to the genus Orthopoxvirus, and the antigenic proteins of the three viruses have a high degree of homology. Considering that monkeypox virus needs to be operated in a biosafety level 3 laboratory, and cowpox virus and monkeypox virus have good cross-reactivity (see, for example, Zeng J et al., Mpox multi-antigen mRNA vaccine candidates by a simplified manufacturing strategy afford efficient protection against lethal orthopoxvirus challenge. Emerg Microbes Infect. 2023 Dec; 12(1): 2204151.), cowpox virus is often used to evaluate the immunogenicity of monkeypox vaccine molecules in currently published scientific research articles. In addition, there is also evidence that vaccination with one orthopoxvirus vaccine can provide cross-protective immunity against other orthopoxviruses (see, for example, Lum FM et al., Monkeypox: disease epidemiology, host immunity and clinical interventions. Nat Rev Immunol. 2022 Oct; 22(10): 597-613.).

[0005] Currently, only two smallpox vaccines developed based on cowpox virus antigens, ACAM2000 and JYNNEOS, have been approved by the U.S. Food and Drug Administration (FDA) for immunization against monkeypox. However, due to poor safety performance of these two vaccines and their association with serious adverse events such as myocarditis and pericarditis, they are only approved for pre-exposure immunization of high-risk populations. Since the smallpox vaccination program was stopped in 1980, the general public's herd immunity to orthopoxviruses has declined. Studies have shown that monkeypox has a higher incidence in unvaccinated populations (see, for example, Shah HH et al., Human monkeypox-After 40 years, an unintended consequence of smallpox eradication. Front Public Health. 2023 Jan 5; 10: 1082586. and Pattnaik H et al., Making Sense of Monkeypox: A Comparison of Other Poxviruses to the Monkeypox. Cureus. 2023 Apr 24; 15(4): e38083.). Therefore, a safe and effective vaccine against monkeypox virus is still urgently needed. Summary of the Invention

[0006] In a first aspect, the present invention provides a polypeptide or polypeptide combination comprising one or more peptide segments, each of which is independently selected from peptide segments in monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins; optionally, the polypeptide or polypeptide combination further comprises one or more linkers, IgE signal peptides and / or HA polypeptide tags.

[0007] In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110, or an amino acid sequence that is at least 80% identical to the amino acid sequence of one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110.

[0008] In some embodiments, the polypeptide or combination of polypeptides comprises two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110, or two or more amino acid sequences that are at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110.

[0009] In a second aspect, the present invention provides a nucleic acid or a combination of nucleic acids comprising a polynucleotide encoding the polypeptide or combination of polypeptides of the present invention.

[0010] In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising a nucleotide sequence of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, or a nucleotide sequence having at least 80% identity to a nucleotide sequence of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22. In some embodiments, the polynucleotide is a DNA comprising the nucleotide sequence of one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44, or a nucleotide sequence that is at least 80% identical to the nucleotide sequence of one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[0011] In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising two or more nucleotide sequences selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, or two or more nucleotide sequences selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 having at least 80% identity. In some embodiments, the polynucleotide is a DNA comprising two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44, or two or more nucleotide sequences that are at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44.

[0012] In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising the nucleotide sequence of one of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, or a nucleotide sequence having at least 80% identity to the nucleotide sequence of one of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66. In some embodiments, the polynucleotide is a DNA comprising the nucleotide sequence of one of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, or a nucleotide sequence that is at least 80% identical to the nucleotide sequence of one of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

[0013] In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, or two or more nucleotide sequences having at least 80% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66. In some embodiments, the polynucleotide is a DNA comprising two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, or two or more nucleotide sequences that are at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

[0014] In a third aspect, the present invention provides a composition comprising a nucleic acid or a combination of nucleic acids of the present invention.

[0015] In some embodiments, the composition comprises a first lipid, a second lipid, a phospholipid, and a steroid, each of which is as defined herein. In some embodiments, the first lipid is a cationic lipid. In some embodiments, the composition is formulated as a lipid nanoparticle in which the nucleic acid or nucleic acid combination of the present invention is encapsulated in a lipid.

[0016] In a fourth aspect, the present invention provides a pharmaceutical composition or kit comprising the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination or composition of the present invention.

[0017] In a fifth aspect, the present invention provides an expression vector comprising the nucleic acid or nucleic acid combination of the present invention.

[0018] In a sixth aspect, the present invention provides a host cell comprising the nucleic acid or nucleic acid combination or expression vector of the present invention.

[0019] In a seventh aspect, the present invention provides use of the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination, composition, pharmaceutical composition or kit of the present invention in the preparation of a vaccine for preventing and / or treating monkeypox virus infection in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 shows the results of testing the titer of cross-neutralizing antibodies against vaccinia virus induced by candidate monkeypox vaccines in mice.

[0021] FIG2A and FIG2B show the results of detecting the immune response of mouse spleen T cells to the monkeypox virus mixed peptide library. Specific implementation plan

[0022] General Definitions and Terminology

[0023] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0024] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are terms widely used in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Meanwhile, for a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0025] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0026] As used herein, unless the context indicates otherwise, the singular forms "a," "an," or "the" include plural references. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0027] As used herein, the term "and / or" connecting multiple elements should be understood to include both individual and combined options. In other words, "and / or" includes "and" and "or." For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements qualified with "and / or" are understood in a similar manner and include any one thereof and any combination thereof.

[0028] Recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless expressly indicated to the contrary, values ​​or ranges recited herein are modified by "about" to mean ±20%, ±10%, ±5%, or ±3% of the recited or claimed value or range.

[0029] As used herein, a "nucleic acid combination" may refer to a combination or collection comprising more than one nucleic acid. Such nucleic acids may be present separately, for example, in different compositions, different parts of a pharmaceutical composition, or a kit, or in different vectors, or they may be present simultaneously in the same part of the same composition, pharmaceutical composition, or kit, or in the same vector. The nucleic acids in a nucleic acid combination may be identical or different, and may each encode the same polypeptide (part) or different polypeptides (parts).

[0030] As used herein, a "polypeptide combination" may refer to a combination or collection comprising more than one polypeptide. Such polypeptides may be present separately, for example, in different pharmaceutical compositions or different portions of a kit, or they may be present simultaneously in the same portion of the same pharmaceutical composition or kit. The polypeptides in the polypeptide combination may be identical or different, and may each comprise the same amino acid sequence (portion) or different amino acid sequences (portions).

[0031] As used herein, the term "wild type" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.

[0032] As used herein, the term "% identity" with respect to sequences refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window." Optimal alignment can be performed manually, or by means of algorithms known in the art, including but not limited to the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the search for similarity method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the publicly available BLASTN or BLASTP algorithms available on the National Center for Biotechnology Information (NCBI) website.

[0033] In some embodiments, the % homogeny of the sequence of interest is as follows: % homogeny ...

[0034] As used herein, "nucleotides" include deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotides" are constituents of ribonucleic acid (RNA), consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotides" are constituents of deoxyribonucleic acid (DNA), also consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen. They are the main chemical components of chromosomes. "Nucleotides" are usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylate containing adenine, "C (c)" refers to deoxycytidine or cytidine containing cytosine, "G (g)" refers to deoxyguanosine or guanylate containing guanine, "U (u)" refers to uridine containing uracil, and "T (t)" refers to deoxythymidylate containing thymine.

[0035] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylic acid in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes.

[0036] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."

[0037] As used herein, "encoding" refers to the inherent property of a specific nucleotide sequence in a nucleic acid, such as a gene, cDNA, or mRNA, which can serve as a template for the synthesis of polymers and macromolecules involved in other biological processes, given a well-defined nucleotide sequence or amino acid sequence. Thus, a gene encoding a protein means that the gene's mRNA, through transcription and translation, produces the protein in a cell or other biological system.

[0038] As used herein, the term "host cell" refers to a cell that is used to receive, maintain, replicate, or express a nucleic acid or vector. In some embodiments, the host cell may be a cell in which a polypeptide or combination of polypeptides of the present invention is expressed.

[0039] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that include both an amino and an acid functional group and that are capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L- optical isomers and peptidomimetics.

[0040] The terms "polypeptide chain," "polypeptide," "peptide," and "protein" (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic methods.

[0041] The terms "signal peptide" and "signal sequence" are used interchangeably herein. Signal peptides are usually located at the amino terminus of proteins and are short peptide chains of about 5-30 amino acids in length that guide newly synthesized proteins to the secretory pathway.

[0042] As used herein, "concatemer" refers to a polypeptide composed of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides connected together, wherein each polypeptide may or may not contain a linker between the other polypeptides.

[0043] As used herein, "antigen" refers to a molecule that can induce an acquired immune response in the body after entering the body. This immune response may involve the production of antibodies, specific immunogenic cells, or both. Those skilled in the art will understand that any macromolecule, including almost all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. Those skilled in the art will understand that any DNA or RNA herein, their nucleotide sequence or partial nucleotide sequence can encode a protein that can induce acquired immunity in the body. Furthermore, those skilled in the art will understand that an antigen does not need to encode the full-length nucleotide sequence of a gene alone. Obviously, the present invention includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences form different mixtures to induce the occurrence of a response. Furthermore, those skilled in the art will understand that antigens do not need to be completely encoded by a gene. Obviously, antigens can be synthesized or derived from biological samples. Biological samples include but are not limited to tissue samples, tumor samples, cells or biological fluids.

[0044] As used herein, "antibody" refers to a protective protein produced by the body in response to antigenic stimulation. It is a type of immunoglobulin produced by B lymphocytes. The antibody monomer is a Y-shaped molecule composed of four polypeptide chains. These chains consist of two identical heavy chains and two identical light chains, linked by disulfide bonds. Each heavy chain is approximately 50 kDa, and each light chain is approximately 25 kDa, with disulfide bonds linking the heavy and light chains. Antibodies are unique in their high affinity and specificity for their binding partners.

[0045] As used herein, "vaccine" refers to a composition comprising an active ingredient (e.g., a polypeptide or polypeptide combination or a nucleic acid or nucleic acid combination of the present invention), which can induce an immune response in a vaccinated subject through vaccination. In a specific embodiment, the immune response it induces can provide immune protection and is sufficient to prevent and / or alleviate at least one symptom associated with a pathogen or disease infection. According to the present invention, the nucleic acid or nucleic acid combination or composition described herein can be used as a vaccine to provide prophylactic and / or therapeutic immunity against monkeypox virus in subjects in need. As used herein, "monkeypox virus RNA vaccine (e.g., mRNA vaccine, circRNA vaccine or saRNA vaccine)" provides a unique therapeutic alternative to peptide-based vaccines or DNA vaccines. The monkeypox virus RNA vaccine (e.g., mRNA vaccine, circRNA vaccine or saRNA vaccine) described herein includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one monkeypox virus antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to monkeypox virus).

[0046] As used herein, the term "neutralizing antibody" refers to an antibody or fragment thereof that is capable of neutralizing, i.e., preventing, inhibiting, reducing, or interfering with the ability of a pathogen to initiate and / or maintain infection in a host (e.g., a host organism or host cell). According to the present invention, neutralizing antibodies against monkeypox virus antigenic proteins (or antigenic polypeptides) can be produced in a subject vaccinated with the vaccine of the present invention, for example, in the subject's immune serum. The level of neutralizing antibody titer in the immune serum can be measured using methods known in the art.

[0047] Terms such as "treating" or "treating" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, alleviate, reduce the symptoms of an existing, diagnosed pathological condition or disorder, and / or arrest or slow the progression of an existing, diagnosed pathological condition or disorder. Terms such as "preventing," "preventing," "avoiding," "containment," and the like refer to preventative or prophylactic measures that prevent the progression of an undiagnosed target pathological condition or disorder. Thus, a "subject in need thereof" can include a subject already suffering from a disease; a subject susceptible to a disease; and a subject in need of prevention of a disease.

[0048] The term "prophylactically or therapeutically effective amount" refers to an amount sufficient to prevent or inhibit the occurrence of a disease or symptom and / or slow, alleviate, or delay the development or severity of a disease or symptom. The prophylactically or therapeutically effective amount is influenced by factors including, but not limited to, the rate of development and severity of the disease or symptom, the age, sex, weight, and physical condition of the subject, the duration of treatment, and the specific route of administration. The prophylactically or therapeutically effective amount can be administered in one or more doses. The prophylactically or therapeutically effective amount can be achieved through continuous or intermittent administration.

[0049] As used herein, the term "subject" describes an organism, such as a mammal, to which a polypeptide or polypeptide combination, nucleic acid or nucleic acid combination, composition or pharmaceutical composition of the present invention can be provided. Preferably, the subject is a rodent or a human.

[0050] As used herein, the term "first polypeptide (chain)" and similarly used "second polypeptide (chain)", "third polypeptide (chain)", "fourth polypeptide (chain)", "fifth polypeptide (chain)", "sixth polypeptide (chain)", etc. are used only for the purpose of distinction and are not intended to limit the order and connection method of these polypeptides (chains). Each polypeptide (chain) itself and each other may be the same or different. If necessary, two or more first polypeptides (chains) may be included, which may be the same or different. The above terms used in different schemes may be the same or different.

[0051] As used herein, the term "first polynucleotide" and similarly used terms "second polynucleotide," "third polynucleotide," "fourth polynucleotide," "fifth polynucleotide," "sixth polynucleotide," etc. are used only for differentiation purposes and are not intended to limit the order or linkage of these polynucleotides. Each polynucleotide may be identical or different in itself or between itself. If desired, two or more first polynucleotides may be included, which may be identical or different. The above terms used in different embodiments may be the same or different.

[0052] As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds that include, but are not limited to, fatty acid esters and are generally characterized by being poorly soluble in water but soluble in many non-polar organic solvents. Although lipids generally have poor water solubility, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and are soluble in water under certain conditions. Known lipid types include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be divided into at least three categories: (1) "simple lipids," which include fats and oils, as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derivatized lipids," such as steroids. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound," also referred to as "lipidoid," refers to lipid-like compounds (e.g., amphiphilic compounds with lipid-like physical properties).

[0053] The term "lipid nanoparticle" or "LNP" refers to a particle with at least one nanometer (nm) size (e.g., 1 to 1,000 nm) containing one or more types of lipid molecules. The LNP provided herein may further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule partially or completely encapsulated in a lipid shell. Specifically, in some embodiments, wherein the payload is a negatively charged molecule (e.g., mRNA, circRNA, or saRNA), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is expected that cationic lipids can interact with negatively charged payload molecules and promote payload to be incorporated into and / or be encapsulated in the LNP during LNP formation. Other lipids that can form a part for LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as steroids, polymer-bound lipids, and various zwitterionic lipids. In certain embodiments, LNPs according to the present disclosure comprise one or more of the lipids of series 01, 02, 03, and 04, for example, one or more of the lipids according to formulas (01-I), (01-II), (02-I), (02-II), (03-I), (04-I), and (04-III) (and subformulas thereof) described herein.

[0054] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or can be positively charged in response to the pH value or hydrogen ion activity of its environment (such as the environment it is intended to use). Therefore, the term "cationic" encompasses "permanent cations" and "cationizable". In certain embodiments, the positive charge in the cationic lipid is caused by the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid includes a zwitterionic lipid that is positively charged in the environment it is intended to use (such as at physiological pH). In certain embodiments, the cationic lipid includes one or more of the lipids of series 01, 02, 03, and 04, such as one or more of the lipids of formula (01-I), (01-II), (02-I), (02-II), (03-I), (04-I), and (04-III) (and subformulas thereof) as described herein. The term "anionic lipid" refers to a lipid that is negatively charged at any pH value or hydrogen ion activity of its environment, or can be negatively charged in response to the pH value or hydrogen ion activity of its environment (such as the environment it is intended to use). Exemplary anionic lipids include one or more phosphate groups that are negatively charged, eg, at physiological pH.

[0055] The term "polymer-bound lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer-bound lipid is a pegylated lipid (PEG-lipid), wherein the polymer portion comprises polyethylene glycol.

[0056] The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of the intended lipid use, such as a physiological pH value. As non-limiting examples, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP); sphingomyelin (SM); ceramides. The neutral lipids provided herein can be synthetic or derived from natural sources or compounds (isolated or modified therefrom).

[0057] The term "steroid" refers to a lipid with a cyclopentane polyhydrophenanthrene as a parent nucleus, including sterols and sterol derivatives. Steroid compounds do not contain bound fatty acids and are non-saponifiable lipids. In this article, steroids can be used as structural lipids in the lipid component of the nanoparticle composition. As non-limiting examples, steroids that can be used in conjunction with the present disclosure include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, and mixtures thereof.

[0058] The term "charged lipid" encompasses any lipid molecule present in a positively charged or negatively charged form under a selected pH value or within a selected pH range. In some embodiments, the selected pH value or range correspond to the pH conditions in the environment of predetermined lipid uses, such as physiological pH. As a non-limiting example, the charged lipid that can be used in conjunction with the present disclosure includes but is not limited to phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (such as DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (such as DC-Chol), 1,2-dioleoyl-sn-glycero-3-phosphoric acid-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phosphoric acid-(1'-racemic-glycerol) sodium salt (DOPG-Na) and 1,2-dioleoyl-sn-glycero-3-phosphoric acid sodium salt (DOPA-Na). The charged lipids provided herein can be synthetic or derived from (isolated or modified from) natural sources or compounds.

[0059] As used herein and unless otherwise indicated, the term "alkyl" refers to a saturated straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms. In one embodiment, an alkyl group has, for example, 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12 The alkyl group is a C1-C8 alkyl group, a C1-C8 alkyl group, or a C1-C6 alkyl group, and is attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, alkyl groups are optionally substituted.

[0060] As used herein and unless otherwise indicated, the term "alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon double bonds. As understood by one of ordinary skill in the art, the term "alkenyl" also encompasses groups having "cis" and "trans" configurations, or alternatively, "E" and "Z" configurations. In one embodiment, an alkenyl group has, for example, 2 to 24 carbon atoms (C2-C 24 alkenyl), 4 to 20 carbon atoms (C4-C 20 alkenyl), 6 to 16 carbon atoms (C6-C 16 alkenyl), 6 to 9 carbon atoms (C6-C9 alkenyl), 2 to 15 carbon atoms (C2-C 15 alkenyl), 2 to 12 carbon atoms (C2-C 12 The term "alkenyl" refers to a group of 2 to 8 carbon atoms (C-C alkenyl), 2 to 8 carbon atoms (C-C alkenyl), or 2 to 6 carbon atoms (C-C alkenyl) attached to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, and the like. Unless otherwise specified, alkenyl groups are optionally substituted.

[0061] As used herein and unless otherwise indicated, the term "alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon triple bonds. In one embodiment, an alkynyl group has, for example, 2 to 24 carbon atoms (C2-C 24 Alkynyl), 4 to 20 carbon atoms (C4-C 20 Alkynyl), 6 to 16 carbon atoms (C6-C 16 alkynyl), 6 to 9 carbon atoms (C6-C9 alkynyl), 2 to 15 carbon atoms (C2-C 15 Alkynyl), 2 to 12 carbon atoms (C2-C 12 Alkynyl groups are 2 to 8 carbon atoms (C2-C8 alkynyl), or 2 to 6 carbon atoms (C2-C6 alkynyl) and are attached to the rest of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specified, alkynyl groups are optionally substituted.

[0062] As used herein and unless otherwise indicated, the term "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a group, consisting solely of carbon and hydrogen and being saturated. In one embodiment, an alkylene group has, for example, 1 to 24 carbon atoms (C1-C 24 alkylene), 1 to 15 carbon atoms (C1-C 15 alkylene), 1 to 12 carbon atoms (C1-C 12C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene). Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule by a single bond and to the radical group by a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbon atoms within the chain. Unless otherwise indicated, the alkylene chain is optionally substituted.

[0063] As used herein and unless otherwise indicated, the term "alkenylene" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical, consisting solely of carbon and hydrogen and containing one or more carbon-carbon double bonds. In one embodiment, an alkenylene group has, for example, 2 to 24 carbon atoms (C2-C 24 alkenyl), 2 to 15 carbon atoms (C2-C 15 alkenyl), 2 to 12 carbon atoms (C2-C 12 Alkenylene is a 2- to 8-carbon-atom (C-C alkenylene), 2 to 6 carbon atoms (C-C alkenylene), or 2 to 4 carbon atoms (C-C alkenylene). Examples of alkenylene include, but are not limited to, vinylene, propenylene, n-butenylene, and the like. Alkenylene is attached to the rest of the molecule by a single or double bond and to the radical group by a single or double bond. The points of attachment of the alkenylene to the rest of the molecule and to the radical group may be through one carbon or any two carbon atoms within the chain. Unless otherwise indicated, alkenylene is optionally substituted.

[0064] As used herein and unless otherwise indicated, the term "cycloalkyl" refers to a non-aromatic saturated monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms. Cycloalkyl groups may include fused or bridged ring systems. In one embodiment, a cycloalkyl group has, for example, 3 to 15 ring carbon atoms (C3-C 15 cycloalkyl), 3 to 10 ring carbon atoms (C3-C 10 The cycloalkyl radical is a cycloalkyl radical having 3 to 8 ring carbon atoms (C3-C8 cycloalkyl). The cycloalkyl radical is connected to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include, but are not limited to, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, and the like. Unless otherwise indicated, cycloalkyl radicals are optionally substituted.

[0065] As used herein, and unless otherwise specified, the term "cycloalkylene" is a divalent cycloalkyl group. Unless otherwise specified, a cycloalkylene group is optionally substituted.

[0066] As used herein and unless otherwise indicated, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms and including one or more carbon-carbon double bonds. Cycloalkenyl groups may include fused or bridged ring systems. In one embodiment, a cycloalkenyl group has, for example, 3 to 15 ring carbon atoms (C3-C 15 cycloalkenyl), 3 to 10 ring carbon atoms (C3-C 10 The cycloalkenyl group is a cycloalkyl group (C-C) or 3 to 8 ring carbon atoms (C-C). The cycloalkenyl group is connected to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise indicated, the cycloalkenyl group is optionally substituted.

[0067] As used herein, and unless otherwise specified, the term "cycloalkenylene" is a divalent cycloalkenyl group. Unless otherwise specified, a cycloalkenylene group is optionally substituted.

[0068] As used herein and unless otherwise indicated, the term "heterocyclyl" refers to a non-aromatic monocyclic or polycyclic moiety containing one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. The heterocyclyl group may be attached to the main structure at any heteroatom or carbon atom. The heterocyclyl group may be a monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic ring system, wherein the polycyclic ring system may be a fused, bridged, or spirocyclic ring system. The heterocyclyl polycyclic ring system may contain one or more heteroatoms in one or more rings. The heterocyclyl group may be saturated or partially unsaturated. Saturated heterocycloalkyl groups may be referred to as "heterocycloalkyl." Partially unsaturated heterocycloalkyl groups may be referred to as "heterocycloalkenyl" when the heterocyclyl group contains at least one double bond, or as "heterocycloalkynyl" when the heterocyclyl group contains at least one triple bond. In one embodiment, the heterocyclyl group has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-membered heterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl). When appearing herein, a numerical range such as "3 to 18" refers to each integer in the given range; for example, "3 to 18 ring atoms" means that the heterocyclyl group can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc. (up to and including 18 ring atoms). Examples of heterocyclic groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thienyl, pyridyl, piperidinyl, quinolyl, and isoquinolyl. Unless otherwise specified, heterocyclic groups are optionally substituted.

[0069] As used herein, and unless otherwise specified, the term "heterocyclylene" is a divalent heterocyclyl group. Unless otherwise specified, a heterocyclylene group is optionally substituted.

[0070] As used herein and unless otherwise indicated, the term "aryl" refers to a monocyclic aromatic group and / or a polycyclic monovalent aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, an aryl group has 6 to 18 ring carbon atoms (C6-C 18 aryl), 6 to 14 ring carbon atoms (C6-C 14 aryl) or 6 to 10 ring carbon atoms (C6-C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthracenyl, phenanthrenyl, pyrenyl, biphenyl, and terphenyl. The term "aryl" also refers to bicyclic, tricyclic, or other polycyclic hydrocarbon rings in which at least one ring is aromatic and the other rings may be saturated, partially unsaturated, or aromatic, such as dihydronaphthyl, indenyl, dihydroindenyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise indicated, aryl groups are optionally substituted.

[0071] As used herein and unless otherwise specified, the term "arylene" is a divalent aromatic group. Unless otherwise specified, an arylene group is optionally substituted.

[0072] As used herein and unless otherwise indicated, the term "heteroaryl" refers to a monocyclic aromatic group and / or a polycyclic aromatic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from O, S, and N. The heteroaryl group can be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, the heteroaryl group has 5 to 20, 5 to 15, or 5 to 10 ring atoms. The term "heteroaryl" also refers to a bicyclic, tricyclic, or other polycyclic ring, wherein at least one ring is an aromatic ring, and the other rings can be saturated, partially unsaturated, or aromatic rings, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. The example of monocyclic heteroaryl includes but is not limited to pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl. The example of bicyclic heteroaryl includes but is not limited to indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolyl, tetrahydroisoquinolyl, isoquinolyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromone radical, coumarin base, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl and tetrahydroquinolyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and xanthenyl.Unless otherwise specified, heteroaryl groups are optionally substituted.

[0073] As used herein, and unless otherwise specified, the term "heteroarylene" is a divalent heteroaryl group. Unless otherwise specified, a heteroarylene group is optionally substituted.

[0074] When a group described herein is referred to as "substituted," it may be substituted with one or more suitable substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as: halogen atoms, such as F, Cl, Br, or I; cyano; oxo (=O); hydroxyl (-OH); alkyl; alkenyl; alkynyl; cycloalkyl; aryl; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) xNR'R', wherein: R' is independently H, C1-C 16 alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments, the substituent is C1-C 12 In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR'R').

[0075] As used herein, and unless otherwise indicated, the term "optionally present" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that the description includes both substituted alkyl groups and alkyl groups without substitution.

[0076] As used herein and unless otherwise indicated, the term "prodrug" of a biologically active compound refers to a compound that can be converted into a biologically active compound under physiological conditions or by solvolysis. In one embodiment, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a biologically active compound. The prodrug may be inactive when administered to a subject in need, but is converted into a biologically active compound in vivo. Prodrugs are generally rapidly transformed in vivo to produce the parent biologically active compound, for example, by hydrolysis in the blood. Prodrug compounds generally provide advantages of solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, pp. 21-24 (Elsevier, Amsterdam)). Discussions on prodrugs are provided in Higuchi, T. et al., ACS Symposium Series, Vol. 14; and Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0077] In one embodiment, the term "prodrug" is also intended to include any covalently bonded carrier that releases the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound can be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved during routine manipulation or in vivo to yield the parent compound. Prodrugs include compounds in which a hydroxyl, amino, or sulfhydryl group is bonded to any group that, when the prodrug of the compound is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or free sulfhydryl group, respectively.

[0078] Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol functional groups or amide derivatives of amine functional groups in the compounds provided herein.

[0079] As used herein, and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

[0080] Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, benzoic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, benzo ... acid), dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0081] Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared by adding an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. In one embodiment, the inorganic salt is an ammonium salt, sodium salt, potassium salt, calcium salt, and magnesium salt. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines, including naturally occurring substituted amines; cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0082] The compounds provided herein may contain one or more asymmetric centers and thus may produce enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R)- or (S)- or as (D)- or (L)- for amino acids based on absolute stereochemistry. Unless otherwise indicated, the compounds provided herein are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors or using, for example, chiral high pressure liquid chromatography (HPLC) to resolve racemates (or racemates of salts or derivatives). When the compounds described herein contain olefinic double bonds or other geometric asymmetric centers, unless otherwise indicated, the compounds are intended to include E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0083] As used herein and unless otherwise indicated, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the arrangement of their atoms in space. "Atropisomers" are stereoisomers resulting from hindered rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any ratio is referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0084] "Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

[0085] "Tautomers" refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment in which the compound is located, and may vary depending on, for example, whether the compound is a solid or in organic or aqueous solution.

[0086] It should also be noted that the compounds described herein may contain unnatural proportions of atomic isotopes at one or more atoms. For example, the compounds may be substituted with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I), sulfur-35( 35 S) or carbon-14 ( 14 C) is radiolabeled, or may be isotopically enriched, such as deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15( 15 N). As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom whose isotopic composition is different from the natural isotopic composition of that atom. "Isotopically enriched" may also refer to a compound containing at least one atom whose isotopic composition is different from the natural isotopic composition of that atom. The term "isotopic composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents, such as cancer therapeutics; research reagents, such as binding assay reagents; and diagnostic agents, such as in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds described herein are provided, for example, isotopologues that are enriched in deuterium, carbon-13, and / or nitrogen-15. As used herein, "deuterated" refers to a compound in which at least one hydrogen (H) has been replaced with deuterium (as D or 2 H represents) substitution, i.e., the compound is enriched in deuterium at at least one position.

[0087] It should be noted that if there is a discrepancy between a depicted structure and the name of that structure, the depicted structure shall prevail.

[0088] As used herein, and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0089] The term "composition" is intended to encompass a product containing the specified ingredients (eg, mRNA molecules provided herein), optionally selected, in the specified amounts.

[0090] Nucleic acid construct name used herein (such as 0201, 0302, 0402, 0505, 0603, 0702, 0803, 0201-J0, 0301-J0, 0501-J0, 0701-J0, 0901-J0, 1101, 1201, 1301, 1401, 1501, 1601, 1701, 1801, 1901, 2001 etc.) is only used for distinguishing or identifying nucleic acid, construct, sequence or product, and is not intended to represent that such mark is the feature of nucleic acid of the present invention or nucleic acid combination, construct, sequence or product. It will be appreciated by those skilled in the art that, for example, for the purpose of distinguishing or identifying, other nucleic acid or nucleic acid combination, construct, sequence or product also may use such mark equally, but do not refer to identical or equivalent nucleic acid or nucleic acid combination, construct, sequence or product. Similarly, similar numbers or identifiers used in the examples are only for illustrative purposes. The nucleic acid or nucleic acid combination, construct, sequence or product of the present invention is defined by the features described in the appended claims.

[0091] Monkeypox virus

[0092] As used herein, the term "Monkeypox Virus (MPXV)" belongs to the genus Orthopoxvirus of the Poxviridae family, which also includes smallpox virus and cowpox virus. MPXV has a similar morphology to other known orthopoxviruses. MPXV is oval or brick-shaped, surrounded by a lipoprotein outer membrane. Its genome is linear double-stranded DNA with a genome size of approximately 197kb. MPXV can enter the host through the oropharynx, nasopharynx or intradermal route. The virus replicates at the site of infection and then spreads to the regional lymph nodes. Although MPXV is a DNA virus, its life cycle occurs in the cytoplasm. The MPXV replication process produces two infectious virus particles: mature virus particles (MV) and enveloped virus particles (EV); among them, MV enters the cell through micropinocytosis or membrane fusion, while EV enters through membrane fusion. After completing viral DNA replication and viral protein assembly in the cytoplasm, MV is released by cell lysis, while EV is released by exocytosis (see, for example, Kumar N et al., The 2022 outbreak and the pathobiology of the monkeypox virus. J Autoimmun. 2022 Jul; 131: 102855.).

[0093] As used herein, the terms "A29L protein" and "A29L peptide", "A35R protein" and "A35R peptide", "B2R protein" and "B2R peptide", "B6R protein" and "B6R peptide", "C15L protein" and "C15L peptide", "M1R protein" and "M1R peptide", "H3L protein" and "H3L peptide", "E8L protein" and "E8L peptide" are each used interchangeably, and are all major antigenic proteins (or antigenic polypeptides) of monkeypox virus. The "A29L protein", "A35R protein", "B2R protein", "B6R protein", "M1R protein", "E8L protein", "H3L protein", and "C15L protein" of monkeypox virus are homologous to the "A27L protein", "A33R protein", "A56R protein", "B5R protein", "L1R protein", "D8L protein", "H3L protein", and "F9L protein" of vaccinia virus, respectively.

[0094] It is reported that "A29L protein" is a surface membrane fusion protein located on mature virions that can bind to cell surface heparan; "A35R protein" is an envelope protein located on enveloped virions that participates in the formation of actin-containing microvilli and the intercellular spread of virions; "B2R protein" is a membrane glycoprotein hemagglutinin located on enveloped virions that prevents cell fusion; "B6R protein" is a palmitoylated glycoprotein located on enveloped virions that is required for efficient cell spread and participates in complement regulation; "M1R protein" is a myristoylated surface membrane protein located on mature virions that plays an important role in the entry of the virus into the host; "E8L protein" is a surface membrane adsorption protein located on mature virions that can bind to chondroitin sulfate; "H3L protein" is a surface membrane protein located on mature virions that can bind to heparan and cell surface proteoglycans; "C15L protein" is located on mature virions (see, for example, Papukashvili D et al., Strategy of developing nucleic acid-based universal monkeypox vaccine candidates. Front Immunol. 2022 Oct 27;13:1050309.).

[0095] As used in this article, the A29L protein is shown as NCBI Accession No. YP_010377135.1; the A35R protein is shown as NCBI Accession No. YP_010377142.1; the B2R protein is shown as NCBI Accession No. YP_010377158.1; the B6R protein is shown as NCBI Accession No. NP_536594.1; the C15L protein is shown as NCBI Accession No. URF91574.1; the M1R protein is shown as NCBI Accession No. URF91574.1 or NP_536507.1; the H3L protein is shown as NCBI Accession No. UVB87333.1; and the E8L protein is shown as NCBI Accession No. YP_010377102.1.

[0096] peptides

[0097] In one aspect, the present invention provides a polypeptide or polypeptide combination comprising one or more peptide segments, each of which is independently selected from peptide segments in monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins.

[0098] In some embodiments, the polypeptide or combination of polypeptides comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 peptide segments, each of which is independently selected from the peptide segments in the monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins.

[0099] In some embodiments, the polypeptide or polypeptide combination comprises one peptide segment selected from the peptide segments in monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins; or the polypeptide or polypeptide combination comprises two, three, four, five, six or seven peptide segments, each derived from a different protein, selected from the peptide segments in monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins.

[0100] In one embodiment, the polypeptide or combination of polypeptides comprises three peptides each derived from a different protein, wherein the peptides are selected from peptides in monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins.

[0101] In a preferred embodiment, the polypeptide or polypeptide combination comprises 6 peptides each derived from a different protein, wherein the peptides are selected from peptides in monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins.

[0102] In some embodiments, the polypeptide or combination of polypeptides further comprises one or more linkers, an IgE signal peptide, and / or an HA polypeptide tag.

[0103] In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the A29L protein. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the A35R protein. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B2R protein. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B6R protein. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the C15L protein. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the M1R protein. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the H3L protein. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the A35R, B2R, and C15L proteins. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the A29L, B6R, and M1R proteins. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B6R, M1R, and H3L proteins. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B6R, M1R, and A35R proteins. In one embodiment, the polypeptide or combination of polypeptides comprises peptides from A29L, B2R, and C15L proteins. In one embodiment, the polypeptide or combination of polypeptides comprises peptides from B2R, H3L, and C15L proteins. In one embodiment, the polypeptide or combination of polypeptides comprises peptides from B2R, A35R, and B6R proteins. In one embodiment, the polypeptide or combination of polypeptides comprises peptides from A29L, M1R, and C15L proteins. In one embodiment, the polypeptide or combination of polypeptides comprises peptides from H3L, M1R, and C15L proteins. In one embodiment, the polypeptide or combination of polypeptides comprises peptides from A29L, A35R, B2R, B6R, C15L, and M1R proteins. In one embodiment, the polypeptide or combination of polypeptides comprises peptides from A35R, B2R, B6R, C15L, M1R, and H3L proteins.

[0104] In one embodiment, the peptide segment in the A29L protein comprises the full-length A29L peptide. In one embodiment, the peptide segment in the A29L protein comprises the full-length A29L peptide, and the full-length A29L peptide comprises the amino acid sequence of SEQ ID NO: 111 or 113.

[0105] In one embodiment, the peptide segment in the A35R protein comprises the full-length A35R peptide. In one embodiment, the full-length A35R peptide comprises the amino acid sequence of SEQ ID NO: 114. In one embodiment, the peptide segment in the A35R protein comprises the extracellular segment of A35R. In one embodiment, the extracellular segment of A35R comprises the amino acid sequence of SEQ ID NO: 115.

[0106] In one embodiment, the peptide segment in the B2R protein comprises a B2R signal peptide. In one embodiment, the B2R signal peptide comprises the amino acid sequence of SEQ ID NO: 116. In one embodiment, the peptide segment in the B2R protein comprises the full-length B2R peptide excluding the B2R signal peptide portion. In one embodiment, the peptide segment in the B2R protein comprises the full-length B2R peptide excluding the B2R signal peptide portion, and the peptide segment in the B2R protein comprises the amino acid sequence of SEQ ID NO: 117. In one embodiment, the peptide segment in the B2R protein comprises the full-length B2R peptide. In one embodiment, the full-length B2R peptide comprises the amino acid sequence of SEQ ID NO: 118. In one embodiment, the peptide segment in the B2R protein comprises the B2R extracellular segment. In one embodiment, the B2R extracellular segment comprises the amino acid sequence of SEQ ID NO: 119. In one embodiment, the peptide segment in the B2R protein comprises the B2R signal peptide and the B2R extracellular segment. In one embodiment, the peptide segment in the B2R protein comprises a B2R signal peptide and a B2R extracellular segment, and the peptide segment in the B2R protein comprises the amino acid sequence of SEQ ID NO:120.

[0107] In one embodiment, the peptide segment in the B6R protein comprises the B6R signal peptide. In one embodiment, the B6R signal peptide comprises the amino acid sequence of SEQ ID NO: 121. In one embodiment, the peptide segment in the B6R protein comprises the full-length B6R peptide excluding the B6R signal peptide portion. In one embodiment, the peptide segment in the B6R protein comprises the full-length B6R peptide excluding the B6R signal peptide portion, and the peptide segment in the B6R protein comprises the amino acid sequence of SEQ ID NO: 122. In one embodiment, the peptide segment in the B6R protein comprises the full-length B6R peptide. In one embodiment, the full-length B6R peptide comprises the amino acid sequence of SEQ ID NO: 123. In one embodiment, the peptide segment in the B6R protein comprises the B6R extracellular segment. In one embodiment, the B6R extracellular segment comprises the amino acid sequence of SEQ ID NO: 124. In one embodiment, the peptide segment in the B6R protein comprises the B6R signal peptide and the B6R extracellular segment. In one embodiment, the peptide segment in the B6R protein comprises a B6R signal peptide and a B6R extracellular segment, and the peptide segment in the B6R protein comprises the amino acid sequence of SEQ ID NO:125.

[0108] In one embodiment, the peptide segment in the C15L protein comprises the full-length C15L peptide. In one embodiment, the full-length C15L peptide comprises the amino acid sequence of SEQ ID NO: 126. In one embodiment, the peptide segment in the C15L protein comprises the extracellular segment of C15L. In one embodiment, the extracellular segment of C15L comprises the amino acid sequence of SEQ ID NO: 127.

[0109] In one embodiment, the peptide segment in the M1R protein comprises the full-length M1R peptide. In one embodiment, the full-length M1R peptide comprises the amino acid sequence of SEQ ID NO: 130. In one embodiment, the peptide segment in the M1R protein comprises the extracellular segment of M1R. In one embodiment, the extracellular segment of M1R comprises the amino acid sequence of SEQ ID NO: 128.

[0110] In one embodiment, the peptide segment in the H3L protein comprises the full-length H3L peptide. In one embodiment, the full-length H3L peptide comprises the amino acid sequence of SEQ ID NO: 131. In one embodiment, the peptide segment in the H3L protein comprises the extracellular segment of H3L. In one embodiment, the extracellular segment of H3L comprises the amino acid sequence of SEQ ID NO: 132.

[0111] In one embodiment, a polypeptide or combination of polypeptides comprises an IgE signal peptide, a peptide from the A29L protein, and an HA polypeptide tag. In one embodiment, a polypeptide or combination of polypeptides comprises a peptide from the A35R protein and an HA polypeptide tag. In one embodiment, a polypeptide or combination of polypeptides comprises a peptide from the B2R protein and an HA polypeptide tag. In one embodiment, a polypeptide or combination of polypeptides comprises a peptide from the B6R protein, a linker, and an HA polypeptide tag. In one embodiment, a polypeptide or combination of polypeptides comprises an IgE signal peptide, a linker, an HA polypeptide tag, and a peptide from the C15L protein. In one embodiment, a polypeptide or combination of polypeptides comprises an IgE signal peptide, a linker, an HA polypeptide tag, and a peptide from the M1R protein. In one embodiment, a polypeptide or combination of polypeptides comprises an IgE signal peptide, a linker, an HA polypeptide tag, and a peptide from the H3L protein. In one embodiment, a polypeptide or combination of polypeptides comprises peptides from the A35R, B2R, and C15L proteins, and a linker. In one embodiment, a polypeptide or combination of polypeptides comprises peptides from the A29L, B6R, and M1R proteins, an IgE signal peptide, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B6R, M1R, and H3L proteins, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B6R, M1R, and A35R proteins, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the A29L, B2R, and C15L proteins, an IgE signal peptide, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B2R, H3L, and C15L proteins, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the B2R, A35R, and B6R proteins, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the A29L, M1R, and C15L proteins, an IgE signal peptide, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide from the H3L, M1R, and C15L proteins, an IgE signal peptide, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide fragment from the A29L, A35R, B2R, B6R, C15L, and M1R proteins, an IgE signal peptide, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide fragment from the A29L, A35R, B2R, B6R, C15L, and M1R proteins, an IgE signal peptide, an HA polypeptide tag, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide fragment from the A35R, B2R, B6R, C15L, M1R, and H3L proteins, an IgE signal peptide, an HA polypeptide tag, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide fragment from the A35R, B2R, B6R, C15L, M1R, and H3L proteins, an IgE signal peptide, and a linker. In one embodiment, the polypeptide or combination of polypeptides comprises a peptide fragment from the A35R, B2R, B6R, C15L, M1R, and H3L proteins, an IgE signal peptide, and a linker.

[0112] The linker may comprise an amino acid sequence of any length, in particular an amino acid sequence of 1-50, preferably 1-30, for example 1-10 amino acid residues. Exemplary linkers may include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S) or combinations thereof, such as GGAS, GGGS, GGGSG or (G4S)n, wherein n is an integer of 1-30, preferably 1-10. The linker may also be a hinge region or a functional equivalent thereof. Other suitable linkers may be organic compounds or polymers generally suitable for use in pharmaceutical proteins, including but not limited to polyethylene glycol.

[0113] As used herein, the terms "HA polypeptide tag" and "HA tag protein" are used interchangeably. The HA polypeptide tag is derived from the surface antigenic determinant of the influenza virus hemagglutinin. It has minimal impact on the spatial structure of the foreign target protein and can be easily constructed as a tag protein fused to the N- or C-terminus. These two tags are widely used for protein detection, separation, and purification.

[0114] In one embodiment, the polypeptide or combination of polypeptides comprises an IgE signal peptide. In one embodiment, the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134. In one embodiment, the polypeptide or combination of polypeptides comprises an HA polypeptide tag. In one embodiment, the HA polypeptide tag comprises the amino acid sequence of SEQ ID NO: 135 or 136. In one embodiment, the polypeptide or combination of polypeptides comprises a linker. In one embodiment, the linker is a GS linker. In a preferred embodiment, the GS linker comprises the amino acid sequence of SEQ ID NO: 137 or 138.

[0115] In one embodiment, the polypeptide or combination of polypeptides comprises an amino acid sequence of one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110.

[0116] In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a peptide from the A29L protein, and an HA polypeptide tag. In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a peptide from the A29L protein, and an HA polypeptide tag; the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the peptide from the A29L protein comprises the amino acid sequence of SEQ ID NO: 111, and the HA polypeptide tag comprises the amino acid sequence of SEQ ID NO: 135. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 89.

[0117] In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, a peptide segment from the A35R protein and an HA polypeptide tag. In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, the full-length A35R peptide and an HA polypeptide tag, wherein the full-length A35R peptide comprises the amino acid sequence of SEQ ID NO: 114, and the HA polypeptide tag comprises the amino acid sequence of SEQ ID NO: 136. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 90.

[0118] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a peptide segment from the B2R protein and an HA polypeptide tag. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, the full-length B2R peptide excluding the signal peptide portion, and an HA polypeptide tag. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, the full-length B2R peptide excluding the signal peptide portion, and an HA polypeptide tag, the B2R signal peptide comprising the amino acid sequence of SEQ ID NO: 116, the full-length B2R peptide excluding the signal peptide portion comprises the amino acid sequence of SEQ ID NO: 117, and the HA polypeptide tag comprises the amino acid sequence of SEQ ID NO: 136. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, the full-length B2R peptide and an HA polypeptide tag. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, the full-length B2R peptide comprising the amino acid sequence of SEQ ID NO: 118, and the HA polypeptide tag comprising the amino acid sequence of SEQ ID NO: 136. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO:91.

[0119] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a peptide segment from the B6R protein, a linker, an HA polypeptide tag, and a peptide segment from the B6R protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B6R signal peptide, a GS linker, an HA polypeptide tag, and the full length of the B6R peptide excluding the signal peptide portion. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B6R signal peptide, a GS linker, an HA polypeptide tag, and the full length of the B6R peptide excluding the signal peptide portion, the B6R signal peptide comprising the amino acid sequence of SEQ ID NO: 121, the GS linker comprising the amino acid sequence of SEQ ID NO: 137, the HA polypeptide tag comprising the amino acid sequence of SEQ ID NO: 136, and the full length of the B6R peptide excluding the signal peptide portion comprising the amino acid sequence of SEQ ID NO: 122. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of SEQ ID NO: 92.

[0120] In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a linker, an HA polypeptide tag, and a peptide segment from the C15L protein. In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a GS linker, an HA polypeptide tag, and the full-length C15L peptide. In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a GS linker, an HA polypeptide tag, and the full-length C15L peptide, wherein the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the GS linker comprises the amino acid sequence of SEQ ID NO: 137, the HA polypeptide tag comprises the amino acid sequence of SEQ ID NO: 136, and the full-length C15L peptide comprises the amino acid sequence of SEQ ID NO: 126. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 93.

[0121] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a linker, an HA polypeptide tag, and a peptide segment from the M1R protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a GS linker, an HA polypeptide tag, and an M1R extracellular segment. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a GS linker, an HA polypeptide tag, and an M1R extracellular segment, the IgE signal peptide comprising the amino acid sequence of SEQ ID NO: 134, the GS linker comprising the amino acid sequence of SEQ ID NO: 137, the HA polypeptide tag comprising the amino acid sequence of SEQ ID NO: 136, and the M1R extracellular segment comprising the amino acid sequence of SEQ ID NO: 128. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of SEQ ID NO: 94.

[0122] In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a linker, an HA polypeptide tag, and a peptide segment from the H3L protein. In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a GS linker, an HA polypeptide tag, and a full-length H3L peptide. In one embodiment, the polypeptide or combination of polypeptides comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a GS linker, an HA polypeptide tag, and a full-length H3L peptide, wherein the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the GS linker comprises the amino acid sequence of SEQ ID NO: 137, the HA polypeptide tag comprises the amino acid sequence of SEQ ID NO: 136, and the full-length H3L peptide comprises the amino acid sequence of SEQ ID NO: 131. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 95.

[0123] In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, an IgE signal peptide, a peptide segment from the A29L protein, a linker, a peptide segment from the A35R protein, a linker, a peptide segment from the B6R protein, a linker, a peptide segment from the B2R protein, a linker, a peptide segment from the C15L protein, a linker, and a peptide segment from the M1R protein. In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, an A35R extracellular segment, a GS linker, a B6R extracellular segment, a GS linker, a B2R extracellular segment, a GS linker, a C15L extracellular segment, a GS linker, and a full-length M1R peptide. In one embodiment, the polypeptide or combination of polypeptides comprises, from N-terminus to C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, an A35R extracellular segment, a GS linker, a B6R extracellular segment, a GS linker, a B2R extracellular segment, a GS linker, a C15L extracellular segment, a GS linker, and a full-length M1R peptide; the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the full-length A29L peptide comprises the amino acid sequence of SEQ ID NO: 113, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the A35R extracellular segment comprises the amino acid sequence of SEQ ID NO: 115, the B6R extracellular segment comprises the amino acid sequence of SEQ ID NO: 124, the B2R extracellular segment comprises the amino acid sequence of SEQ ID NO: 119, the C15L extracellular segment comprises the amino acid sequence of SEQ ID NO: 127, and the full-length M1R peptide comprises the amino acid sequence of SEQ ID NO: 130. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 101.

[0124] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a peptide segment from the B2R protein, a linker, a peptide segment from the A35R protein, a linker, and a peptide segment from the C15L protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, a B2R extracellular segment, a GS linker, an A35R extracellular segment, a GS linker, and the full-length C15L peptide. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, a B2R extracellular segment, a GS linker, an A35R extracellular segment, a GS linker, and the full-length C15L peptide; the B2R signal peptide comprises the amino acid sequence of SEQ ID NO: 116, the B2R extracellular segment comprises the amino acid sequence of SEQ ID NO: 119, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the A35R extracellular segment comprises the amino acid sequence of SEQ ID NO: 115, and the full-length C15L peptide comprises the amino acid sequence of SEQ ID NO: 126. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 102.

[0125] In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, an IgE signal peptide, a peptide segment from the A29L protein, a linker, a peptide segment from the M1R protein, a linker, and a peptide segment from the B6R protein. In one embodiment, the polypeptide or polypeptide combination comprises, from N-terminus to C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, an extracellular segment of the M1R, a GS linker, and a full-length B6R peptide excluding the signal peptide portion. In one embodiment, the polypeptide or combination of polypeptides comprises, from N-terminus to C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, an extracellular segment of M1R, a GS linker, and a full-length B6R peptide excluding the signal peptide portion; the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the full-length A29L peptide comprises the amino acid sequence of SEQ ID NO: 113, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the extracellular segment of M1R comprises the amino acid sequence of SEQ ID NO: 128, and the full-length B6R peptide excluding the signal peptide portion comprises the amino acid sequence of SEQ ID NO: 122. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 103.

[0126] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a peptide segment from the B6R protein, a linker, a peptide segment from the M1R protein, a linker, and a peptide segment from the H3L protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B6R signal peptide, a B6R extracellular segment, a GS linker, an M1R extracellular segment, a GS linker, and the full-length H3L peptide. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B6R signal peptide, a B6R extracellular segment, a GS linker, an M1R extracellular segment, a GS linker, and the full-length H3L peptide; the B6R signal peptide comprises the amino acid sequence of SEQ ID NO: 121, the B6R extracellular segment comprises the amino acid sequence of SEQ ID NO: 124, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the M1R extracellular segment comprises the amino acid sequence of SEQ ID NO: 128, and the full-length H3L peptide comprises the amino acid sequence of SEQ ID NO: 131. In one embodiment, the polypeptide or polypeptide combination comprises the amino acid sequence of SEQ ID NO: 104.

[0127] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a peptide segment from the B6R protein, a linker, a peptide segment from the A35R protein, a linker, and a peptide segment from the M1R protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B6R signal peptide, a B6R extracellular segment, a GS linker, an A35R extracellular segment, a GS linker, and a full-length M1R peptide. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B6R signal peptide, a B6R extracellular segment, a GS linker, an A35R extracellular segment, a GS linker, and a full-length M1R peptide; the B6R signal peptide comprises the amino acid sequence of SEQ ID NO: 121, the B6R extracellular segment comprises the amino acid sequence of SEQ ID NO: 124, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the A35R extracellular segment comprises the amino acid sequence of SEQ ID NO: 115, and the full-length M1R peptide comprises the amino acid sequence of SEQ ID NO: 130. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 105.

[0128] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a peptide segment from the A29L protein, a linker, a peptide segment from the B2R protein, a linker, and a peptide segment from the C15L protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, a B2R extracellular segment, a GS linker, and a full-length C15L peptide. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, a B2R extracellular segment, a GS linker, and a full-length C15L peptide; the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the full-length A29L peptide comprises the amino acid sequence of SEQ ID NO: 113, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the B2R extracellular segment comprises the amino acid sequence of SEQ ID NO: 119, and the full-length C15L peptide comprises the amino acid sequence of SEQ ID NO: 126. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 106.

[0129] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a peptide segment from the B2R protein, a linker, a peptide segment from the H3L protein, a linker, and a peptide segment from the C15L protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, a B2R extracellular segment, a GS linker, an H3L extracellular segment, a GS linker, and the full-length C15L peptide. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, a B2R extracellular segment, a GS linker, an H3L extracellular segment, a GS linker, and the full-length C15L peptide; the B2R signal peptide comprises the amino acid sequence of SEQ ID NO: 116, the B2R extracellular segment comprises the amino acid sequence of SEQ ID NO: 119, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the H3L extracellular segment comprises the amino acid sequence of SEQ ID NO: 132, and the full-length C15L peptide comprises the amino acid sequence of SEQ ID NO: 126. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 107.

[0130] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a peptide segment from the B2R protein, a linker, a peptide segment from the A35R protein, a linker, and a peptide segment from the B6R protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, a B2R extracellular segment, a GS linker, an A35R extracellular segment, a GS linker, and the full length of the B6R peptide excluding the signal peptide portion. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, a B2R signal peptide, a B2R extracellular segment, a GS linker, an A35R extracellular segment, a GS linker, and the full length of the B6R peptide excluding the signal peptide portion; the B2R signal peptide comprises the amino acid sequence of SEQ ID NO: 116, the B2R extracellular segment comprises the amino acid sequence of SEQ ID NO: 119, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the A35R extracellular segment comprises the amino acid sequence of SEQ ID NO: 115, and the full length of the B6R peptide excluding the signal peptide portion comprises the amino acid sequence of SEQ ID NO: 122. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 108.

[0131] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a peptide segment from the A29L protein, a linker, a peptide segment from the M1R protein, a linker, and a peptide segment from the C15L protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, an extracellular segment of the M1R, a GS linker, and a full-length C15L peptide. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a full-length A29L peptide, a GS linker, an extracellular segment of the M1R, a GS linker, and a full-length C15L peptide; the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the full-length A29L peptide comprises the amino acid sequence of SEQ ID NO: 113, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the extracellular segment of the M1R comprises the amino acid sequence of SEQ ID NO: 128, and the full-length C15L peptide comprises the amino acid sequence of SEQ ID NO: 126. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO: 109.

[0132] In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, a peptide segment from the H3L protein, a linker, a peptide segment from the C15L protein, a linker, and a peptide segment from the M1R protein. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, an H3L extracellular segment, a GS linker, a C15L extracellular segment, a GS linker, and a full-length M1R peptide. In one embodiment, the polypeptide or polypeptide combination comprises, from the N-terminus to the C-terminus, an IgE signal peptide, an H3L extracellular segment, a GS linker, a C15L extracellular segment, a GS linker, and a full-length M1R peptide; the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134, the H3L extracellular segment comprises the amino acid sequence of SEQ ID NO: 132, the GS linker comprises the amino acid sequence of SEQ ID NO: 138, the C15L extracellular segment comprises the amino acid sequence of SEQ ID NO: 127, and the full-length M1R peptide comprises the amino acid sequence of SEQ ID NO: 130. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of SEQ ID NO:110.

[0133] In one embodiment, the polypeptide or combination of polypeptides comprises two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110, or two or more amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110.

[0134] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide and a sixth polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:89, the second polypeptide comprises the amino acid sequence of SEQ ID NO:90, the third polypeptide comprises the amino acid sequence of SEQ ID NO:91, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO:92, the fifth polypeptide comprises the amino acid sequence of SEQ ID NO:93, and the sixth polypeptide comprises the amino acid sequence of SEQ ID NO:94.

[0135] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide and a sixth polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:90, the second polypeptide comprises the amino acid sequence of SEQ ID NO:91, the third polypeptide comprises the amino acid sequence of SEQ ID NO:92, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO:93, the fifth polypeptide comprises the amino acid sequence of SEQ ID NO:94, and the sixth polypeptide comprises the amino acid sequence of SEQ ID NO:95.

[0136] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 102 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 103.

[0137] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 105 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 106.

[0138] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 108 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 109.

[0139] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 102 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 104.

[0140] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 105 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 107.

[0141] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 108.

[0142] The polypeptide or polypeptide combination of the present invention can be used as a polypeptide antigen to induce a protective immune response against monkeypox virus infection in a subject.

[0143] Nucleic Acids

[0144] On the one hand, the present invention provides nucleic acids or nucleic acid combinations encoding polypeptides or polypeptide combinations described herein. In some embodiments, nucleic acids or nucleic acid combinations comprise polynucleotides encoding polypeptides or polypeptide combinations described herein. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include but are not limited to DNA, cDNA, RNA (e.g., mRNA, circRNA or saRNA), recombinantly produced and chemically synthesized nucleic acids. The nucleic acids or nucleic acid combinations of the present invention may be contained in a vector. The nucleic acids of the present invention may include naturally occurring, synthetic and modified nucleotides. In some embodiments, the nucleic acids or nucleic acid combinations of the present invention are used to express polypeptides or polypeptide combinations described herein in cells to provide polypeptide antigens. In some embodiments, polypeptide antigens can induce an immune response against monkeypox virus in suitable subjects.

[0145] The nucleic acid may comprise one or more segments (nucleotide fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more segments). The nucleic acid may comprise a segment encoding a polypeptide of interest (e.g., a polypeptide and polypeptide antigen described herein). In a specific embodiment, the nucleic acid may comprise a coding sequence for a polypeptide of interest and a regulatory sequence (including but not limited to transcriptional and translational regulatory sequences). In one embodiment, the regulatory sequence comprises one or more of the following: a promoter sequence, a 5' untranslated region (5'UTR) sequence, a 3' untranslated region (3'UTR) sequence, and a poly(A) sequence.

[0146] Coding sequence

[0147] As used herein, "coding sequence" refers to a nucleotide sequence in a nucleic acid that can be used as a template for synthesizing a nucleotide sequence having a defined nucleotide sequence (e.g., tRNA and mRNA) or a defined amino acid sequence in a biological process. A coding sequence can be a DNA sequence or an RNA sequence. If an mRNA corresponding to a DNA sequence (including a coding strand identical to an mRNA sequence and a template strand complementary thereto) is translated into a polypeptide in a biological process, the DNA sequence or mRNA sequence can be considered to encode the polypeptide.

[0148] As used herein, "codon" refers to a sequence of three consecutive nucleotides (also known as a triplet code) in a nucleic acid that encodes a specific amino acid. Synonymous codons (codons encoding the same amino acid) are used at different frequencies in different species, which is called "codon preference". It is generally believed that for a given species, a coding sequence using the codons of its preference can have higher translation efficiency and accuracy in the species expression system. Therefore, nucleic acids can be "codon optimized", i.e., the codons in the nucleic acid are changed to reflect the codons of the host cell preference, while preferably not changing the amino acid sequence encoded by it. It will be understood by those skilled in the art that due to the degeneracy of codons, the nucleic acids or nucleic acid combinations of the present invention may include coding sequences that are different from the coding sequences described herein (e.g., having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous to the coding sequences described herein) but encode the same amino acid sequence. In a specific embodiment, the nucleic acids of the present invention include codons optimized for host (e.g., subject, particularly human) cells so that the polypeptide of the present invention is optimally expressed in the host (e.g., subject, particularly human). In a specific embodiment, the RNA of the present invention comprises codons optimized for host (eg, subject, particularly human) cells, such that the polypeptide of the present invention is optimally expressed in the host (eg, subject, particularly human).

[0149] In some embodiments, nucleic acid or nucleic acid combination of the present invention comprises the coding sequence of a polypeptide as described herein. In some embodiments, nucleic acid or nucleic acid combination of the present invention comprises the coding sequence of a polypeptide antigen as described herein. In some embodiments, nucleic acid or nucleic acid combination of the present invention comprises a nucleotide sequence complementary to the coding sequence as described herein. In some embodiments, the coding sequence comprises a start codon at its 5' end and a stop codon at its 3' end. In some embodiments, the coding sequence comprises an open reading frame (ORF) as described herein.

[0150] In some embodiments, the coding sequences of the invention encode any of the polypeptide antigens of the invention.

[0151] In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen comprising:

[0152] (1) an amino acid sequence of one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110; (2) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110; (3) an amino acid sequence of SEQ ID NOs: NO:89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109 and 110; (4) an immunogenic fragment of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of one of SEQ ID NO:89, 90, 91, 92, 93, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, 109 and 110.

[0153] In one embodiment, the coding sequence of the present invention encodes a polypeptide comprising the amino acid sequence of a polypeptide antigen as described above.

[0154] In one embodiment, the coding sequence of a polypeptide or polypeptide antigen described herein comprises a nucleotide sequence comprising: (1) a nucleotide sequence of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22; (2) a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to a nucleotide sequence of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22; (3) a nucleotide sequence of one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44; or (4) a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to a nucleotide sequence of one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44. The nucleotide sequence of one of NO:23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical.

[0155] In one embodiment, the coding sequence of a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising: (1) two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22; (2) two or more nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22; (3) two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: NO:23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44; or (4) two or more nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence selected from SEQ ID NO:23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44.

[0156] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 1; the nucleotide sequence of the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 2; and the nucleotide sequence of the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 3. NO:3 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:4; the nucleotide sequence of the fourth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:4; the nucleotide sequence of the fifth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:5; the nucleotide sequence of the sixth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:6 The nucleotide sequence of NO:6 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0157] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 23; the nucleotide sequence of the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 24; and the nucleotide sequence of the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:25; the nucleotide sequence of the fourth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:26 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:26; the nucleotide sequence of the fifth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:27 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:27; the nucleotide sequence of the sixth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:28 The nucleotide sequence of NO:28 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0158] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 2; the nucleotide sequence of the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 3; the nucleotide sequence of the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: NO:4 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:5; the nucleotide sequence of the fourth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:5; the nucleotide sequence of the fifth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:6; the nucleotide sequence of the sixth polynucleotide comprises: the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:7 The nucleotide sequence of NO:7 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0159] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 24; the nucleotide sequence of the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 25; and the nucleotide sequence of the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 27. NO:26 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:27; the nucleotide sequence of the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:28; the nucleotide sequence of the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of SEQ ID NO:29. The nucleotide sequence of NO:29 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0160] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 14; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 15.

[0161] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO:36 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:36; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO:37 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:37.

[0162] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 17; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 18.

[0163] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO:39 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:39; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO:40 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:40.

[0164] In one embodiment, the coding sequence of the polypeptide or polypeptide combination comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 20; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence of SEQ ID NO: 21.

[0165] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO:42 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:42; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO:43 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:43.

[0166] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 14; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 16.

[0167] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO:36 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:36; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:38.

[0168] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 17; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 19.

[0169] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO:39 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:39; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO:41 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:41.

[0170] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:22; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:20.

[0171] In one embodiment, the coding sequence of the polypeptide or combination of polypeptides comprises the nucleotide sequence of a first polynucleotide and a second polynucleotide; wherein the nucleotide sequence of the first polynucleotide comprises: the nucleotide sequence of SEQ ID NO:44 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:44; the nucleotide sequence of the second polynucleotide comprises: the nucleotide sequence of SEQ ID NO:42 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:42.

[0172] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, and a sixth polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 90, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 91, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 92, the fifth polypeptide comprises the amino acid sequence of SEQ ID NO: 93, and the sixth polypeptide comprises the amino acid sequence of SEQ ID NO: 94; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the fifth polypeptide, and the sixth polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 1; and the nucleotide sequence encoding the second polypeptide comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 1. NO:2 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:2; the nucleotide sequence encoding the third polypeptide comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:3; the nucleotide sequence encoding the fourth polypeptide comprises the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:4; the nucleotide sequence encoding the fifth polypeptide comprises the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:5 NO:5 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:5; the nucleotide sequence encoding the sixth polypeptide comprises: the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:6.

[0173] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, and a sixth polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 90, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 91, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 92, the fifth polypeptide comprises the amino acid sequence of SEQ ID NO: 93, and the sixth polypeptide comprises the amino acid sequence of SEQ ID NO: 94; and the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the fifth polypeptide, and the sixth polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 23; and the nucleotide sequence encoding the second polypeptide comprises the nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: NO:24 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:25; the nucleotide sequence encoding the third polypeptide comprises the nucleotide sequence of SEQ ID NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:25; the nucleotide sequence encoding the fourth polypeptide comprises the nucleotide sequence of SEQ ID NO:26 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:26; the nucleotide sequence encoding the fifth polypeptide comprises the nucleotide sequence of SEQ ID NO:27 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:27. The nucleotide sequence of NO:27 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:27; the nucleotide sequence encoding the sixth polypeptide comprises: the nucleotide sequence of SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:28.

[0174] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, and a sixth polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:90, the second polypeptide comprises the amino acid sequence of SEQ ID NO:91, the third polypeptide comprises the amino acid sequence of SEQ ID NO:92, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO:93, the fifth polypeptide comprises the amino acid sequence of SEQ ID NO:94, and the sixth polypeptide comprises the amino acid sequence of SEQ ID NO:95; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the fifth polypeptide, and the sixth polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:2; and the nucleotide sequence encoding the second polypeptide comprises the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:4. NO:3 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:3; the nucleotide sequence encoding the third polypeptide comprises the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:4; the nucleotide sequence encoding the fourth polypeptide comprises the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:5; the nucleotide sequence encoding the fifth polypeptide comprises the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:6. NO:6 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:6; the nucleotide sequence encoding the sixth polypeptide comprises: the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:7.

[0175] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, and a sixth polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:90, the second polypeptide comprises the amino acid sequence of SEQ ID NO:91, the third polypeptide comprises the amino acid sequence of SEQ ID NO:92, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO:93, the fifth polypeptide comprises the amino acid sequence of SEQ ID NO:94, and the sixth polypeptide comprises the amino acid sequence of SEQ ID NO:95; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the fifth polypeptide, and the sixth polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises the nucleotide sequence of SEQ ID NO:24, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:24; and the nucleotide sequence encoding the second polypeptide comprises the nucleotide sequence of SEQ ID NO:25, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:25. NO:25 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:25; the nucleotide sequence encoding the third polypeptide comprises the nucleotide sequence of SEQ ID NO:26 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:26; the nucleotide sequence encoding the fourth polypeptide comprises the nucleotide sequence of SEQ ID NO:27 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:27; the nucleotide sequence encoding the fifth polypeptide comprises the nucleotide sequence of SEQ ID NO:28 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:28. The nucleotide sequence of NO:28 has a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:29; the nucleotide sequence encoding the sixth polypeptide comprises: the nucleotide sequence of SEQ ID NO:29 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:29.

[0176] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 102 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 103; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 14; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 15.

[0177] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 102 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 103; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: a nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 36; and the nucleotide sequence encoding the second polypeptide comprises: a nucleotide sequence of SEQ ID NO: 37, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 37.

[0178] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 105 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 106; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: a nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 17; and the nucleotide sequence encoding the second polypeptide comprises: a nucleotide sequence of SEQ ID NO: 18, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 18.

[0179] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 105 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 106; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 39, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 39; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 40, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 40.

[0180] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 108 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 109; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 20; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 21, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 21.

[0181] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 108 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 109; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 42, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 42; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 43, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 43.

[0182] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 102 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 104; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 14; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 16.

[0183] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 102 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 104; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: a nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 36; and the nucleotide sequence encoding the second polypeptide comprises: a nucleotide sequence of SEQ ID NO: 38, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 38.

[0184] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 105 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 107; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: a nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 17; and the nucleotide sequence encoding the second polypeptide comprises: a nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 19.

[0185] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 105 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 107; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 39, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 39; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 41, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 41.

[0186] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 108; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 22, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 22; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 20.

[0187] In one embodiment, the polypeptide or combination of polypeptides comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 110 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 108; the coding sequence described herein comprises a nucleotide sequence encoding the first polypeptide and the second polypeptide; wherein the nucleotide sequence encoding the first polypeptide comprises: the nucleotide sequence of SEQ ID NO: 44, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 44; and the nucleotide sequence encoding the second polypeptide comprises: the nucleotide sequence of SEQ ID NO: 42, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 42.

[0188] RNA

[0189] In some embodiments, the nucleic acid or nucleic acid combination of the present invention is RNA. In some embodiments, the polynucleotide of the present invention is RNA. As used herein, the definition of "RNA" encompasses single-stranded, double-stranded, linear and circular RNA. The RNA of the present invention can be non-amplified RNA or self-amplified RNA. The RNA of the present invention can be RNA produced by chemical synthesis, recombinant production and in vitro transcription. In some embodiments, the RNA of the present invention is used to express the polypeptide of the present invention in a host cell. In some embodiments, the RNA of the present invention is used to express the polypeptide combination of the present invention in a host cell.

[0190] In some embodiments, the RNA of the present invention is single-stranded RNA. In some embodiments, the RNA of the present invention is in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription using a DNA template by RNA polymerase (e.g., as described herein).

[0191] In some embodiments, the RNA of the present invention is a messenger RNA (mRNA). Generally speaking, the mRNA may comprise a 5'-UTR sequence, a coding sequence for a polypeptide (e.g., a polypeptide or polypeptide combination of the present invention), a 3'-UTR sequence, and an optional poly(A) sequence. The mRNA can be produced, for example, by in vitro transcription or chemical synthesis. In one embodiment, the mRNA of the present invention is obtained by in vitro transcription using an RNA polymerase (e.g., T7 RNA polymerase) using a DNA template. In one embodiment, the mRNA of the present invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3'-UTR, and (4) an optional poly(A) sequence. The 5'-UTR, coding sequence, 3'-UTR, and poly(A) sequence are as described herein. In one embodiment, the mRNA of the present invention is a nucleoside-modified mRNA. In one embodiment, the mRNA of the present invention comprises an optional 5' cap. In one embodiment, the mRNA of the present invention comprises a cap-proximal sequence.

[0192] In some embodiments, the RNA of the present invention is a circular RNA (circRNA). As used herein, the term "circRNA" or "circular RNA" refers to an RNA that forms a circular structure by covalent bonds. Generally speaking, circRNA is a single-stranded RNA that may include a UTR (5'-UTR and / or 3'-UTR) and an open reading frame, but does not include a 5' cap and a poly (A) tail. CircRNA may include elements in the UTR that promote cap-independent translation, such as an internal ribosome entry site (IRES) and an optional polyadenylic acid (polyA) bundle or polyadenylic acid (polyAC) interspersed with cytidylic acid. CircRNA can be produced using enzymatic or chemical methods. In general, enzymatic methods can include, for example, the end connection of a linear precursor RNA catalyzed by T4 ligase and the formation of circRNA using ribozymes during IVT. Methods for preparing circRNA using ribozymes can include, for example, group I intron-based methods, group II intron-based methods, and hairpin ribozyme-based methods (see, for example, Wesselhoeft RA et al., Nature Communications, (2018) 9: 2629; Petkovic, S. et al., Nucleic Acids Research, 2015, Vol. 43, No. 4, 2454-2465; Chen, XJ et al., Front. Bioeng. Biotechnol., 30 November 2021; and Lambowitz, AM et al., Cold Spring Harb Perspect Biol. 2011 Aug; 3(8): a003616).

[0193] In some embodiments, the RNA of the present invention is a self-amplifying RNA (saRNA). As used herein, the terms "self-replicating RNA," "self-amplifying RNA (saRNA)," and "replicon" are used interchangeably to refer to RNA that can self-amplify and translate. In general, saRNA can be derived from or comprise a self-replicating subgenome of a viral genome (e.g., a positive-sense single-stranded RNA virus, such as an alphavirus, such as Venezuelan equine encephalitis virus (VEEV)), which includes viral genes encoding non-structural proteins nsP1, nsP2, nsP3, and nsP4 that form RNA-dependent RNA polymerase (RdRP), while the viral genes encoding structural proteins are replaced by genes encoding polypeptides of interest. The saRNA can comprise two open reading frames (ORFs) separated by a subgenomic promoter, i.e., a second ORF encoding a polypeptide of interest (e.g., a polypeptide or polypeptide combination described herein) at the 3' end of the first ORF encoding nsP1-nsP4, driven by the subgenomic promoter. The saRNA can also comprise a 5' cap and a 5'-UTR at the 5' end, and a 3'-UTR and a poly(A) sequence at the 3' end. In addition, the untranslated region of saRNA may also contain 5' and 3' conserved sequence elements (CSEs) required for self-amplification recognized by RdRP. Similar to mRNA, saRNA can also be produced by in vitro transcription or chemical synthesis. For a detailed description of saRNA, see, for example, WO2016135675A1 and WO2021183564A1.

[0194] In some embodiments, the RNA of the present invention (e.g., mRNA, circRNA, and saRNA) comprises a coding sequence for a polypeptide as described herein. In some embodiments, the RNA of the present invention (e.g., mRNA, circRNA, and saRNA) comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the RNA of the present invention (e.g., mRNA, circRNA, and saRNA) comprises a coding sequence for a combination of polypeptides as described herein.

[0195] In some embodiments, the RNA of the present invention further comprises structural elements that help improve the stability and / or translation efficiency of the RNA, including but not limited to a 5' cap, a cap-proximal sequence, a 5'-UTR, a 3'-UTR, and a poly(A) sequence.

[0196] As used herein, the term "untranslated region (UTR)" generally refers to a region (non-coding region) in RNA (such as mRNA) that is not translated into an amino acid sequence, or a corresponding region in DNA. Generally, the UTR located at the 5' end (upstream) of the open reading frame (start codon) can be referred to as the 5' untranslated region 5'-UTR; the UTR located at the 3' end (downstream) of the open reading frame (stop codon) can be referred to as the 3'-UTR. In the presence of a 5' cap, the 5'-UTR is located downstream of the 5' cap, for example, directly adjacent to the 5' cap. In a specific embodiment, an optimized "Kozak sequence" can be included in the 5'-UTR, for example, near the start codon, to improve translation efficiency. In the presence of a poly (A) sequence, the 3'-UTR is located upstream of the poly (A) sequence, for example, directly adjacent to the poly (A) sequence.

[0197] In some embodiments, the RNA of the present invention comprises a 5'-UTR. In a preferred embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 139 or 148. In some embodiments, the RNA of the present invention comprises a 3'-UTR. In a preferred embodiment, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 140, 141, or 149. In some embodiments, the RNA of the present invention comprises a 5'-UTR and a 3'-UTR. In one embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 139 or 148, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 140, 141, or 149. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 139, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 140 or 141. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 148, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 149.

[0198] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to a nucleotide sequence containing continuous or discontinuous adenylate nucleotides. The poly(A) sequence is typically located at the 3' end of the RNA, for example, at the 3' end (downstream) of the 3'-UTR. In some embodiments, the poly(A) sequence does not contain nucleotides other than adenylate nucleotides at its 3' end. The poly(A) sequence can be transcribed from the coding sequence of the DNA template by a DNA-dependent RNA polymerase during the preparation of the IVT-RNA, or can be attached to the free 3' end of the IVT-RNA, for example, at the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).

[0199] In some embodiments, the RNA of the present invention comprises a poly(A) sequence. In one embodiment, the poly(A) sequence comprises contiguous adenylate nucleotides. In one embodiment, the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100, and up to 120, 150, 180, 200, or 300 adenylate nucleotides. In one embodiment, the contiguous adenylate sequence in the poly(A) sequence is interrupted by a sequence comprising U, C, or G nucleotides. In one embodiment, the poly(A) sequence comprises at least 50 nucleotides. In one embodiment, the poly(A) sequence comprises at least 80 nucleotides. In one embodiment, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises approximately 70, 80, 90, 100, 120, or 150 nucleotides. In a preferred embodiment, the poly(A) sequence comprises 121 nucleotides. In a preferred embodiment, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:145.

[0200] As used herein, the term "5' cap" generally refers to an N7-methylguanosine structure (also known as an "m7G cap," "m7Gppp-") attached to the 5' end of an mRNA via a 5' to 5' triphosphate bond. The 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or it can be attached to the RNA post-transcriptionally using a capping enzyme. In some embodiments, RNA (e.g., IVT-RNA) is modified to "Cap0 RNA" using a capping enzyme (e.g., vaccinia virus capping enzyme). In some embodiments, the 5' cap immediately adjacent to the m in Cap0 RNA is 5'-methylguanosine. 7 Additional methylation occurs at the 2'-O position of the ribose of the nucleotide of the G cap (e.g., by a 2'-O-methyltransferase), resulting in "Cap1 RNA". In some embodiments, cap analogs are used to generate 5' cap-modified RNA. For a description of "cap analogs", see, for example, Contreas, R. et al. (1982). Nucl. Acids Res. 10, 6353-6363 and US7074596B2. Examples of cap analogs include, but are not limited to, N7-methylguanosine-5'-triphosphate-5' guanosine (m 7 G(5')ppp(5')G), N7-methylguanosine-5'-triphosphate-5'-adenosine (m 7 G(5')ppp(5')A) and 3'-O-Me-m 7 G(5')ppp(5')G(ARCA).

[0201] The RNA of the present invention (e.g., mRNA and saRNA) may be a RNA comprising Cap0 (m 7 The ribose of the adjacent nucleotides of G is not methylated), Cap1(m 7 Methylation of the ribose of the adjacent nucleotide of G) or Cap2 (m 7 In a preferred embodiment, the mRNA of the present invention is Cap1 mRNA. In a preferred embodiment, the mRNA of the present invention comprises 7Me GpppG 2’OMe .

[0202] In some embodiments, the RNA of the present invention further comprises a cap-proximal sequence. In some embodiments, the 5'UTR comprises a cap-proximal sequence. In some embodiments, the 5'UTR does not comprise a cap-proximal sequence. In some embodiments, the cap-proximal sequence comprises a sequence adjacent to the 5' cap. In one embodiment, the cap-proximal sequence comprises the nucleotide sequence of SEQ ID NO: 147. In one embodiment, one or more residues in the cap-proximal sequence may be included in the RNA because they are already included in the cap entity (e.g., Cap1 structure, etc.); alternatively, in one embodiment, at least some of the residues in the cap-proximal sequence may be enzymatically added (e.g., by a polymerase such as T7 polymerase).

[0203] In some embodiments, a nucleic acid or combination of nucleic acids of the invention comprises a polynucleotide encoding a polypeptide or combination of polypeptides described herein.

[0204] In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising the nucleotide sequence of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22. In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising the nucleotide sequence of one of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of one of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66.

[0205] In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising two or more nucleotide sequences selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, or two or more nucleotide sequences selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, comprising two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, or two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0206] In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6. In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 45, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 46, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 47, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 48, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 49, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 50.

[0207] In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7. In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 46, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 47, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 48, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 49, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 50, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 51.

[0208] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 14, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 58, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 59.

[0209] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 61 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 62.

[0210] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 20 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 21. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 64 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 65.

[0211] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 14, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 58, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 60.

[0212] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 19. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 61, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 63.

[0213] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 22, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 20. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 66, and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 64.

[0214] Modified nucleotides

[0215] In some embodiments, the nucleotides in the RNA (e.g., mRNA) of the present invention can be naturally occurring nucleotides (e.g., naturally occurring ribonucleotides) and modified nucleotides. Modified nucleotides can be, for example, nucleotides that are not present in naturally occurring RNA, such as non-standard nucleotides or deoxynucleotides. Modification of nucleotides can occur on the nucleoside, for example, on the ribose moiety and / or the nucleobase moiety. Modified nucleotides can be incorporated during transcription (e.g., in vitro transcription) or added during RNA chemical synthesis.

[0216] In some embodiments, RNA is modified by comprising one or more modified nucleosides. In some embodiments, RNA (e.g., mRNA) of the present invention is modified by comprising one or more modified nucleobases. In some embodiments, the modified nucleobases include modified cytosine, modified uracil, modified adenine, modified guanine, or a combination thereof.

[0217] Exemplary nucleobases and nucleosides having modified uracils can include, but are not limited to, pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thiouracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyluracil (m3U), 5-methoxy-uracil (m o5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5-aminomethyl-2-thio-uracil (nm5s2U), 5-methylaminomethyl-uracil (mnm5U), 5- Methylaminomethyl-2-thiouracil (mnm5s2U), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouracil (τm55s2U), 1-taurinomethyl Sulfonic acid methyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uracil (m5U), 5-(isopentenylaminomethyl)-2-thio-uracil (m5s2U), 5,2'-O-dimethyl-uridine (m5Um), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonyl 5-(Isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyl-uracil, 5-methoxy-2-thiouracil and 5-[3-(1-E-propenylamino)]uracil.

[0218] Exemplary nucleobases and nucleosides having modified cytosines can include, but are not limited to, 5-azacytosine, 6-azacytosine, pseudoisocytidine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolocytosine, pyrrolopseudoisocytidine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thiopseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, ), 5-aza-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), 5,2'-O-dimethyl-cytidine (m5Cm), N4- Acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (fSCm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine and 5-(2-azidoethyl)-cytosine.

[0219] Exemplary nucleobases and nucleosides having a modified adenine can include, but are not limited to, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza- Azo-8-aza-2,6-diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycyl N6-methylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxahedronadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine and N6-hydroxymethyl-adenine.

[0220] Exemplary nucleobases and nucleosides having modified guanines can include, but are not limited to, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q), epoxyqueuosine (oQ), galactosylqueuosine (galQ), mannosylqueuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-Methyl-guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl- 6-Thio-guanine, N2,N2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 1-thio-guanine and O-6-methyl-guanine.

[0221] In some embodiments, the proportion of modified nucleobases in the RNA of the present invention is 10%-100%, that is, the RNA of the present invention can be modified by replacing 10%-100% of the nucleobases therein with modified nucleobases.

[0222] In some embodiments, the RNA (e.g., mRNA) of the present invention is modified by replacing one or more uracils with modified uracils. In some embodiments, the modified uracils include 1-methylpseudouracil, pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the modified uracils include pseudouracils. In one embodiment, the modified uracils include 5-methyl-uracils. In one embodiment, the modified uracils include 1-methyl-pseudouracils.

[0223] In one embodiment, the RNA is modified by replacing at least one uracil with a modified uracil. In one embodiment, the RNA is modified by replacing all uracils with modified uracils. In one embodiment, the proportion of modified uracils in the RNA is 10%-100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In one embodiment, the proportion of modified uracils in the RNA is 20%-100%. In one embodiment, 20%-100% of the uracils in the RNA are replaced by 1-methylpseudouracil. In a preferred embodiment, 100% of the uracils in the RNA are replaced by 1-methylpseudouracil.

[0224] In some embodiments, the RNA (e.g., mRNA) of the present invention is modified by replacing one or more uridines with modified uridines. In some embodiments, the modified uridines include 1-methylpseudouridine, pseudouridine, 5-methyl-uridine, or a combination thereof. In one embodiment, the modified uridines include pseudouridines. In one embodiment, the modified uridines include 5-methyl-uridines. In one embodiment, the modified uridines include 1-methyl-pseudouridine.

[0225] In one embodiment, the RNA is modified by replacing at least one uridine with a modified uridine. In one embodiment, the RNA is modified by replacing all uridines with modified uridines. In one embodiment, the proportion of modified uridines in the RNA is 10%-100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In one embodiment, the proportion of modified uridines in the RNA is 20%-100%. In one embodiment, 20%-100% of the uridines in the RNA are replaced by 1-methylpseudouridine. In a preferred embodiment, 100% of the uridines in the RNA are replaced by 1-methylpseudouridine.

[0226] 1-Methyl-pseudouridine has the following structure:

[0227] In a specific embodiment, the RNA of the present invention comprises the nucleotide sequence of any one of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, and wherein 100% of the uracil is replaced by 1-methylpseudouracil.

[0228] In a specific embodiment, the RNA of the present invention comprises the nucleotide sequence of any one of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, and wherein 100% of the uridine is replaced by 1-methylpseudouridine.

[0229] In a specific embodiment, the mRNA of the present invention comprises the nucleotide sequence of any one of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, and wherein 100% of the uracil is replaced by 1-methylpseudouracil.

[0230] In a specific embodiment, the mRNA of the present invention comprises the nucleotide sequence of any one of SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66, and wherein 100% of the uridine is replaced by 1-methylpseudouridine.

[0231] DNA

[0232] In some embodiments, nucleic acid of the present invention or nucleic acid combination is DNA. In some embodiments, polynucleotide of the present invention is DNA. Such DNA can be, for example, a DNA template for in vitro transcription of RNA of the present invention or a DNA for expressing polypeptide or polypeptide antigen in a host cell. DNA can be double-stranded, single-stranded, linear and circular DNA.

[0233] The DNA template can be provided in a suitable transcription vector. Generally speaking, the DNA template can be a double-stranded complex, which comprises a nucleotide sequence identical to the coding sequence described herein (coding strand) and a nucleotide sequence complementary to the coding sequence described herein (template strand). As known to those skilled in the art, the DNA template can include a promoter, a DNA sequence corresponding to a 5'-UTR, a coding sequence, a 3'-UTR, and an optional poly (A) sequence. The promoter can be an available promoter for a suitable RNA polymerase (particularly DNA-dependent RNA polymerase) known to those skilled in the art, including but not limited to promoters of SP6, T3, and T7 RNA polymerases. In some embodiments, the 5'-UTR, the coding sequence, the 3'-UTR, and the poly (A) sequence in the DNA template are the corresponding sequences included in the RNA described herein or are complementary thereto. DNA can be provided in a plasmid vector (e.g., a circular plasmid vector).

[0234] In some embodiments, the DNA of the present invention comprises the coding sequence of a polypeptide as described herein. In some embodiments, the DNA of the present invention comprises the coding sequence of a polypeptide antigen as described herein. In some embodiments, the DNA of the present invention comprises the coding sequence of a combination of polypeptides as described herein.

[0235] In some embodiments, the DNA of the present invention comprises a DNA sequence corresponding to a 5'-UTR sequence. In a preferred embodiment, the DNA sequence corresponding to the 5'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 142 or 150. In some embodiments, the DNA of the present invention comprises a DNA sequence corresponding to a 3'-UTR sequence. In a preferred embodiment, the DNA sequence corresponding to the 3'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 143, 144, or 151. In some embodiments, the DNA of the present invention comprises DNA sequences corresponding to both the 5'-UTR and 3'-UTR sequences. In one embodiment, the DNA sequence corresponding to the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 142 or 150, and the DNA sequence corresponding to the 3'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 143, 144, or 151. In a specific embodiment, the DNA sequence corresponding to the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 142, and the DNA sequence corresponding to the 3'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 143 or 144. In a specific embodiment, the DNA sequence corresponding to the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 150, and the DNA sequence corresponding to the 3'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 151.

[0236] In some embodiments, the DNA of the present invention comprises a DNA sequence corresponding to a poly(A) sequence. In one embodiment, the DNA sequence corresponding to the poly(A) sequence comprises contiguous deoxyadenylic acids. In one embodiment, the DNA sequence corresponding to the poly(A) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 deoxyadenylic acids, and up to 120, 150, 180, 200, or 300 deoxyadenylic acids. In one embodiment, the DNA sequence corresponding to the poly(A) sequence comprises at least 50 nucleotides. In one embodiment, the DNA sequence corresponding to the poly(A) sequence comprises at least 80 nucleotides. In one embodiment, the DNA sequence corresponding to the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the DNA sequence corresponding to the poly(A) sequence comprises approximately 70, 80, 90, 100, 120, or 150 nucleotides. In a preferred embodiment, the DNA sequence corresponding to the poly(A) sequence comprises 121 nucleotides. In a preferred embodiment, the DNA sequence corresponding to the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 146.

[0237] In some embodiments, the polynucleotide is a DNA comprising a nucleotide sequence of one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44. In some embodiments, the polynucleotide is a DNA comprising a nucleotide sequence of one of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of one of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

[0238] In some embodiments, the polynucleotide is DNA and comprises two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44, or two or more nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44. In some embodiments, the polynucleotide is DNA and comprises two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, or two or more nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

[0239] In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 23, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 24, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 26, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 27, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 28. In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 67, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 68, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 69, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 70, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 71, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 72.

[0240] In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 24, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 25, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 26, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 27, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 28, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 29. In one embodiment, the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide, and a sixth polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 68, the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 69, the third polynucleotide comprises the nucleotide sequence of SEQ ID NO: 70, the fourth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 71, the fifth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 72, and the sixth polynucleotide comprises the nucleotide sequence of SEQ ID NO: 73.

[0241] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 36 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 37. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 80 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 81.

[0242] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 39 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 40. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 83 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 84.

[0243] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 42 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 43. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 86 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 87.

[0244] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 36 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 38. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 80 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 82.

[0245] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 39 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 41. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 83 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 85.

[0246] In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 44 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 42. In one embodiment, the polynucleotide comprises a first polynucleotide and a second polynucleotide; wherein the first polynucleotide comprises the nucleotide sequence of SEQ ID NO: 88 and the second polynucleotide comprises the nucleotide sequence of SEQ ID NO: 86.

[0247] Vectors and host cells

[0248] In yet another aspect, the present invention also provides an expression vector comprising a nucleic acid or nucleic acid combination of the present invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The nucleic acid or nucleic acid combination of the present invention may be present in one or more expression vectors.

[0249] The present invention also provides a host cell comprising the nucleic acid or nucleic acid combination or expression vector of the present invention. Nucleic acid or nucleic acid combination or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include but are not limited to liposome transfection, electroporation, viral transduction and calcium phosphate transfection, etc. In a preferred embodiment, the host cell is used to express the polypeptide, polypeptide antigen or polypeptide combination of the present invention. Examples of host cells include but are not limited to prokaryotic cells (e.g., bacteria, e.g., Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for expression include but are not limited to human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, e.g., HEK293 cells), Chinese hamster ovary (CHO) cells and other mammalian cells suitable for expression.

[0250] Composition

[0251] On the one hand, nucleic acid or nucleic acid combination as described herein is formulated for in vitro and in vivo delivery. On the one hand, the present invention provides a composition comprising nucleic acid or nucleic acid combination of the present invention. In particular, in some embodiments, nucleic acid or nucleic acid combination is formulated into lipid-containing composition. In some embodiments, composition comprises lipids that encapsulate nucleic acid or nucleic acid combination. In some embodiments, lipid-containing composition forms lipid nanoparticles that enclose nucleic acid or nucleic acid combination in lipid shell. In some embodiments, lipid shell protects nucleic acid or nucleic acid combination from degradation. In some embodiments, lipid nanoparticles also promote the transport of enclosed nucleic acid or nucleic acid combination to intracellular compartments and / or mechanisms to exert expected therapeutic functions. In certain embodiments, when present in lipid nanoparticles, nucleic acid resists degradation by nucleases in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods for their preparation are known in the art, such as those disclosed in, for example, U.S. Patent Publication No. 2004 / 0142025, U.S. Patent Publication No. 2007 / 0042031, PCT Publication No. WO 2017 / 004143, PCT Publication No. WO 2015 / 199952, PCT Publication No. WO 2013 / 016058, PCT Publication No. WO 2013 / 086373, and PCT Publication No. WO 2023 / 098842, the complete disclosure of each of these publications being incorporated herein by reference in their entirety for all purposes.

[0252] Nanoparticle compositions that can be used in conjunction with the present disclosure include, for example, lipid nanoparticles (LNPs), nanolipoprotein particles, liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more ligands, proteins, or channels.

[0253] In some embodiments, the composition includes at least the first lipid. In some embodiments, the composition includes at least the first lipid, and the first lipid is a cationic lipid. In some embodiments, the composition includes at least the first lipid (such as a cationic lipid), and the first lipid includes one or more of the compounds of lipid series 01, 02, 03 and 04 as described herein, for example, according to one or more of the compounds of formula (01-I), (01-II), (02-I), (02-II), (03-I), (04-I) and (04-III) (and its subformula). In some schemes, the first lipid includes one or more of the compounds listed in Tables 01-1, 02-1, 03-1 and 04-1. In some embodiments, the composition also includes the second lipid, and the second lipid is a polymer-bound lipid. In some schemes, the second lipid includes one or more of the compounds according to Formula 05-I. In some embodiments, the composition is formulated as a lipid nanoparticle encapsulated in a lipid by nucleic acid or a combination of nucleic acids.

[0254] In some embodiments, the nanoparticle composition comprises a lipid component comprising at least one lipid, such as one or more of the compounds of lipid series 01, 02, 03, 04, and 05 as described herein, for example, one or more of the compounds according to formula (01-I), (01-II), (02-I), (02-II), (03-I), (04-I), (04-III), and (05-I) (and subformulae thereof). For example, in some embodiments, the nanoparticle composition may comprise a lipid component comprising one of the compounds provided herein. The nanoparticle composition may further comprise one or more other lipid or non-lipid components, exemplary of which can be found in, for example, PCT Publication No. WO 2023 / 098842.

[0255] Cationic lipids

[0256] Cationic lipids include the following lipid series 01-04 (and subformulas thereof).

[0257] 1) Lipid Series 01

[0258] In one embodiment, the cationic lipid comprises a compound of formula (01-1):

[0259] or one or more of its pharmaceutically acceptable salts, prodrugs or stereoisomers, wherein:

[0260] G 1 and G 2 Each is independently a bond, C2-C 12 Alkylene or C2-C 12 Alkenylene, wherein one or more -CH2- in the alkylene or alkenylene is optionally replaced by -O-;

[0261] L 1 Yes-OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c )、-(C6-C 10 arylene)-R 1 , -(6- to 10-membered heteroarylene)-R 1 or R 1 ;

[0262] L 2 Yes-OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f )、-(C6-C 10 arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0263] R 1 and R 2 Each independently is C6-C 32 Alkyl or C6-C 32 alkenyl;

[0264] R a 、R b 、R d and R e Each independently is H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0265] R c and R f Each is independently C1-C 32 Alkyl or C2-C 32 alkenyl;

[0266] G 3 It is C2-C 24 Alkylene, C2-C 24 Alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0267] R3 Yes-N(R 4 )R 5 ;

[0268] R 4 is a C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4 to 8 membered heterocyclic group or C6-C 10 Aryl; or R 4 , G 3 or G 3 A portion of together with the nitrogen to which they are attached forms a cyclic portion;

[0269] R 5 It is C1-C 12 Alkyl or C3-C8 cycloalkyl; or R 4 、R 5 Together with the nitrogen to which they are attached, they form a cyclic moiety;

[0270] x is 0, 1, or 2; and

[0271] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0272] In one embodiment, the cationic lipid comprises a compound of formula (01-II):

[0273] or one or more of its pharmaceutically acceptable salts, prodrugs or stereoisomers, wherein:

[0274] Is a single bond or a double bond;

[0275] G 1 and G 2 Each is independently a bond, C2-C 12 Alkylene or C2-C 12 Alkenylene, wherein one or more -CH2- in the alkylene or alkenylene is optionally replaced by -O-;

[0276] L 1 Yes-OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NRa C(=O)R 1 、 -C(=O)NR b R c 、 -NR a C(=O)NR b R c 、 -OC(=O)NR b R c 、 -NR a C(=O)OR 1 、 -SC(=S)R 1 、 -C(=S)SR 1 、 -C(=S)R 1 、 -CH(OH)R 1 、 -P(=O)(OR b )(OR c )、 -(C6 - C 10 arylene)-R 1 、 -(6 - to 10 - membered heteroarylene)-R 1 or R 1 ;

[0277] L 2 is - OC(=O)R 2 、 -C(=O)OR 2 、 -OC(=O)OR 2 、 -C(=O)R 2 、 -OR 2 、 -S(O) x R 2 、 -S - SR 2 、 -C(=O)SR 2 、 -SC(=O)R 2 、 -NR d C(=O)R 2 、 -C(=O)NR e R f 、 -NR d C(=O)NR e R f 、 -OC(=O)NR e R f 、 -NR d C(=O)OR 2 、 -SC(=S)R 2 、 -C(=S)SR 2 、 -C(=S)R 2 、 -CH(OH)R 2 、 -P(=O)(OR e )(OR f )、 -(C6 - C 10 arylene)-R2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0278] R 1 and R 2 Each independently is C6-C 32 Alkyl or C6-C 32 alkenyl;

[0279] R a 、R b 、R d and R e Each independently is H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0280] R c and R f Each is independently C1-C 32 Alkyl or C2-C 32 alkenyl;

[0281] G 4 is a key, C1-C 23 Alkylene, C2-C 23 Alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0282] R 3 Yes-N(R 4 )R 5 ;

[0283] R 4 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4 to 8 membered heterocyclic group or C6-C 10 Aryl; or R 4 , G 3 or G 3 A portion of together with the nitrogen to which they are attached forms a cyclic portion;

[0284] R 5 It is C1-C 12 Alkyl or C3-C8 cycloalkyl; or R 4 、R 5 Together with the nitrogen to which they are attached, they form a cyclic moiety;

[0285] x is 0, 1, or 2; and

[0286] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0287] In one embodiment, the cationic lipid comprises a compound in Table 01-1, or one or more pharmaceutically acceptable salts, prodrugs or stereoisomers thereof.

[0288] Table 01-1.

[0289] 2) Lipid Series 02

[0290] In one embodiment, the cationic lipid comprises a compound of formula (02-1):

[0291] or one or more of its pharmaceutically acceptable salts, prodrugs or stereoisomers, wherein:

[0292] G 1 and G 2 Each is independently C2-C 12 Alkylene or C2-C 12 Alkenylene, where G 1 and G 2 One or more -CH2- in is optionally replaced by -O-, -C(=O)O- or -OC(=O)-;

[0293] Each L 1 is independently -OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(ORc ),-NR a P(=O)(OR b )(OR c );

[0294] Each L 2 is independently -OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f ),-NR d P(=O)(OR e )(OR f );

[0295] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0296] R a 、R b 、R d and R e Each independently is H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0297] R c and R f Each is independently C1-C24 Alkyl or C2-C 24 alkenyl;

[0298] G 3 It is C2-C 12 Alkylene or C2-C 12 Alkenylene, wherein a part or all of the alkylene or alkenylene group is optionally replaced by a C3-C8 cycloalkylene group or a C3-C8 cycloalkenylene group;

[0299] R 3 Yes-N(R 4 )R 5 、-OR 6 or -SR 6 ;

[0300] R 4 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0301] R 5 It is H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0302] R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 aryl;

[0303] x is 0, 1, or 2; and

[0304] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0305] In one embodiment, the cationic lipid is a compound of formula (02-II):

[0306] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0307] G 1 and G 2 Each independently is C2-C 12 Alkylene or C2-C 12 Alkenylene, where G 1 and G 2One or more -CH2- in is optionally replaced by -O-, -C(=O)O- or -OC(=O)-;

[0308] Each L 1 are independently -OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c ),-NR a P(=O)(OR b )(OR c );

[0309] Each L 2 are independently -OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR dC(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f ),-NR d P(=O)(OR e )(OR f );

[0310] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0311] R a 、R b 、R d and R e Each independently represents H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0312] R c and R f Each independently is C1-C 24 Alkyl or C2-C 24 alkenyl;

[0313] G 3 C2-C 12 Alkylene or C2-C 12 Alkenylene, wherein a part or all of the alkylene or alkenylene group is optionally replaced by a C3-C8 cycloalkylene group or a C3-C8 cycloalkenylene group;

[0314] R 3 -N(R 4 )R 5 、-OR 6 or -SR 6 ;

[0315] R 4 C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0316] R 5 H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0317] R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 aryl;

[0318] x is 0, 1, or 2; and

[0319] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0320] In one embodiment, the cationic lipid comprises a compound in Table 02-1, or one or more pharmaceutically acceptable salts, prodrugs or stereoisomers thereof.

[0321] Table 02-1.

[0322] 3) Lipid Series 03

[0323] In one embodiment, the cationic lipid comprises a compound of formula (03-1):

[0324] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0325] G 1 and G 2 Each is independently a bond, C2-C 12 Alkylene or C2-C 12 Alkenylene, where G 1 and G 2 One or more -CH2- in are optionally replaced by -O-;

[0326] Each L 1 is independently -OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c ),-NR a P(=O)(OR b )(OR c )、-(C6-C 10 arylene)-R 1 , -(6- to 10-membered heteroarylene)-R 1 、-(4- to 8-membered heterocyclylene)-R 1 or R 1 ;

[0327] Each L 2 is independently -OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f ),-NR d P(=O)(OR e )(OR f )、-(C6-C 10 arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 、-(4- to 8-membered heterocyclylene)-R 2 or R 2 ;

[0328] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0329] R a 、R b 、R d and R e Each independently is H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0330] R c and R f Each is independently C1-C 24 Alkyl or C2-C 24 alkenyl;

[0331] G 3 It is C2-C 12 Alkylene or C2-C 12 Alkenylene, wherein part or all of the alkylene or alkenylene group is optionally substituted by C3-C8 cycloalkylene, C3-C8 cycloalkenylene, C3-C8 cycloalkynylene, 4 to 8 membered heterocyclylene, C6-C8 10 substituted by an arylene group or a 5- to 10-membered heteroarylene group;

[0332] R 3 is hydrogen, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4- to 8-membered heterocyclic group, C6-C 10 aryl or 5- to 10-membered heteroaryl; or R 3 , G 1 or G 1 together with the nitrogen to which they are attached, form a cyclic moiety; or R 3 , G3 or G 3 A portion of together with the nitrogen to which they are attached forms a cyclic portion;

[0333] R 4 It is C1-C 12 Alkyl or C3-C8 cycloalkyl;

[0334] x is 0, 1, or 2;

[0335] n is 1 or 2;

[0336] m is 1 or 2; and

[0337] wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0338] In one embodiment, the cationic lipid comprises a compound in Table 03-1, or one or more of its pharmaceutically acceptable salts, prodrugs or stereoisomers.

[0339] Table 03-1.

[0340] 4) Lipid Series 04

[0341] In one embodiment, the cationic lipid comprises a compound of formula (04-1):

[0342] or one or more of its pharmaceutically acceptable salts, prodrugs or stereoisomers, wherein:

[0343] G 1 and G 2 Each is independently a bond, C2-C 12 Alkylene or C2-C 12 alkenylene;

[0344] L 1 Yes-OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R1 、 -C(=O)NR b R c 、 -NR a C(=O)NR b R c 、 -OC(=O)NR b R c 、 -NR a C(=O)OR 1 、 -SC(=S)R 1 、 -C(=S)SR 1 、 -C(=S)R 1 、 -CH(OH)R 1 、 -P(=O)(OR b )(OR c )、 -(C6 - C 10 arylene)-R 1 、 -(6 - to 10 - membered heteroarylene)-R 1 or R 1 ;

[0345] L 2 is - OC(=O)R 2 、 -C(=O)OR 2 、 -OC(=O)OR 2 、 -C(=O)R 2 、 -OR 2 、 -S(O) x R 2 、 -S - SR 2 、 -C(=O)SR 2 、 -SC(=O)R 2 、 -NR d C(=O)R 2 、 -C(=O)NR<00006​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​, -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0346] R 1 and R 2 Each independently is C5-C 32 Alkyl or C5-C 32 alkenyl;

[0347] R a 、R b 、R d and R e Each independently is H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0348] R c and R f Each is independently C1-C 32 Alkyl or C2-C 32 alkenyl;

[0349] R 0 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0350] G 3 It is C2-C 12 Alkylene or C2-C 12 alkenylene;

[0351] R 4 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0352] R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0353] x is 0, 1, or 2;

[0354] s is 0 or 1; and

[0355] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene is independently optionally substituted.

[0356] In one embodiment, the cationic lipid is a compound of formula (04-III):

[0357] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0358] R 1 and R 2 Each independently is C5-C 32 Alkyl or C5-C 32 alkenyl;

[0359] R 0 C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0360] G 3 C2-C 12 Alkylene or C2-C 12 alkenylene;

[0361] G 4 C2-C 12 Alkylene or C2-C 12 alkenylene;

[0362] R 3 -N(R 4 )R 5 OR 6 ;

[0363] R 4 C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0364] R 5 C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocycloalkyl; or R 4 、R 5 Together with the nitrogen to which it is attached, it forms a cyclic moiety;

[0365] R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 aryl; and

[0366] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene and cyclic moiety is independently optionally substituted.

[0367] In one embodiment, the cationic lipid comprises a compound in Table 04-1, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0368] Table 04-1.

[0369] In a preferred embodiment, the cationic lipid comprises Compound 01-1.

[0370] It should be understood that any embodiment of the compound provided herein as stated above, and any specific substituent and / or variable of the compound provided herein as stated above can be independently combined with other embodiments and / or substituents and / or variables of the compound to form the above embodiments not specifically stated. In addition, in the case of listing the substituents and / or variable lists of any specific groups or variables, it should be understood that each individual substituent and / or variable can be deleted from the specific embodiments and / or technical solutions, and the remaining substituents and / or variable lists will be considered to be within the scope of the embodiments provided herein.

[0371] It is understood that in this specification, combinations of substituents and / or variables in the depicted formulas are permissible only if such contributions result in stable compounds.

[0372] Other ionizable lipids

[0373] As described herein, in some embodiments, the nanoparticle compositions provided herein comprise one or more charged or ionizable lipids in addition to one or more of the lipids of series 01, 02, 03, and 04, such as one or more of the lipids according to formula (01-I), (01-II), (02-I), (02-II), (03-I), (04-I), and (04-III) (and subformulas thereof). Without being bound by theory, it is contemplated that certain charged or zwitterionic lipid components of the nanoparticle compositions are similar to lipid components in cell membranes, thereby improving cellular uptake of the nanoparticles. Exemplary additional charged lipids, ionizable lipids, or cationic lipids that can form part of the nanoparticle compositions of the present invention can be found, for example, in PCT Publication No. WO 2023 / 098842, the contents of which are incorporated herein by reference in their entirety.

[0374] In addition, in some embodiments, the charged or ionizable lipids that may form part of the nanoparticle compositions of the present invention are lipids comprising cyclic amine groups. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO2015199952A1, WO2016176330A1, and WO2015011633A1, the entire contents of which are incorporated herein by reference in their entirety. In addition, in some embodiments, the charged or ionizable lipids that may form part of the nanoparticle compositions of the present invention are lipids comprising cyclic amine groups. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO2015199952A1, WO2016176330A1, and WO2015011633A1, the entire contents of which are incorporated herein by reference in their entirety.

[0375] polymer-bound lipids

[0376] In some embodiments, the lipid component of nanoparticle composition can comprise the lipid of one or more polymer bonds, such as pegylated lipid (PEG lipid).Not bound by theory, it is expected that the polymer-bonded lipid component in nanoparticle composition can improve colloidal stability and / or reduce the protein absorption of nanoparticle.The lipid that can be combined with the exemplary polymer bonded of the disclosure includes but is not limited to the diacylglycerol, dialkylglycerol and mixture thereof of the phosphatidylethanolamine, phosphatidic acid, ceramide, PEG modification, PEG modification and PEG modification.For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000 or Chol-PEG2000.

[0377] In one embodiment, the polymer-bound lipid is a pegylated lipid. For example, some embodiments include pegylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); pegylated phosphatidylethanolamine (PEG-PE); PEG succinate diacylglycerols (PEG-S-DAG), such as 4-O-(2',3'-di-tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); pegylated ceramide (PEG-cer); or PEG dialkoxypropylcarbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di-tetradecanoyloxy)propyl)carbamate or 2,3-di-tetradecanoyloxypropyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate.

[0378] In some embodiments, the polymer-bound lipid (preferably, a PEGylated lipid) is present at a concentration in the range of 0.5 mol% to 5 mol%. In one embodiment, the polymer-bound lipid is present at a concentration in the range of 1.0 mol% to 2.5 mol%. In one embodiment, the polymer-bound lipid is present at a concentration in the range of about 1.2 mol% to 2 mol%. In one embodiment, the polymer-bound lipid is present at a concentration of about 1.7 mol%. In one embodiment, the polymer-bound lipid is present at a concentration of about 1.5 mol%.

[0379] In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 35: 1 to about 25: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 100: 1 to about 20: 1.

[0380] In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 35: 1 to about 25: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 100: 1 to about 20: 1.

[0381] In one embodiment, the PEGylated lipid has the formula:

[0382] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0383] R 12 and R 13 are each independently a linear or branched saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester linkages; and

[0384] w has an average value ranging from 30 to 60.

[0385] In one embodiment, R 12 and R 13 Each independently represents a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average value of w is in the range of 42 to 55, for example, the average value of w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some specific embodiments, the average value of w is about 49.

[0386] In one embodiment, the PEGylated lipid has the formula:

[0387] The average value of w is about 49.

[0388] Polymer-bound lipids also include one or more of the following compounds according to lipid series 05 (and its subformulas).

[0389] 5) Lipid Series 05

[0390] In one embodiment, provided herein is a compound of formula (05-1):

[0391] or one or more of its pharmaceutically acceptable salts or stereoisomers, wherein:

[0392] L is lipid;

[0393] X is a linker;

[0394] Each R 3 are independently H or C1-C6 alkyl;

[0395] Each Y 1 is independently a bond, O, S or NR a ;

[0396] Each G 4 are independently a bond or C1-C 12 Alkylene, wherein one or more -CH2- are independently optionally substituted by -O-, -S- or -NR a - Substitution;

[0397] Each G 5 are independently a bond or C1-C 12 Alkylene, wherein one or more -CH2- are independently optionally substituted by -O-, -S- or -NR a - Substitution;

[0398] Each R a are independently H, C1-C12 Alkyl or C2-C 12 alkenyl;

[0399] Z 1 and Z 2 One of them is positively charged, and Z 1 and Z 2 The other part is the negatively charged part;

[0400] n is an integer from 2 to 100;

[0401] T is hydrogen, halogen, alkyl, alkenyl, -OR", -SR", -COOR", -OCOR", -NR"R", -N + (R”)3, -P + (R")3, -SC(=S)-SR", -SC(=S)-OR", -SC(=S)-NR"R", -SC(=S)-aryl, cyano, azido, aryl, heteroaryl, or a targeting group, wherein R" at each occurrence is independently hydrogen or alkyl; and

[0402] Wherein each alkyl, alkenyl, alkylidene, aryl and heteroaryl are independently optionally substituted.Should be understood that any embodiment of the compound provided herein as stated above, and any specific substituent and / or variable of the compound provided herein as stated above can be independently combined with other embodiments and / or substituents and / or variables of the compound to form the above embodiment not specifically stated.In addition, in the case of listing the substituent and / or variable list of any particular group or variable, it should be understood that each individual substituent and / or variable can be deleted from specific embodiments and / or claims, and the remaining substituents and / or variable lists will be considered within the scope of the embodiment provided herein.

[0403] It is understood that in this specification, combinations of substituents and / or variables in the depicted formulas are permissible only if such contributions result in stable compounds.

[0404] Structured lipids

[0405] In some embodiments, the lipid component of the nanoparticle composition may include one or more structural lipids. Without being bound by theory, it is expected that structural lipids can stabilize the amphiphilic structure of the nanoparticle, such as, but not limited to, the lipid bilayer structure of the nanoparticle. Exemplary structural lipids that can be used in conjunction with the present disclosure include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0406] In some embodiments, the structured lipids are present at a concentration ranging from 20 mol% to 50 mol%. In some embodiments, the structured lipids are present at a concentration ranging from 30 mol% to 50 mol%. Preferably, the structured lipids are present at a concentration ranging from 32 mol% to 46 mol%. More preferably, the structured lipids are present at a concentration ranging from 34 mol% to 44 mol%. Even more preferably, the structured lipids are present at a concentration ranging from 36 mol% to 42 mol%.

[0407] In some embodiments, the structured lipid is present at a concentration of about 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 38.5 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol%.

[0408] In one embodiment, the lipid nanoparticles provided herein comprise a steroid or a steroid analog. In one embodiment, the steroid or steroid analog is cholesterol.

[0409] In one embodiment, the steroid is present in a concentration ranging from 39 mol% to 49 mol%, 40 mol% to 46 mol%, 40 mol% to 44 mol%, 40 mol% to 42 mol%, 42 mol% to 44 mol% or 44 mol% to 46 mol%. In one embodiment, the steroid is present in a concentration of 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol% or 46 mol%.

[0410] In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5:1 to 1:1. In one embodiment, the steroid is present at a concentration in the range of 32 mol% to 40 mol% steroid.

[0411] In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5:1 to 1:1. In one embodiment, the steroid is present at a concentration in the range of 32 mol% to 40 mol% steroid.

[0412] phospholipids

[0413] In some embodiments, the lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Without being bound by theory, it is expected that phospholipids can be assembled into one or more lipid bilayer structures. Exemplary phospholipids that can form part of the nanoparticle composition of the present invention include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), etc. Exemplary phospholipids can be found, for example, in PCT Publication No. WO 2023 / 098842, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0414] Additional exemplary phospholipids include, for example, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In one embodiment, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the phospholipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0415] In one embodiment, the phospholipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).

[0416] Additionally, phospholipids that may form part of the nanoparticle compositions of the present invention also include those described in WO 2017 / 112865, the entire contents of which are incorporated herein by reference.

[0417] In some embodiments, the phospholipid is present at a concentration ranging from 5 mol% to 40 mol%. In some embodiments, the phospholipid is present at a concentration ranging from 5 mol% to 15 mol%. Preferably, the phospholipid is present at a concentration ranging from 7 mol% to 13 mol%. More preferably, the phospholipid is present at a concentration ranging from 9 mol% to 11 mol%.

[0418] In some embodiments, the phospholipid is present at a concentration of about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol%.

[0419] Preparations

[0420] According to the present disclosure, the nanoparticle compositions described herein can comprise at least one lipid component and one or more additional components, such as a nucleic acid or nucleic acid combination (e.g., a therapeutic nucleic acid or nucleic acid combination as described herein). Nanoparticle compositions can be designed for one or more specific applications or targets. The ingredients of the nanoparticle compositions can be selected based on the specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more ingredients. Similarly, a specific formulation of the nanoparticle composition can be selected for a specific application or target based on, for example, the efficacy and toxicity of a specific combination of each ingredient.

[0421] The lipid component of the nanoparticle composition can include, for example, one or more of the lipids of series 01, 02, 03, and 04 described herein, for example, one or more of the lipids according to formula (01-I), (01-II), (02-I), (02-II), (03-I), (04-I), and (04-III) (and subformulas thereof), phospholipids (such as unsaturated lipids, e.g., DOPE or DSPC), PEG lipids, and structural lipids. The ingredients of the lipid component can be provided in specific fractions.

[0422] In one embodiment, provided herein are nanoparticle compositions comprising a cationic or ionizable lipid compound, a nucleic acid or combination of nucleic acids, and one or more other lipids as provided herein. In one embodiment, the cationic or ionizable lipid compound comprises one or more of the compounds of series 01, 02, 03, and 04 as described herein, for example, one or more of the compounds according to formula (01-I), (01-II), (02-I), (02-II), (03-I), (04-I), and (04-III) (and subformulas thereof), and optionally one or more other ionizable lipid compounds. In one embodiment, the one or more other lipids are selected from neutral lipids, steroids, and polymer-bound lipids. In one embodiment, the nucleic acid or combination of nucleic acids is encapsulated within or associated with the lipid nanoparticle.

[0423] In one embodiment, provided herein are nanoparticle compositions (lipid nanoparticles) comprising:

[0424] i) between 40 mol% and 50 mol% cationic lipids;

[0425] ii) neutral lipids;

[0426] iii) steroids;

[0427] iv) polymer-bound lipids; and

[0428] v) Nucleic acid or a combination of nucleic acids.

[0429] As used herein, "mol%" refers to the molar percentage of a component relative to the total moles of all lipid components in the LNP (i.e., the total moles of cationic lipids, neutral lipids, steroids, and polymer-bound lipids). Unless otherwise specified, the sum of the amounts (mol%) of each lipid in the composition is 100 mol%, i.e., the sum of the amounts (mol%) of cationic lipids, phospholipids, steroids, and polyethylene glycol-bound lipids is 100 mol%.

[0430] In one embodiment, the lipid nanoparticle comprises 20 mol% to 65 mol% of the cationic lipid. More preferably, the lipid nanoparticle comprises 25 mol% to 60 mol% of the cationic lipid. Even more preferably, the lipid nanoparticle comprises 30 mol% to 55 mol% of the cationic lipid. Even more preferably, the lipid nanoparticle comprises 35 mol% to 55 mol% of the cationic lipid. Even more preferably, the lipid nanoparticle comprises 40 mol% to 52 mol% of the cationic lipid.

[0431] In one embodiment, the lipid nanoparticle comprises 41 mol% to 49 mol%, 41 mol% to 48 mol%, 42 mol% to 48 mol%, 43 mol% to 48 mol%, 44 mol% to 48 mol%, 45 mol% to 48 mol%, 46 mol% to 48 mol% or 47.2 mol% to 47.8 mol% of cationic lipid. In one embodiment, the lipid nanoparticle comprises about 47.0 mol%, 47.1 mol%, 47.2 mol%, 47.3 mol%, 47.4 mol%, 47.5 mol%, 47.6 mol%, 47.7 mol%, 47.8 mol%, 47.9 mol% or 48.0 mol% of cationic lipid.

[0432] In one embodiment, the neutral lipid is present at a concentration in the range of 5 mol% to 15 mol%, 7 mol% to 13 mol%, or 9 mol% to 11 mol%. In one embodiment, the neutral lipid is present at a concentration of about 9.5 mol%, 10 mol%, or 10.5 mol%. In one embodiment, the molar ratio of cationic lipid to neutral lipid is in the range of about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0.

[0433] In one embodiment, the steroid is present at a concentration within the range of 39mol% to 49mol%, 40mol% to 46mol%, 40mol% to 44mol%, 40mol% to 42mol%, 42mol% to 44mol% or 44mol% to 46mol%. In one embodiment, the steroid is present at a concentration of 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol% or 46mol%. In one embodiment, the molar ratio of the cationic lipid to the steroid is within the range of 1.0:0.9 to 1.0:1.2 or 1.0:1.0 to 1.0:1.2. In one embodiment, the steroid is cholesterol.

[0434] In one embodiment, the nucleic acid or nucleic acid combination in the LNP: lipid ratio (i.e., N / P, where N represents the number of moles of cationic lipid and P represents the number of moles of phosphate present as part of the nucleic acid backbone) is in the range of 2: 1 to 30: 1, e.g., 3: 1 to 22: 1. In one embodiment, N / P is in the range of 6: 1 to 20: 1 or 2: 1 to 12: 1. Exemplary N / P ranges include about 3: 1, about 6: 1, about 12: 1, and about 22: 1.

[0435] In one embodiment, provided herein is a lipid nanoparticle comprising:

[0436] i) cationic lipids with an effective pKa greater than 6.0;

[0437] ii) 5 mol% to 15 mol% neutral lipids;

[0438] iii) 1 mol% to 15 mol% of anionic lipids;

[0439] iv) 30 mol% to 45 mol% of a steroid;

[0440] v) polymer-bound lipids; and

[0441] vi) a nucleic acid or a combination of nucleic acids,

[0442] Wherein mol % is determined based on the total moles of lipid present in the lipid nanoparticles.

[0443] In one embodiment, the compositions provided herein include:

[0444] (a) (i) 20 mol% to 65 mol% cationic lipid; (ii) 5 mol% to 40 mol% phospholipid; (iii) 20 mol% to 50 mol% steroid; and (iv) polymer-bound lipid;

[0445] (b) (i) 40 mol% to 55 mol% cationic lipid; (ii) 5 mol% to 15 mol% phospholipid; (iii) 35 mol% to 50 mol% steroid; and (iv) 2 mol% to 10 mol% polymer-bound lipid;

[0446] or

[0447] (c) (i) 45 mol% to 55 mol% cationic lipid; (ii) 6 mol% to 10 mol% phospholipid; (iii) 40 mol% to 48 mol% steroid; and (iv) 1 mol% to 2.5 mol% polymer-bound lipid.

[0448] In one embodiment, the cationic lipid can be any of a variety of lipid species that carry a net positive charge at a selected pH (such as physiological pH). Exemplary cationic lipids are described below. In one embodiment, the cationic lipid has a pKa greater than 6.25. In one embodiment, the cationic lipid has a pKa greater than 6.5. In one embodiment, the cationic lipid has a pKa greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65, or greater than 6.7.

[0449] In one embodiment, the lipid nanoparticle comprises 40 mol% to 45 mol% of cationic lipid.In one embodiment, the lipid nanoparticle comprises 45 mol% to 50 mol% of cationic lipid.

[0450] In one embodiment, the molar ratio of cationic lipid to neutral lipid is in the range of about 2: 1 to about 8: 1. In one embodiment, the lipid nanoparticle comprises 5 mol% to 10 mol% neutral lipid.

[0451] Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), or 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG).

[0452] In one embodiment, the lipid nanoparticles comprise 1 mol% to 10 mol% anionic lipids. In one embodiment, the lipid nanoparticles comprise 1 mol% to 5 mol% anionic lipids. In one embodiment, the lipid nanoparticles comprise 1 mol% to 9 mol%, 1 mol% to 8 mol%, 1 mol% to 7 mol%, or 1 mol% to 6 mol% anionic lipids. In one embodiment, the molar ratio of anionic lipids to neutral lipids is in the range of 1:1 to 1:10.

[0453] In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5:1 to 1:1. In one embodiment, the lipid nanoparticles comprise 32 mol% to 40 mol% of the steroid.

[0454] In one embodiment, the sum of the mol% of the neutral lipid and the mol% of the anionic lipid is in the range of 5mol% to 15mol%. In one embodiment, the sum of the mol% of the neutral lipid and the mol% of the anionic lipid is in the range of 7mol% to 12mol%.

[0455] In one embodiment, the molar ratio of anionic lipid to neutral lipid is in the range of 1 : 1 to 1 : 10. In one embodiment, the sum of the mol% of neutral lipid and the mol% of steroid is in the range of 35 mol% to 45 mol%.

[0456] In one embodiment, the lipid nanoparticle comprises:

[0457] i) 45 mol% to 55 mol% of a cationic lipid;

[0458] ii) 5 mol% to 10 mol% neutral lipids;

[0459] iii) 1 mol% to 5 mol% of anionic lipids; and

[0460] iv) 32 mol% to 40 mol% of a steroid.

[0461] In one embodiment, the lipid nanoparticle comprises 1.0 mol% to 2.5 mol% bound lipid.In one embodiment, the polymer-bound lipid is present at a concentration of about 1.5 mol%.

[0462] In one embodiment, the neutral lipid is present at a concentration in the range of 5 mol% to 15 mol%, 7 mol% to 13 mol%, or 9 mol% to 11 mol%. In one embodiment, the neutral lipid is present at a concentration of about 9.5 mol%, 10 mol%, or 10.5 mol%. In one embodiment, the molar ratio of cationic lipid to neutral lipid is in the range of about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0.

[0463] In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present in a concentration within the range of 39mol% to 49mol%, 40mol% to 46mol%, 40mol% to 44mol%, 40mol% to 42mol%, 42mol% to 44mol% or 44mol% to 46mol%. In one embodiment, the steroid is present in a concentration of 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol% or 46mol%. In certain embodiments, the molar ratio of cationic lipid to steroid is within the range of 1.0:0.9 to 1.0:1.2 or 1.0:1.0 to 1.0:1.2.

[0464] In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 5:1 to 1:1.

[0465] In one embodiment, the lipid nanoparticle comprises 1.0 mol% to 2.5 mol% bound lipid.In one embodiment, the polymer-bound lipid is present at a concentration of about 1.5 mol%.

[0466] In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 100: 1 to about 20: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 35: 1 to about 25: 1.

[0467] In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 100: 1 to about 20: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-bound lipid is in the range of about 35: 1 to about 25: 1.

[0468] In some embodiments, the lipid nanoparticles have an average diameter in the range of 50 nm to 180 nm. In some embodiments, the lipid nanoparticles have an average diameter in the range of 50 nm to 150 nm. In some embodiments, the lipid nanoparticles have an average diameter in the range of 50 nm to 120 nm. In one embodiment, the lipid nanoparticles have an average diameter in the range of 50 nm to 100 nm or 60 nm to 85 nm.

[0469] In one embodiment, compositions comprises cationic lipid, DSPC, cholesterol and PEG-lipid provided herein and nucleic acid or nucleic acid combination.In one embodiment, cationic lipid, DSPC, cholesterol and PEG-lipid provided herein are in the molar ratio of 50:10:38.5:1.5.

[0470] In a preferred embodiment, the cationic lipid comprises Compound 01-1.

[0471] In a preferred embodiment, the PEG-lipid comprises DMG-PEG.

[0472] In a preferred embodiment, the lipid nanoparticles comprise 30-55 mol% of compound 01-1, 0.5-5 mol% of DMG-PEG (eg, DMG-PEG2000), 5-40 mol% of DSPC, and 20-50 mol% of cholesterol.

[0473] In a most preferred embodiment, the composition comprises 50 mol% Compound 01-1, 1.5 mol% DMG-PEG, 10 mol% DSPC, and 38.5 mol% cholesterol.

[0474] Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver nucleic acids or nucleic acid combinations, such as RNA or RNA combinations, to specific cells, tissues, organs, or systems or groups thereof in a mammal. The physicochemical properties of the nanoparticle compositions can be altered to increase selectivity for specific body targets. For example, the particle size can be adjusted based on the window size of different organs. The nucleic acids or nucleic acid combinations contained in the nanoparticle compositions can also be selected based on one or more desired delivery targets. For example, the nucleic acids or nucleic acid combinations can be selected for specific indications, disorders, diseases, or conditions and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, the nanoparticle compositions can include nucleic acids or nucleic acid combinations (particularly mRNA) encoding a polypeptide of interest that can be translated intracellularly to produce the polypeptide of interest. Such compositions can be designed to be specifically delivered to a specific organ. In certain embodiments, the compositions can be designed to be specifically delivered to the liver of a mammal.

[0475] In nanoparticle compositions, the amount of nucleic acid or nucleic acid combination can depend on the size, composition, desired target and / or application of nanoparticle compositions, or other characteristics, and the characteristics of nucleic acid or nucleic acid combination. For example, the amount of RNA that can be used in nanoparticle compositions can depend on the size, sequence and other characteristics of RNA. The relative amounts of nucleic acid or nucleic acid combination and other ingredients (such as lipid) can also change in nanoparticle compositions. In some embodiments, the wt / wt ratio of lipid component and nucleic acid or nucleic acid combination in nanoparticle compositions can be about 5:1 to about 60:1, such as about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 22:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1 and 60:1. For example, the wt / wt ratio of the lipid component to the nucleic acid or combination of nucleic acids can be from about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of nucleic acid or combination of nucleic acids in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0476] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of the nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N:P ratio is selected. The one or more RNAs, lipids, and amounts thereof can be selected to provide an N:P ratio of about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N:P ratio may be about 5.67:1.

[0477] Additional characterization methods and various characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential, can be found in, for example, PCT Publication No. WO 2023 / 098842, the contents of which are incorporated herein by reference in their entirety.

[0478] The encapsulation efficiency of a nucleic acid or nucleic acid combination describes the amount of nucleic acid or nucleic acid combination that is encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. Encapsulation efficiency is expected to be high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of nucleic acid or nucleic acid combination in a solution containing the nanoparticle composition before and after disruption of the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free nucleic acid or nucleic acid combination (e.g., RNA) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the nucleic acid or nucleic acid combination can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0479] The nanoparticle compositions can optionally include one or more coatings. For example, the nanoparticle compositions can be formulated into capsules, films, or tablets having a coating. The capsules, films, or tablets containing the compositions described herein can have any useful size, tensile strength, hardness, or density.

[0480] In one aspect, the present invention also provides a vaccine preparation (also referred to as a "vaccine agent") comprising the nucleic acid or nucleic acid combination described herein.

[0481] In some embodiments, a vaccine formulation comprises a composition described herein.

[0482] In some embodiments, the vaccine formulation comprises Compound 01-1, DMG-PEG, DSPC, and cholesterol; wherein the nucleic acid or nucleic acid combination encodes a polypeptide or polypeptide combination described herein. In one embodiment, the nucleic acid or nucleic acid combination comprises the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 or SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65, and 66.

[0483] In some embodiments, the vaccine formulation comprises compound 01-1, DMG-PEG, DSPC, and cholesterol; wherein the nucleic acid or nucleic acid combination encodes a polypeptide or polypeptide combination described herein. In one embodiment, the nucleic acid or nucleic acid combination comprises two or more nucleotide sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 or SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65, and 66.

[0484] In one embodiment, the vaccine formulation comprises a nucleic acid or a combination of nucleic acids encoding a polypeptide or a combination of polypeptides as described herein and a lipid encapsulating the nucleic acid or the combination of nucleic acids, the lipid comprising compound 01-1, DMG-PEG, DSPC and cholesterol, and the nucleic acid or the combination of nucleic acids comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 or SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66.

[0485] In one embodiment, the vaccine formulation comprises a nucleic acid or a combination of nucleic acids encoding a polypeptide or a combination of polypeptides as described herein and a lipid encapsulating the nucleic acid or the combination of nucleic acids, the lipid comprising compound 01-1, DMG-PEG, DSPC and cholesterol, and the nucleic acid or the combination of nucleic acids comprising two or more nucleotide sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 or SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66.

[0486] In one embodiment, the vaccine formulation comprises a nucleic acid or a combination of nucleic acids encoding a polypeptide or a combination of polypeptides as described herein and a lipid encapsulating the nucleic acid or the combination of nucleic acids, the lipid comprising 50 mol% Compound 01-1, 1.5 mol% DMG-PEG, 10 mol% DSPC and 38.5 mol% cholesterol, and the nucleic acid or the combination of nucleic acids comprises a nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 or SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66.

[0487] In one embodiment, the vaccine formulation comprises a nucleic acid or a combination of nucleic acids encoding a polypeptide or a combination of polypeptides as described herein and a lipid encapsulating the nucleic acid or the combination of nucleic acids, the lipid comprising 50 mol% Compound 01-1, 1.5 mol% DMG-PEG, 10 mol% DSPC and 38.5 mol% cholesterol, and the nucleic acid or the combination of nucleic acids comprises two or more nucleotide sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 or SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 and 66.

[0488] Pharmaceutical composition / kit

[0489] According to the present disclosure, nanoparticle compositions can be formulated in whole or in part as pharmaceutical compositions. A pharmaceutical composition can comprise one or more nanoparticle compositions. For example, a pharmaceutical composition can comprise one or more nanoparticle compositions comprising one or more different nucleic acids or combinations of nucleic acids. A pharmaceutical composition can also comprise one or more pharmaceutically acceptable carriers, diluents, excipients, or auxiliary ingredients, such as those described herein. General guidance regarding the formulation and manufacture of pharmaceutical compositions and agents can be found, for example, in Remington, The Science and Practice of Pharmacy, 21st ed., A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional carriers, diluents, excipients, and auxiliary ingredients can be used in any pharmaceutical composition, unless any conventional carrier, diluent, excipient, or auxiliary ingredient is incompatible with one or more components of the nanoparticle composition. A carrier, diluent, excipient, or adjunct ingredient is incompatible with a component of the nanoparticle composition if the combination of the carrier, diluent, excipient, or adjunct ingredient with the components of the nanoparticle composition would result in any undesirable biological or other deleterious effect.

[0490] In some embodiments, one or more carriers, diluents, excipients, or adjuvants may comprise greater than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, one or more carriers, diluents, excipients, or adjuvants may comprise 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the carrier, diluent, excipient is approved for human and veterinary use. In some embodiments, the carrier, diluent, excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the carrier, diluent, excipient is pharmaceutical grade. In some embodiments, the carrier, diluent, excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0491] The relative amounts of the one or more nanoparticle compositions, one or more pharmaceutically acceptable carriers, diluents, excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated and further on the route of administration of the composition. For example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of the one or more nanoparticle compositions.

[0492] In some embodiments, the nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are stored and / or transported refrigerated or frozen. Refrigerated or frozen storage and / or transport conditions can be found, for example, in PCT Publication No. WO 2023 / 098842, the contents of which are incorporated herein by reference in their entirety.

[0493] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient selected from one or more of the following: Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pH of the pharmaceutical compositions of the present disclosure is between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8, or between 7 and 7.8). For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, which is suitable for storage and / or transportation at, for example, about -20°C. For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS and has a pH of about 7-7.8, which is suitable for storage and / or transportation at, for example, about 4° C. or lower. In the context of the present disclosure, “stability,” “stabilization,” and “stable” refer to the nanoparticle compositions and / or pharmaceutical compositions disclosed herein being resistant to chemical or physical changes (e.g., degradation, changes in particle size, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transportation, storage, and / or use conditions, for example, when stress is applied, such as shear force, freeze / thaw stress, etc.

[0494] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including patients or subjects who may benefit from the delivery of a nucleic acid or combination of nucleic acids to one or more specific cells, tissues, organs, or systems or groups thereof. Although the descriptions of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein are primarily directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications to compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals are well known, and veterinary pharmacologists of ordinary skill can design and / or perform such modifications with only ordinary experimentation (if any). It is contemplated that subjects to whom the compositions may be administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0495] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or later developed in the art of pharmacology. Generally, such preparation methods comprise combining the active ingredient with a carrier, diluent, excipient, and / or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping, and / or packaging the product into desired single-dose or multi-dose units.

[0496] Pharmaceutical compositions according to the present disclosure can 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 containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of this dose, such as half or one-third of this dose.

[0497] Pharmaceutical compositions can be prepared into various forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared into liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0498] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and / or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, for example, water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also contain additional therapeutic and / or prophylactic agents, additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and / or fragrances. In certain embodiments for parenteral administration, the composition is mixed with a solubilizer, such as Cremophor TM , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof.

[0499] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in non-toxic parenteral acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution (USP) and isotonic sodium chloride solution. Sterile fixed oils are typically used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injectables.

[0500] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0501] The present invention provides methods for delivering nucleic acids or nucleic acid combinations to mammalian cells or organs, producing antigenic polypeptides in mammalian cells, and preventing and / or treating diseases or disorders in mammals in need thereof, the methods comprising administering to the mammal a nanoparticle composition comprising the nucleic acid or nucleic acid combination and / or contacting mammalian cells with the nanoparticle composition.

[0502] The polypeptide / polypeptide combination, nucleic acid / nucleic acid combination or composition of the present disclosure can also be provided in the form of a kit. Therefore, the present disclosure also relates to a kit comprising the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination or composition described herein. The kit can also include instructions for use or a suitable container as needed. In certain embodiments, the kit also includes a device for administration. The kit generally includes a label indicating the intended use and / or method of use of the contents of the kit. The term "label" includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0503] Therapeutic applications

[0504] The present invention provides polypeptides or polypeptide combinations, nucleic acids or nucleic acid combinations (particularly RNA), compositions, vaccine preparations, pharmaceutical compositions or kits of the present invention for preventing and / or treating monkeypox virus infection in a subject.

[0505] The present invention provides use of the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition, vaccine formulation, pharmaceutical composition or kit of the present invention in the preparation of a medicament for preventing and / or treating monkeypox virus infection in a subject.

[0506] The present invention provides use of the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition, vaccine formulation, pharmaceutical composition or kit of the present invention in the preparation of a vaccine for preventing and / or treating monkeypox virus infection in a subject.

[0507] The present invention provides a method for preventing and / or treating monkeypox virus infection in a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide or polypeptide combination, a nucleic acid or nucleic acid combination (particularly RNA), a composition, a vaccine formulation, a pharmaceutical composition, or a kit of the present invention. In one embodiment, the method comprises administering a therapeutically effective amount of a composition comprising a nucleic acid or nucleic acid combination of the present invention. In one embodiment, the method comprises administering a therapeutically effective amount of a composition comprising an RNA or RNA combination of the present invention, particularly a composition comprising an LNP as described herein. In one embodiment, the method comprises administering a therapeutically effective amount of a composition comprising an mRNA or mRNA combination of the present invention, particularly a composition comprising an LNP as described herein.

[0508] In some embodiments, the therapeutically effective amount is provided in one or more administrations. In some embodiments, the therapeutically effective amount is provided in two administrations. In some embodiments, the therapeutically effective amount is provided in three administrations. In some embodiments, the therapeutically effective amount is provided in four administrations. In some embodiments, the therapeutically effective amount is provided in five administrations. In some embodiments, the therapeutically effective amount is provided in six administrations. Preferably, in some embodiments, the therapeutically effective amount is provided in two administrations.

[0509] In some embodiments, the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (particularly RNA), composition or pharmaceutical composition of the present invention can be administered to a subject by any method known to those skilled in the art, such as parenteral, oral, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous or intraperitoneal. Preferably, the composition of the present invention is administered by intramuscular injection. Beneficial effects

[0510] The polypeptide or polypeptide combination, nucleic acid or nucleic acid combination (especially RNA), composition, pharmaceutical composition or kit of the present invention can exhibit excellent effects, such as but not limited to: (1) high in vitro expression efficiency; (2) inducing a higher level of binding antibodies against monkeypox virus antigen protein in the subject; (3) inducing a higher level of cowpox virus cross-neutralizing antibodies in the subject; (4) inducing a higher level of T cell immune response in the subject.

[0511] Example

[0512] The present invention is further described with reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the present invention. The following materials and instruments are commercially available or prepared according to methods known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0513] Example 1 Preparation of mRNA

[0514] 1.1. Design and linearization of DNA template plasmid

[0515] The designs encode different monkeypox virus antigen polypeptide single bodies (including antigen polypeptide single bodies whose target polypeptide is A29L, A35R, B2R, B6R, C15L, M1R or H3L) or antigen polypeptide concatemers (including target polypeptide is A29L+A35R+B6R+B2R+C15L+M1R, B2R+A35R+C15L, A29L+M1R+B6R, B6R+M1R+H3L, B6R+A35R+M1R, A29L+B2R+C15L, B2R+H3L+C15L, B2R+A35R+B6R+C15L, R, A29L+M1R+C15L or H3L+C15L+M1R antigen polypeptide concatemers), the constructs were named 0201, 0302, 0402, 0505, 0603, 0702, 0803, 1101, 1201, 1301, 1401, 1501, 1601, 1701, 1801, 1901, 2001; the DNA open reading frame (ORF) sequence of each construct and the amino acid sequence encoded by it are shown in Tables 2 and 1, respectively.

[0516] Table 1. Amino acid sequences encoded by each construct

[0517] Table 2. Nucleic acid sequences of each construct (SEQ ID NO:)

[0518] The 5'-UTR sequence (corresponding DNA sequence is shown in SEQ ID NO: 142 or 150), DNA ORF, 3'-UTR sequence (corresponding DNA sequence is shown in SEQ ID NO: 143, 144 or 151) was inserted into the plasmid pJ241 for IVT (developed in-house, containing a kanamycin resistance gene, a T7 promoter sequence, a poly (A) marker (corresponding DNA sequence is shown in SEQ ID NO: 146) and a unique IIS type restriction site downstream of the poly (A) sequence) to construct an IVT vector.

[0519] Linearization was performed using the IIS type restriction enzyme Esp3I / BsmBI (Thermo, FD0294) or Bsa I (Kaixia, GMP-BSA-EE101-11). Specifically, each 10 μg of plasmid was mixed with 10 U of restriction enzyme and incubated at 37 ° C for 4 hours to ensure complete linearization. The reaction was terminated by adding 1 / 10 volume of 3M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, mixed thoroughly, and cooled at -20 ° C for 1 hour. Centrifuge at 13800g for 15 minutes at 4 ° C to precipitate the linearized DNA, wash twice with 70% ethanol, and resuspend in nuclease-free H2O.

[0520] 1.2. In vitro transcription of mRNA from DNA template

[0521] Table 3 shows a 20 μL in vitro transcription reaction system, according to which a reaction mixture was prepared for in vitro transcription, wherein UTP was modified with 1-methylpseudouridine.

[0522] Table 3. 20 μL in vitro transcription reaction system

[0523] In vitro transcription: The reaction mixture was incubated at 37°C for 6 hours, and then 1 μL of DNase I (RNase-free, 1 U / μL, Kaixia, GMP-DNI-EE001-12) was added to remove the DNA template and incubated at 37°C for 30 minutes. The synthesized RNA was purified by adding 0.5 volumes of 7.5M LiCl, 50mM EDTA and incubating at -20°C for 45 minutes, followed by centrifugation at 4°C, 13800g for 15 minutes to precipitate the mRNA. The supernatant was removed and the pellet was rinsed twice with 500 μL of ice-cold 70% ethanol. After rinsing, the mRNA was resuspended in nuclease-free H2O, the concentration was adjusted to 1 mg / mL, and stored at -20°C.

[0524] mRNA capping: Each 10 μg of uncapped mRNA was heated at 65°C for 10 minutes, placed on ice for 5 minutes, and then mixed with 10U of vaccinia virus capping enzyme (Kaixia, GMP-VCS-VE101-13), 50U of mRNA cap 2'-O-methyltransferase (Kaixia, GMP-MEH-VE101-13), 0.2mM SAM, 0.5mM GTP and 1U of RNase inhibitor, and incubated at 37°C for 60 minutes to generate Cap 1 modification structure. The modified mRNA was precipitated by LiCl as described above, and the RNA was resuspended in nuclease-free HO and stored at -20°C.

[0525] HPLC purification: RNA was purified by high performance liquid chromatography (HPLC) using a C4 column (5 μm) (10 mm × 250 mm column) (data not shown). Buffer A contained 0.1 M triethylammonium acetate (TEAA) (pH = 7.0), and buffer B contained 0.1 M TEAA (pH = 7.0) and 25% acetonitrile.

[0526] The mRNA sequences shown in Table 2 were obtained through the above in vitro transcription, capping and purification processes.

[0527] Furthermore, constructs encoding the A29L, A35R, B6R, M1R, and E8L antigenic polypeptides were prepared using the above method and designated 0201-J0, 0301-J0, 0501-J0, 0701-J0, and 0901-J0, respectively. The nucleic acid sequences of each construct and the encoded amino acid sequences are shown in Tables 2 and 1, respectively.

[0528] Example 2 In vitro transfection

[0529] In this example, Lipofectamine 2000 transfection reagent was used to transfect mRNA encoding different monkeypox virus antigen polypeptides (single or concatemers) prepared in Example 1 into HEK293T cells. Flow cytometry was then used to assess the in vitro expression of the different mRNA constructs. The specific in vitro transfection methods and flow cytometric assays are described below.

[0530] In vitro transfection: HEK293T cells (Shanghai Cell Bank, Chinese Academy of Sciences, SCSP-502) were cultured at 1.5×10 5Cells / well were seeded in a 24-well cell culture plate. Cells were cultured overnight at 37°C, 5% CO2, and transfection was performed after the cell confluence reached 70%-90%. An exemplary transfection method is as follows: 1 μg of mRNA was added to 25 μL of serum-free Opti-MEM medium, and the mixture was mixed by pipetting to form mixture A. Another 25 μL of serum-free Opti-MEM medium was taken, and 1 μL of Lipofectamine 2000 transfection reagent was added thereto, and the mixture was mixed thoroughly to form mixture B. Mixture A was added to mixture B, gently mixed, and then allowed to stand at room temperature for 5 minutes. Finally, the mixture was added dropwise to HEK293T cells and incubated at 37°C, 5% CO2 for 18-24 hours. Each mRNA was transfected in duplicate wells. In addition, a mixture containing only Lipofectamine 2000 but no mRNA was prepared to transfect HEK293T cells under the same conditions and cultured under the same conditions to serve as a blank control.

[0531] Sample collection: Aspirate the cell culture supernatant, gently dislodge and resuspend the HEK239T cells in 200 μL of staining buffer (PBS containing 2% FBS), then transfer the cells to a 96-well plate. For cells transfected with mRNA encoding a concatemer of antigenic peptides, divide the cells from one well of the 24-well plate equally into the corresponding number of wells of the 96-well plate. For example, cells transfected with 1101 mRNA (encoding 6 antigenic peptides) should be divided equally into 6 wells of the 96-well plate, and cells transfected with 1201, 1301, 1401, 1501, 1601, 1701, 1801, 1901, or 2001 mRNA (each encoding 3 antigenic peptides) should be divided equally into 3 wells of the 96-well plate. After transfer, centrifuge at 300 g, discard the supernatant, and wash the cells twice with 300 μL of staining buffer.

[0532] Staining: Select primary and secondary antibodies corresponding to the antigenic peptide encoded by each mRNA to detect whether the mRNA can successfully express the antigenic peptide in vitro. Table 4-1 shows the staining antibodies used. Table 4-2 shows the name, supplier, and catalog number of the staining antibodies used.

[0533] Table 4-1. Antibodies used for staining in flow cytometry

[0534] Table 4-2. Antibody names, suppliers, and product numbers of staining antibodies used in flow cytometry

[0535] The primary antibody was diluted to the working concentration in staining buffer and the cells were resuspended in 50 μL / well. The cells were stained in the dark for 30 minutes at 4°C. After incubation, the cells were centrifuged at 300 g, the primary antibody dilution was discarded, and the cells were washed three times with staining buffer, and the supernatant was discarded. The secondary antibody was diluted to the working concentration in staining buffer and the cells were resuspended in 50 μL / well. The cells were stained in the dark for 30 minutes at 4°C. After incubation, the cells were centrifuged at 300 g, the secondary antibody dilution was discarded, and the cells were washed three times with staining buffer, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of staining buffer and analyzed using a flow cytometer (BD biosciences, LSRFortessa).

[0536] Flow cytometry was used to detect in vitro expression: the blank control group cells were used as the benchmark to draw the gate, and the ratio of the number of HEK293T cells that successfully expressed the target polypeptide to the total number of cells was detected.

[0537] The results of flow cytometry are shown in Table 5 (data are shown as the average value of the expression positive rate of two replicate wells). Except for 0201, 1101 and 1301, the remaining mRNAs can successfully express the target polypeptide in vitro. Among them, the in vitro expression levels of 0302, 1201 and 1401 are higher; the in vitro expression levels of 0402, 0505, 0603, 0702, 0803, 1601, 1701 and 1901 are medium, and the in vitro expression levels of 1501, 1801 and 2001 are lower. Although the successful expression of the target polypeptides of 0201 and 1101 was not detected in this embodiment, in subsequent experiments, mRNA was encapsulated into liposomes to prepare mRNA vaccines and then immunized mice. Specific binding antibodies to the corresponding antigen polypeptides can be detected in the serum of the immunized mice, suggesting that the failure to detect the expression of the target polypeptide in this embodiment does not necessarily mean that the mRNA molecule does not express, but may be due to insufficient sensitivity of the detection method or false negatives caused by other reasons. However, 1301 neither detected the expression of the target protein at the in vitro level nor detected specific binding antibodies to the antigen protein in the serum of immunized mice, suggesting that 1301 could not successfully express the target protein.

[0538] Table 5. In vitro expression results

[0539] “—” indicates not applicable or not tested

[0540] Example 3 Preparation of mRNA-LNP Preparation

[0541] Experimental materials

[0542] The lipids of series 01 were synthesized according to the method described in patent WO2021204175; the lipids of series 02 were synthesized according to the method described in patent WO 2023 / 138611; the lipids of series 03 were synthesized according to the method described in patent WO2022152109; and the lipids of series 04 were synthesized according to the method described in patent WO2022247755.

[0543] DSPC, cholesterol, and mPEG2000-DMG (ie, DMG-PEG 2000) were purchased through conventional commercial channels.

[0544] 3.2. Exemplary Methods for Preparation of Lipid Nanoparticles

[0545] In brief, cationic lipid (such as Compound 01-1), DSPC, cholesterol and PEG-lipid are dissolved in ethanol with a molar ratio of 50:10:38.5:1.5, and the mRNA prepared in Example 1 is diluted in 10 to 50mM citrate buffer (pH=4) according to the individual or combination mode shown in Table 6. By using a microfluidic device (self-developed equipment) with a total flow rate in the range of 9-30mL / min, the ethanol lipid solution is mixed with the mRNA aqueous solution with a volume ratio of 1:3, and LNP is prepared with a total lipid and mRNA weight ratio of about 10:1 to 30:1. Ethanol is removed by dialysis and replaced with DPBS. Finally, lipid nanoparticles are filtered through a 0.2 μm sterile filter. The mRNA-LNP preparations (lipid nanoparticles comprising mRNA) finally obtained are respectively named as mRNA-LNP preparations G1, G2, G3, G4, G5, G6, G7, G8, G9 and G10. Among them, the antigen polypeptide encoded by G10 is consistent with the antigen polypeptide encoded by the mRNA vaccine in Zhang RR et al., Rational development of multicomponent mRNA vaccine candidates against mpox. Emerg Microbes Infect. 2023Dec;12(1):2192815. Therefore, G10 was used as the mRNA vaccine reference for subsequent experiments.

[0546] Table 6. Composition of each mRNA-LNP formulation

[0547] Example 4 Immunogenicity Evaluation of Candidate Vaccines

[0548] In this example, the immunogenicity of the mRNA-LNP preparations (vaccines) G1, G2, G3, G4, G5, G6, G7, G8, G9, and G10 prepared in Example 3 was evaluated.

[0549] In this example, 55 female BALB / c mice (Zhejiang Weitonglihua) aged 6-8 weeks were randomly divided into 11 groups (G1, G2, G3, G4, G5, G6, G7, G8, G9 and G10 groups named after the administered preparations, and G11 group administered with PBS as a placebo) according to body weight, with 5 mice in each group. According to the dosing design in Table 7, each mouse was injected with a total dose of 2 μg of monkeypox virus mRNA vaccine or placebo, 50 μL / mouse, by intramuscular injection. At week 3 after the primary immunization, that is, on the 21st day after the primary immunization, an equal dose of booster immunization was performed. On the 28th day after the primary immunization, the test mice were subjected to orbital bleeding. The serum was separated and inactivated at 56°C for 30 min and frozen at -80°C for use.

[0550] Table 7. Drug administration design

[0551] 4.1 Detection of vaccine-induced serum binding antibody titers

[0552] The serum binding antibody titer induced by the vaccine was detected using the mouse serum collected above, and the specific detection method is as follows.

[0553] Coating: Dilute each monkeypox antigen protein (A29L antigen protein: nearshore protein, DRA207; A35R antigen protein: nearshore protein, DRA209; B2R antigen protein: Antibody System, EVV12501; B6R antigen protein: nearshore protein, DRA211; C15L antigen protein: custom protein from Qingke Bio; M1R antigen protein: nearshore protein, DRA210; H3L antigen protein: Antibody System, EVV12701; E8L antigen protein: Antibody System, YVV13201) to 1 μg / mL in 1× PBS buffer and add 100 μL / well to a 96-well microtiter plate. Cover with a sealing film and coat overnight at 4°C. After coating, discard the liquid from the wells and wash the plate three times with PBST (300 μL / well).

[0554] Blocking: Add 5% BSA to the wells to block nonspecific binding sites (100 μL / well), apply sealing film, and incubate at 37°C for 1 hour. After blocking, discard the liquid in the wells and wash the plate three times with PBST (300 μL / well).

[0555] Add the test sample: Dilute heat-inactivated test mouse serum with 5% BSA in three-fold increments, starting at an appropriate dilution, into four concentration gradients. Add the diluted serum to the blocked ELISA plate at 100 μL / well. Cover with a sealing film and incubate at 37°C for 1 hour. After incubation, discard the liquid from the wells and wash the plate five times with PBST (300 μL / well).

[0556] Incubation with secondary antibody: Dilute Peroxidase-conjugated Affini Pure Donkey Anti-Mouse IgG (H+L) (Jackson, 715-035-151) 50,000-fold with 5% BSA and add 100 μL / well to the ELISA plate. Cover with film and incubate at 37°C for 1 hour. After incubation, discard the liquid in the wells and wash the plate five times with PBST (300 μL / well).

[0557] Color development and termination: Add 100 μL / well of TMB color development solution to each well and incubate at room temperature in the dark for 5 min. After incubation, add 100 μL / well of ELISA stop solution (Solarbio, C1058), mix thoroughly, and read the absorbance at 450 nm using a multi-function microplate reader (Molecular Devices, ID5).

[0558] Result determination: 2.1 times the average absorbance value of serum of mice in PBS group (G11) was used as the critical value, and OD 450 The highest dilution factor at which the reading is greater than the critical value, the binding antibody titer in the serum of each group of mice was calculated according to the following formula: serum binding antibody titer (IgG titer) = OD reading at 450nm / critical value × serum dilution factor. 450 For the groups whose readings were still below the critical value, the serum binding antibody titer was artificially defined as half of the starting dilution, i.e., 50, which means that no specific binding antibody titer against the coated antigen protein was detected in the mouse serum.

[0559] The test results are shown in Table 8. Except for the mice in groups G4 and G8, which did not produce specific binding antibodies against some antigens, the mice in other groups could produce specific binding antibodies against the antigen proteins encoded by the vaccinated vaccines.

[0560] In the in vitro expression test (Example 2), no successful expression of the A29L, M1R, and B6R antigenic polypeptides encoded by the 1301 mRNA molecule was detected. Similarly, in the serum of the G4 group of mice immunized with the 1301 component, no specific binding antibodies to the A29L, M1R, and B6R proteins were detected. It is reasonable to speculate that this is due to the lack of expression of the 1301 mRNA. However, although the successful expression of the 0201 and 1101 mRNA molecules was not detected in the in vitro expression test (Example 2), the binding antibody titer against each antigenic polypeptide encoded by 0201 or 1101 was detected in the serum of mice immunized with the 0201 or 1101 components, indicating that the 0201 and 1101 mRNA molecules were successfully expressed but at a low level, below the detection limit of the test method.

[0561] Furthermore, the serum of mice in groups G6 and G7 had the highest titers of M1R antigen-specific binding antibodies among all groups. Furthermore, the serum of mice in group G7 had the highest titers of B6R-binding antibodies.

[0562] Table 8. Serum binding antibody titers of tested mice

[0563] “—” indicates not tested (because the mRNA contained in the vaccine does not encode the antigenic polypeptide)

[0564] 4.2 Detection of vaccine-induced serum neutralizing antibody titers

[0565] The collected mouse serum was used to detect the vaccine-induced serum neutralizing antibody titer, and the specific detection method is as follows.

[0566] Prepare cells: Vero cells (Shanghai Cell Bank, Chinese Academy of Sciences, SCSP-520) were seeded into 96-well cell culture plates at 2 × 10 4 Cells were cultured overnight at 37°C, 5% CO2 for 8-16 hours using maintenance medium (DMEM containing 2% FBS). When the cell confluence reached 90%-95% on the second day, they were used for neutralizing antibody detection.

[0567] Neutralization of Viruses: In separate sterile 96-well plates, dilute the heat-inactivated serum sample in maintenance medium at an appropriate dilution, followed by three-fold serial dilutions through six concentrations. Vaccinia virus (Elstree strain) (ATCC, VR-1549) is then diluted to 2000 PFU / mL in maintenance medium and mixed with the diluted serum sample in equal volumes. Set up two replicate wells for each serum dilution. Also, set up virus-infected wells without serum sample as a virus control (12 replicates) and wells with maintenance medium as a cell control (12 replicates). Incubate all the above mixtures or dilutions at 37°C for 1 hour.

[0568] Infect cells: Aspirate the cell culture supernatant and add 100 μL of the incubated virus / serum mixture, virus dilution, or maintenance medium to a 96-well plate containing Vero cells. Infect at 37°C for 1 hour. During infection, shake the plate every 15 minutes to ensure uniform viral infection. After infection, aspirate the supernatant and add 200 μL / well of semi-solid medium (DMEM containing 1.8% sodium carboxymethylcellulose and 2% FBS) preheated to 37°C. Incubate the cells at 37°C, 5% CO2 for 15-17 hours.

[0569] Fixation and Permeabilization: After incubation, discard the cell supernatant and wash the cells once with PBS buffer (200 μL / well). Then, add 4% tissue fixative (50 μL / well) to the cells and incubate at room temperature for 30 minutes. Then, replace the cell medium with 0.25% Triton X (50 μL / well) and permeabilize at room temperature for 30 minutes. Discard the liquid in the wells and wash the cells three times with PBST (300 μL / well).

[0570] Blocking: Add 50 μL of 10% BSA to the wells to block nonspecific binding sites and incubate at 37°C for 1 hour. After blocking, discard the liquid in the wells and wash the cells 5 times with PBST (300 μL / well).

[0571] Antibody Incubation: Dilute HRP-conjugated anti-vaccinia virus antibody (Antibodies-Online, ABIN288495) 2000-fold with 5% BSA and add 100 μL / well to a 96-well plate. Incubate at 37°C for 1 h. After incubation, discard the solution and wash the cells five times with PBST (300 μL / well).

[0572] Color development and reading: According to the instructions, add 50 μL / well of TrueBlue™ Peroxidase Substrate (KPL, 5510-0030) to a 96-well plate and incubate at room temperature in the dark for 15 minutes to develop viral spots. After development, discard the color development solution and allow the 96-well plate to dry overnight at room temperature, protecting it from light. The next day, count the spots using a fluorescent ELISpot reader (CTL).

[0573] Calculation of results: The plaque area of ​​the test well was taken as A x The mean plaque area of ​​the virus control wells is A v The mean plaque area of ​​the cell control wells is A c The serum neutralization inhibition rate (%) was calculated according to the following formula: serum neutralization inhibition rate (%) = 1-(A x -A c ) / (A v -A c The calculated serum neutralization inhibition rate and its corresponding concentration logarithm (Log) value were imported into Graphpad Prism 9.0 software to calculate the neutralizing antibody titer NT in the serum sample. 50 , which is the reciprocal of the serum dilution required to reduce 50% of plaque production. One-way ANOVA was used to determine whether there were significant differences between the groups.

[0574] The experimental results are shown in Figure 1. For fusion-expressed mRNA vaccines, different vaccine designs have different effects on the immunogenicity of candidate vaccines. For example, the mRNA vaccines (G1, G3, G4, G5, G6 mRNA vaccines) used to immunize mice in the G1, G3, G4, G5, and G6 groups all targeted six antigenic peptides (A29L, A35R, B2R, B6R, C15L, and M1R). Among them, the G1 mRNA vaccine contains six individually expressed mRNA molecules. In comparison, the fusion expression design (1501+1601) used in the G5 mRNA vaccine did not significantly affect the immunogenicity of the candidate vaccine, while the fusion expression design (1801+1901) used in the G6 mRNA vaccine significantly improved the candidate vaccine's ability to induce neutralizing antibodies.

[0575] Similarly, the mRNA vaccines immunized in the G2, G7, G8 and G9 groups of mice (G2, G7, G8, G9 mRNA vaccines) also targeted six antigenic peptides (A35R, B2R, B6R, C15L, M1R and H3L). Among them, the fusion expression design method (1201+1401) adopted by the G7 mRNA vaccine can significantly enhance the ability of the candidate vaccine to induce the production of neutralizing antibodies.

[0576] Furthermore, among mRNA vaccines G1-G10, candidate vaccines G6 and G7 induced the highest serum neutralizing antibody titers, with neutralization levels significantly higher than those achieved by co-encapsulating individually expressed mRNA molecules in the multivalent vaccines G1 and / or G2, as well as the reference vaccine G10. Furthermore, the fusion expression design reduces the number of mRNA sequences required for vaccine preparation, significantly reducing the workload and difficulty required during vaccine production and release.

[0577] 4.3 Detection of vaccine-induced cellular immune responses

[0578] The test mice were euthanized on the 42nd day after the primary immunization, and the spleen tissues of the mice were collected and ground into single cell suspensions to detect the vaccine-induced cellular immune response. The specific detection method is as follows.

[0579] Prepare a spleen cell suspension and count viable cells: Prepare an appropriate number of 6-well plates. Add 3 mL of pre-chilled 1640 medium containing 2% FBS to each well. Place a sterile cell strainer in each well. Place freshly harvested spleen tissue on the cell strainer. Gently triturate the spleen tissue using the plunger of a 5 mL syringe and filter through the cell strainer to remove fat and other tissues to form a spleen cell suspension. Transfer the spleen cell suspension to a 15 mL centrifuge tube and centrifuge at 300 g for 5 minutes at 4°C. Discard the supernatant. Resuspend the spleen cells in 2 mL of 1X red blood cell lysis buffer and incubate at room temperature for 3-5 minutes. Immediately add 4 mL of PBS containing 10% FBS and mix by inversion to terminate the lysis reaction. Filter the lysed spleen cells through a cell strainer into a fresh 15 mL centrifuge tube to remove cell clumps. Centrifuge at 300 g for 5 minutes at 4°C and thoroughly remove the supernatant. Resuspend the cells in 1 mL of pre-cooled 1640 medium containing 10% FBS, gently pipette to mix, and then take 10 μL of cells and mix with an equal volume of AO / PI staining solution (Count Star, RE010212) in a 1:1 ratio to calculate the viable cell concentration. The cell concentration was adjusted to 1.5×10 7 Then 0.1 mL of cells were added to a U-bottom 96-well cell culture plate, i.e. 1.5 × 10 cells per well. 6 Two replicate wells were made for each sample.

[0580] Prepare antigen stimulator working solution, negative control culture medium working solution, and cell activation stimulator working solution (positive control):

[0581] 1) Antigen stimulator working solution: CD28 antibody (Biolegend, 122002), CD49d antibody (BD Biosciences, 553313), and monkeypox antigen mixed peptide library (GenScript) were added to pre-chilled 1640 culture medium containing 10% FBS to a final concentration of 4 μg / mL for each of the CD28 antibody, CD49d antibody, and each individual monkeypox antigen peptide library.

[0582] 2) Negative control culture medium working solution: CD28 antibody and CD49d antibody were added to pre-chilled 1640 culture medium containing 10% FBS, so that the final concentration of both antibodies was 4 μg / mL.

[0583] 3) Cell activation stimulator working solution: 500× cell activation cocktail (Biolegend, 423302) was diluted 250-fold with pre-chilled 1640 culture medium containing 10% FBS to a concentration of 2×.

[0584] Stimulation: For each sample's duplicate wells, add 100 μL of antigen stimulator working solution to one of the duplicate wells, achieving a final concentration of 2 μg / mL for the CD28 antibody, CD49d antibody, and each individual monkeypox antigen peptide library. Add 100 μL of negative control culture medium working solution to the other duplicate well, achieving a final concentration of 2 μg / mL for both the CD28 antibody and CD49d. For the positive control wells, add 100 μL of cell activation stimulator working solution to each well, achieving a working concentration of 1× Cell Activation Cocktail in each well. Stimulate the cell samples at 37°C, 5% CO2 for 2 hours.

[0585] Blocking: Dilute 1000× Brefeldin A blocking agent (Biolegend, 420601) 50-fold to 20× with pre-chilled 1640 medium containing 10% FBS. Add 10.5 μL of 20× Brefeldin A to each 200 μL cell suspension for a working concentration of 1× and mix thoroughly by gentle pipetting. Block the cell samples with blocking agent at 37°C, 5% CO₂ for 4 hours. After blocking, centrifuge the cells at 300 g for 5 minutes at 4°C and discard the supernatant. Wash the cells twice with PBS buffer (200 μL / well) at 300 g for 5 minutes at 4°C and discard the supernatant.

[0586] Live-Death Dye Staining: Dilute the live-death dye Zombie Green (Biolegend, 423112) 1000-fold in PBS buffer, add to the cells, resuspend the cells, and incubate at 4°C in the dark for 30 min. After incubation, centrifuge the cells at 300 g for 5 min at 4°C, and discard the supernatant. Wash the cells three times with flow cytometry staining buffer (PBS containing 2% FBS), centrifuging at 300 g for 5 min at 4°C, and discard the supernatant.

[0587] Surface staining: Add surface staining antibodies anti-mouse CD45 (Biolegend, 103138), anti-mouse CD3 (Biolegend, 100216), anti-mouse CD4 (Biolegend, 100434), and anti-mouse CD8a (Biolegend, 100714) to the flow staining buffer in order to make the final concentration of each antibody 2 μg / mL. Take 50 μL of the surface staining antibody mixture and add it to the well and resuspend the cells. Incubate at 4°C in the dark for 30 minutes. After incubation, place the cells in a centrifuge and centrifuge at 4°C, 300g for 5 minutes, and discard the supernatant. Wash the cells three times with flow staining buffer, 200 μL / well, centrifuge at 4°C, 300g for 5 minutes, and discard the supernatant.

[0588] Fixation, Permeabilization, and Intracellular Staining: Add 50 μL of the fixative solution from the Fixation and Permeabilization Kit (BD Biosciences, 554714) to the cells and resuspend the cells. Incubate at 4°C in the dark for 20 min. After fixation, centrifuge the cells at 300 g for 5 min at 4°C and discard the supernatant. Dilute the 10× Permeabilization Buffer from the Fixation and Permeabilization Kit to 1× with double-distilled water. Wash the cells three times with 1× Permeabilization Buffer (200 μL / well) at 300 g for 5 min at 4°C and discard the supernatant. Intracellular staining antibodies anti-mouse IFN-γ (Biolegend, 505808), anti-mouse TNF-α (Biolegend, 506328), anti-mouse IL-2 (Biolegend, 503810) and anti-mouse IL-4 (Biolegend, 504118) were added to the 1× permeabilization buffer in sequence to give a final concentration of 2 μg / mL for each antibody. 50 μL of the intracellular staining antibody mixture was added to the wells and the cells were resuspended. The cells were incubated at 4°C in the dark for 30 minutes. After incubation, the cells were centrifuged at 4°C, 300g for 5 minutes, and the supernatant was discarded. The cells were washed three times with 1× permeabilization buffer, 200 μL / well, 4°C, 300g for 5 minutes, and the supernatant was discarded. The cells were resuspended in 200 μL of flow staining buffer and the samples were detected using a flow cytometer (BD biosciences, LSRFortessa).

[0589] The test results are shown in Figure 2. Compared with the multivalent vaccine G1 made by co-encapsulating individually expressed mRNA molecules, the fusion expression designed mRNA vaccine G6 induced a higher level of cellular immune response. For mRNA vaccines expressing another combination of antigenic polypeptides, the fusion expression mRNA vaccine G7 induced a comparable level of cellular immune response to the multivalent vaccine G2 made by co-encapsulating individually expressed mRNA molecules (data not shown). In addition, all mRNA vaccines prepared in Example 3 induced a Th1 immune response (some data not shown).

[0590] Sequence Listing

Claims

1. A polypeptide or polypeptide combination, which comprises one or more peptide segments, and each of the peptide segments is independently selected from peptide segments of monkeypox virus A29L, A35R, B2R, B6R, C15L, M1R and H3L proteins; Optionally, the polypeptide or polypeptide combination further comprises one or more linkers, IgE signal peptides and / or HA polypeptide tags.

2. The polypeptide or polypeptide combination according to claim 1, wherein (1) the polypeptide or polypeptide combination comprises peptide segments of A35R, B2R, B6R, C15L, M1R and H3L proteins; (2) the polypeptide or polypeptide combination comprises peptide segments of A29L, A35R, B2R, B6R, C15L and M1R proteins; (3) the polypeptide or polypeptide combination comprises peptide segments of A35R, B2R and C15L proteins; (4) the polypeptide or polypeptide combination comprises peptide segments of B6R, M1R and H3L proteins; (5) the polypeptide or polypeptide combination comprises peptide segments of B2R, A35R and B6R proteins; (6) the polypeptide or polypeptide combination comprises peptide segments of A29L, M1R and C15L proteins; (7) the polypeptide or polypeptide combination comprises peptide segments of A29L, B6R and M1R proteins; (8) the polypeptide or polypeptide combination comprises peptide segments of B6R, M1R and A35R proteins; (9) the polypeptide or polypeptide combination comprises peptide segments of A29L, B2R and C15L proteins; (10) the polypeptide or polypeptide combination comprises peptide segments of B2R, H3L and C15L proteins; (11) the polypeptide or polypeptide combination comprises peptide segments of H3L, M1R and C15L proteins; (12) the polypeptide or polypeptide combination comprises peptide segments of A29L protein; (13) the polypeptide or polypeptide combination comprises peptide segments of A35R protein; (14) the polypeptide or polypeptide combination comprises peptide segments of B2R protein; (15) the polypeptide or polypeptide combination comprises peptide segments of B6R protein; (16) the polypeptide or polypeptide combination comprises peptide segments of C15L protein; (17) the polypeptide or polypeptide combination comprises peptide segments of M1R protein; or (18) the polypeptide or polypeptide combination comprises peptide segments of H3L protein.

3. The polypeptide according to claim 1 or 2, wherein the peptide segment of A29L protein comprises the amino acid sequence of SEQ ID NO: 111 or 113; the peptide segment of A35R protein comprises the amino acid sequence of SEQ ID NO: 114 or 115; the peptide segment of B2R protein comprises the amino acid sequence of SEQ ID NO: 116, 117, 118, 119 or 120; the peptide segment of B6R protein comprises the amino acid sequence of SEQ ID NO: 121, 122, 123, 124 or 125; the peptide segment of C15L protein comprises the amino acid sequence of SEQ ID NO: 126 or 127; the peptide segment of M1R protein comprises the amino acid sequence of SEQ ID NO: 128 or 130; or The peptide segment in the H3L protein comprises the amino acid sequence of SEQ ID NO: 131 or 132.

4. The polypeptide or polypeptide combination according to any one of claims 1-3, wherein (1) the polypeptide or polypeptide combination comprises peptide segments in A35R, B2R, B6R, C15L, M1R and H3L proteins, and a linker; (2) the polypeptide or polypeptide combination comprises peptide segments in A29L, A35R, B2R, B6R, C15L and M1R proteins, an IgE signal peptide and a linker; (3) the polypeptide or polypeptide combination comprises peptide segments in A29L, A35R, B2R, B6R, C15L and M1R proteins, an IgE signal peptide, an HA polypeptide tag and a linker; (4) the polypeptide or polypeptide combination comprises peptide segments in A35R, B2R, B6R, C15L, M1R and H3L proteins, an IgE signal peptide, an HA polypeptide tag and a linker; (5) the polypeptide or polypeptide combination comprises peptide segments in A35R, B2R, B6R, C15L, M1R and H3L proteins, an IgE signal peptide and a linker; (6) the polypeptide or polypeptide combination comprises peptide segments in A35R, B2R and C15L proteins and a linker; (7) the polypeptide or polypeptide combination comprises peptide segments in B6R, M1R and H3L proteins and a linker; (8) the polypeptide or polypeptide combination comprises peptide segments in B2R, A35R and B6R proteins and a linker; (9) the polypeptide or polypeptide combination comprises peptide segments in A29L, M1R and C15L proteins, an IgE signal peptide and a linker; (10) the polypeptide or polypeptide combination comprises peptide segments in A29L, B6R and M1R proteins, an IgE signal peptide and a linker; (11) the polypeptide or polypeptide combination comprises peptide segments in B6R, M1R and A35R proteins and a linker; (12) the polypeptide or polypeptide combination comprises peptide segments in A29L, B2R and C15L proteins, an IgE signal peptide and a linker; (13) the polypeptide or polypeptide combination comprises peptide segments in B2R, H3L and C15L proteins and a linker; (14) the polypeptide or polypeptide combination comprises peptide segments in H3L, M1R and C15L proteins, an IgE signal peptide and a linker; (15) the polypeptide or polypeptide combination comprises an IgE signal peptide, peptide segments in A29L protein and an HA polypeptide tag; (16) the polypeptide or polypeptide combination comprises peptide segments in A35R protein and an HA polypeptide tag; (17) the polypeptide or polypeptide combination comprises peptide segments in B2R protein and an HA polypeptide tag; (18) the polypeptide or polypeptide combination comprises peptide segments in B6R protein, a linker and an HA polypeptide tag; (19) the polypeptide or polypeptide combination comprises an IgE signal peptide, a linker, an HA polypeptide tag and peptide segments in C15L protein; (20) the polypeptide or polypeptide combination comprises an IgE signal peptide, a linker, an HA polypeptide tag and peptide segments in M1R protein; or (21) the polypeptide or polypeptide combination comprises an IgE signal peptide, a linker, an HA polypeptide tag and peptide segments in H3L protein.

5. The polypeptide or polypeptide combination according to any one of claims 1-4, wherein the IgE signal peptide comprises the amino acid sequence of SEQ ID NO: 134; the HA polypeptide tag comprises the amino acid sequence of SEQ ID NO: 135 or 136; and / or the linker is a GS linker, preferably, the GS linker comprises the amino acid sequence of SEQ ID NO: 137 or 138.

6. The polypeptide or polypeptide combination according to any one of claims 1-5, wherein the polypeptide or polypeptide combination comprises the amino acid sequence of any one of SEQ ID NO: 102, 104, 108, 109, 89, 90, 91, 92, 93, 94, 95, 101, 103, 105, 106, 107 and 110 or an amino acid sequence having at least 80% identity with the amino acid sequence of any one of SEQ ID NO: 102, 104, 108, 109, 89, 90, 91, 92, 93, 94, 95, 101, 103, 105, 106, 107 and 110.

7. The polypeptide or polypeptide combination according to any one of claims 1-5, wherein the polypeptide or polypeptide combination comprises two or more amino acid sequences selected from SEQ ID NO: 102, 104, 108, 109, 89, 90, 91, 92, 93, 94, 95, 101, 103, 105, 106, 107 and 110 or two or more amino acid sequences having at least 80% identity with the amino acid sequences selected from SEQ ID NO: 102, 104, 108, 109, 89, 90, 91, 92, 93, 94, 95, 101, 103, 105, 106, 107 and 110.

8. The polypeptide or polypeptide combination according to claim 7, wherein (1) the polypeptide or polypeptide combination comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise the amino acid sequences of SEQ ID NO: 102 and 104, respectively; (2) the polypeptide or polypeptide combination comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise the amino acid sequences of SEQ ID NO: 108 and 109, respectively; (3) the polypeptide or polypeptide combination comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide and a sixth polypeptide, wherein the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the fifth polypeptide and the sixth polypeptide comprise the amino acid sequences of SEQ ID NO: 89, 90, 91, 92, 93 and 94, respectively; (4) the polypeptide or polypeptide combination comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide and a sixth polypeptide, wherein the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the fifth polypeptide and the sixth polypeptide comprise the amino acid sequences of SEQ ID NO: 90, 91, 92, 93, 94 and 95, respectively; (5) The polypeptide or polypeptide combination comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise the amino acid sequences of SEQ ID NO: 102 and 103, respectively; (6) The polypeptide or polypeptide combination comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise the amino acid sequences of SEQ ID NO: 105 and 106, respectively; (7) The polypeptide or polypeptide combination comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise the amino acid sequences of SEQ ID NO: 105 and 107, respectively; or (8) The polypeptide or polypeptide combination comprises a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise the amino acid sequences of SEQ ID NO: 110 and 108, respectively.

9. A nucleic acid or nucleic acid combination, which comprises a polynucleotide encoding the polypeptide or polypeptide combination according to any one of claims 1-8.

10. The nucleic acid or nucleic acid combination of claim 9, wherein the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NO: 14, 16, 20, 21, 1, 2, 3, 4, 5, 6, 7, 13, 15, 17, 18, 19 and 22 or a nucleotide sequence having at least 80% identity with the nucleotide sequence of any one of SEQ ID NO: 14, 16, 20, 21, 1, 2, 3, 4, 5, 6, 7, 13, 15, 17, 18, 19 and 22; or wherein the polynucleotide is DNA, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NO: 36, 38, 42, 43, 23, 24, 25, 26, 27, 28, 29, 35, 37, 39, 40, 41 and 44 or a nucleotide sequence having at least 80% identity with the nucleotide sequence of any one of SEQ ID NO: 36, 38, 42, 43, 23, 24, 25, 26, 27, 28, 29, 35, 37, 39, 40, 41 and 44.

11. The nucleic acid or nucleic acid combination of claim 9, wherein the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, the polynucleotide comprises two or more nucleotide sequences selected from SEQ ID NO: 14, 16, 20, 21, 1, 2, 3, 4, 5, 6, 7, 13, 15, 17, 18, 19 and 22 or two or more nucleotide sequences having at least 80% identity with the nucleotide sequences selected from SEQ ID NO: 14, 16, 20, 21, 1, 2, 3, 4, 5, 6, 7, 13, 15, 17, 18, 19 and 22; or wherein the polynucleotide is DNA, The polynucleotide comprises two or more nucleotide sequences selected from SEQ ID NO: 36, 38, 42, 43, 23, 24, 25, 26, 27, 28, 29, 35, 37, 39, 40, 41 and 44, or two or more nucleotide sequences having at least 80% identity with the nucleotide sequences selected from SEQ ID NO: 36, 38, 42, 43, 23, 24, 25, 26, 27, 28, 29, 35, 37, 39, 40, 41 and 44.

12. The nucleic acid or nucleic acid combination of claim 11, wherein the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, (1) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 14 and 16; (2) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 20 and 21; (3) the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5 and 6; (4) the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 2, 3, 4, 5, 6 and 7; (5) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 14 and 15; (6) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 17 and 18; (7) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 17 and 19; or (8) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 22 and 20; or the polynucleotide is DNA, (1’) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide respectively comprise the nucleotide sequences of SEQ ID NO: 36 and 38; (2’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:42 and 43, respectively; (3’) The polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise the nucleotide sequences of SEQ ID NO:23, 24, 25, 26, 27 and 28, respectively; (4’) The polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise the nucleotide sequences of SEQ ID NO:24, 25, 26, 27, 28 and 29, respectively; (5’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:36 and 37, respectively; (6’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:39 and 40, respectively; (7’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:39 and 41, respectively; or (8’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:44 and 42, respectively.

13. The nucleic acid or nucleic acid combination according to any one of claims 9-12, wherein the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, the polynucleotide further comprises a 5’-UTR sequence; preferably, the 5’-UTR sequence comprises the nucleotide sequence of SEQ ID NO:139 or 148; the polynucleotide further comprises a 3’-UTR sequence; preferably, the 3’-UTR sequence comprises the nucleotide sequence of SEQ ID NO:140, 141 or 149; and / or the polynucleotide further comprises a poly(A) sequence; preferably, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:145; or wherein the polynucleotide is DNA, the polynucleotide further comprises a DNA sequence corresponding to the 5’-UTR sequence; preferably, the DNA sequence corresponding to the 5’-UTR sequence comprises the nucleotide sequence of SEQ ID NO:142 or 150; The polynucleotide further comprises a DNA sequence corresponding to a 3'-UTR sequence; preferably, the DNA sequence corresponding to the 3'-UTR sequence comprises the nucleotide sequence of SEQ ID NO: 143, 144 or 151; and / or The polynucleotide further comprises a DNA sequence corresponding to a poly(A) sequence; preferably, the DNA sequence corresponding to the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:

146.

14. The nucleic acid or nucleic acid combination according to any one of claims 9-13, wherein the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NO: 58, 60, 64, 65, 45, 46, 47, 48, 49, 50, 51, 57, 59, 61, 62, 63 and 66 or a nucleotide sequence having at least 80% identity with the nucleotide sequence of any one of SEQ ID NO: 58, 60, 64, 65, 45, 46, 47, 48, 49, 50, 51, 57, 59, 61, 62, 63 and 66; or wherein the polynucleotide is DNA, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NO: 80, 82, 86, 87, 67, 68, 69, 70, 71, 72, 73, 79, 81, 83, 84, 85 and 88 or a nucleotide sequence having at least 80% identity with the nucleotide sequence of any one of SEQ ID NO: 80, 82, 86, 87, 67, 68, 69, 70, 71, 72, 73, 79, 81, 83, 84, 85 and 88.

15. The nucleic acid or nucleic acid combination according to any one of claims 9-13, wherein the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, the polynucleotide comprises two or more nucleotide sequences selected from SEQ ID NO: 58, 60, 64, 65, 45, 46, 47, 48, 49, 50, 51, 57, 59, 61, 62, 63 and 66 or two or more nucleotide sequences having at least 80% identity with the nucleotide sequences selected from SEQ ID NO: 58, 60, 64, 65, 45, 46, 47, 48, 49, 50, 51, 57, 59, 61, 62, 63 and 66; or wherein the polynucleotide is DNA, The polynucleotide comprises two or more nucleotide sequences selected from SEQ ID NO:80, 82, 86, 87, 67, 68, 69, 70, 71, 72, 73, 79, 81, 83, 84, 85 and 88, or two or more nucleotide sequences having at least 80% identity to the nucleotide sequences selected from SEQ ID NO:80, 82, 86, 87, 67, 68, 69, 70, 71, 72, 73, 79, 81, 83, 84, 85 and 88.

16. The nucleic acid or nucleic acid combination of claim 15, wherein the polynucleotide is RNA, preferably mRNA, circRNA or saRNA, more preferably mRNA, (1) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:58 and 60, respectively; (2) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:64 and 65, respectively; (3) the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise the nucleotide sequences of SEQ ID NO:45, 46, 47, 48, 49 and 50, respectively; (4) the polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise the nucleotide sequences of SEQ ID NO:46, 47, 48, 49, 50 and 51, respectively; (5) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:58 and 59, respectively; (6) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:61 and 62, respectively; (7) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:61 and 63, respectively; or (8) the polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:66 and 64, respectively; or the polynucleotide is DNA, (1’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:80 and 82, respectively; (2’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:86 and 87, respectively; (3’) The polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise the nucleotide sequences of SEQ ID NO:67, 68, 69, 70, 71 and 72, respectively; (4’) The polynucleotide comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, a fifth polynucleotide and a sixth polynucleotide, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise the nucleotide sequences of SEQ ID NO:68, 69, 70, 71, 72 and 73, respectively; (5’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:80 and 81, respectively; (6’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:83 and 84, respectively; (7’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:83 and 85, respectively; or (8’) The polynucleotide comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences of SEQ ID NO:88 and 86, respectively.

17. A composition comprising the nucleic acid or nucleic acid combination of any one of claims 9-16.

18. The composition of claim 17, which comprises a lipid encapsulating the nucleic acid or nucleic acid combination.

19. The composition of claim 17 or 18, which comprises at least a first lipid, wherein the first lipid comprises one or more of the compounds according to formula 01-I or 01-II, preferably the first lipid comprises one or more of the compounds listed in Table 01-1; wherein the first lipid comprises one or more of the compounds according to formula 02-I or 02-II, preferably the first lipid comprises one or more of the compounds listed in Table 02-1; wherein the first lipid comprises one or more of the compounds according to formula 03-I, preferably the first lipid comprises one or more of the compounds listed in Table 03-1; or Wherein the first lipid comprises one or more of the compounds according to Formula 04-I or 04-III, preferably the first lipid comprises one or more of the compounds listed in Table 04-1; More preferably, the first lipid comprises Compound 01-1.

20. The composition of claim 19, further comprising a second lipid, Wherein the second lipid is a polymer-bound lipid; preferably, it comprises one or more of the compounds according to Formula 05-I.

21. The composition of any one of claims 17-20, comprising A first lipid, a second lipid, a phospholipid, and a steroid.

22. The composition of any one of claims 17-21, wherein the composition is formulated as a lipid nanoparticle encapsulating the nucleic acid or nucleic acid combination in a lipid.

23. A pharmaceutical composition or kit, comprising the polypeptide or polypeptide combination of any one of claims 1-8, the nucleic acid or nucleic acid combination of any one of claims 9-16, or the composition of any one of claims 17-22.

24. An expression vector, comprising the nucleic acid or nucleic acid combination of any one of claims 9-16.

25. A host cell, comprising the nucleic acid or nucleic acid combination of any one of claims 9-16 or the expression vector of claim 24.

26. Use of the polypeptide or polypeptide combination of any one of claims 1-8, the nucleic acid or nucleic acid combination of any one of claims 9-16, the composition of any one of claims 17-22, the pharmaceutical composition or kit of claim 23 in the preparation of a vaccine for preventing and / or treating monkeypox virus infection in a subject.

27. The use of claim 26, wherein the subject is a mammal; preferably, the subject is a human.

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