Novel adjuvant polypeptide sequence and use thereof

Abstract

The present invention relates to the field of vaccines, and in particular to a novel adjuvant polypeptide sequence and a use thereof. The adjuvant polypeptide sequence provided by the present invention comprises amino acid sequences of tetanus toxoid epitopes P2 and P16 and further comprises amino acid sequences of P30 and / or PX. The adjuvant polypeptide sequence aims to expand the coverage population of immune responses by increasing epitope diversity. In addition, the present invention uses an LNP (lipid nanoparticle) delivery system to introduce an mRNA encoding an adjuvant polypeptide and an initial antigen protein into a human body, thereby improving the immunostimulatory ability of the initial antigen. The adjuvant polypeptide sequence provided by the present invention and the initial antigen sequence are preferably fused, thereby simplifying the composition of mRNA in a nucleic acid vaccine and reducing the complexity of vaccine preparation.

Description

A novel adjuvant polypeptide sequence and its application Technical Field This invention relates to the field of vaccines, and in particular to a novel adjuvant polypeptide sequence and its application. Background Technology Tetanus toxoid (TT), as a potent immunogen of T helper (Th) cells, possesses intrinsic adjuvant properties. It primarily triggers Th immune responses through its specific epitopes (peptide sequences), thus demonstrating potential in the treatment of infectious diseases, inflammatory diseases, immune system diseases, and tumors. Traditionally, common epitopes such as P2 and P16 of TT have been widely used to activate CD4. + Although these epitopes can partially break immune tolerance, their coverage of T helper cells is limited due to individual variability in MHC II alleles. Furthermore, the adjuvant effect elicited by the P2 and P16 epitopes is insufficient to fully activate or amplify memory CD4+ cells. + T helper cells, thereby limiting the enhancement of antigen-stimulated immunity and the effectiveness of preventive / therapeutic drugs (such as vaccines). Summary of the Invention In view of this, the present invention provides a novel adjuvant polypeptide sequence and its application, aiming to overcome the limitations of existing TT epitopes in terms of immune tolerance breaking ability and population coverage. To achieve the above and other related objectives, the present invention provides an adjuvant polypeptide sequence comprising the amino acid sequences of tetanus toxoid epitopes P2 and P16, and also comprising the amino acid sequences of P30 and / or PX. Preferably, the adjuvant nucleic acid molecule further comprises an amino acid sequence encoding an MHC class I transport domain (MITD). The present invention also provides an adjuvant nucleic acid molecule, characterized in that the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding the aforementioned polypeptide sequence, the nucleotide fragment being located in one or more nucleic acid molecules. The present invention also provides a nucleic acid vaccine, characterized in that the nucleic acid vaccine comprises mRNA and lipid nanoparticles, wherein the mRNA is selected from one of the following: I) Multiple mRNAs, each having one or more open reading frames encoding 1-500 peptide epitopes and the aforementioned adjuvant nucleic acid molecule, wherein the open reading frames encoding 1-500 peptide epitopes and the aforementioned adjuvant nucleic acid molecule are not on the same mRNA; II) One or more mRNAs, each having one or more open reading frames encoding 1-500 peptide epitopes, wherein the mRNAs further comprise the aforementioned adjuvant nucleic acid molecule. The present invention also provides the use of the aforementioned adjuvant polypeptide sequence, adjuvant polypeptide nucleic acid molecule, or the aforementioned nucleic acid vaccine in the preparation of preventive or therapeutic drugs. As described above, the novel adjuvant polypeptide sequence and its application of the present invention have the following beneficial effects: 1) The adjuvant polypeptide sequence of the present invention can expand the coverage of the immune response by increasing epitope diversity. 2) This invention utilizes an LNP (lipid nanoparticle) delivery system to efficiently introduce mRNA encoding adjuvant peptides and initial antigen proteins into the body, effectively enhancing the immunostimulatory capacity of the initial antigen, thereby achieving or exceeding the immunogenicity of traditional doses while reducing the dosage of the initial antigen. 3) The adjuvant polypeptide sequence and the initial antigen sequence provided by the present invention are preferably fused together, which simplifies the composition of mRNA in nucleic acid vaccines, reduces the complexity of vaccine preparation, and provides a new approach for vaccine process development. Attached Figure Description Figure 1 shows the antitumor effects of P2P16P30PX auxiliary epitope fusion with E6 and E7 antigens compared to P2P16 auxiliary epitope fusion with E6 and E7 antigens, respectively. Figure 2a shows the antibody titer data of P2P16P30PX auxiliary epitope fusion with E6 and E7 antigens, respectively, compared with the antibody titer data of P2P16 auxiliary epitope fusion with E6 and E7 antigens. Figure 2b shows the percentage of IFN-gamma-positive CTL cells in the Model group compared to those with E6 and E7 antigens fused with P2P16P30PX helper epitope. Figure 2c shows the percentage of CTL cells with antigen specificity in the Model group compared to the E6 and E7 antigens fused with P2P16P30PX helper epitope. Figure 3 shows the antitumor effect of P2P16P30PX auxiliary epitope fusion with E6 and E7 antigens simultaneously fused with MITD. Figure 4a shows the antibody titer data after the P2P16P30PX auxiliary epitope was fused with both E6 and E7 antigens and simultaneously fused with MITD. Figure 4b shows the percentage of IFN-gamma-positive CTL cells after P2P16P30PX helper epitope fusion with E6 and E7 antigens and simultaneous fusion with MITD. Figure 4c shows the percentage of antigen-specific CTL cells after the P2P16P30PX helper epitope is fused with both E6 and E7 antigens and simultaneously fused with MITD. Figure 5 shows the percentage of CD8+H-2Db+ cells specific to E7 antigen in the Model group after P2P16P30PX helper epitope fusion with E6 and E7 antigens and simultaneous fusion with MITD. Figure 6a shows the efficacy verification of the P2P16P30PX auxiliary epitope fusion with E6 and E7 antigen mutants, which are simultaneously fused with MITD. Figure 6b shows the long-term survival of the P2P16P30PX helper epitope fusion with the E6 and E7 antigen mutants after simultaneous fusion with MITD. Figure 7 shows the inhibitory effects of different mRNA-LNPs on tumor growth. Figure 8 shows the results of long-term survival studies for different mRNA-LNPs. Detailed Implementation To address the shortcomings of existing technologies, the present invention aims to provide a novel adjuvant polypeptide sequence and its applications. The adjuvant polypeptide sequence provided by the present invention comprises the amino acid sequences of tetanus toxoid epitopes P2 and P16, and also contains the amino acid sequences of P30 and / or PX. The mRNA encoding the above epitopes and the initial antigen protein is delivered into the body via LNP to exert its effects, thereby enhancing the immunostimulatory capacity of the initial antigen, breaking immune tolerance, and thus being used to treat infectious diseases, inflammatory diseases, immune system diseases, or tumors. To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides an adjuvant polypeptide sequence comprising the amino acid sequences of tetanus toxoid epitopes P2 and P16, and further comprising the amino acid sequences of P30 and / or PX. In some specific embodiments, the adjuvant polypeptide sequence comprises the amino acid sequences of tetanus toxoid epitopes P2, P16, and P30; or, the adjuvant polypeptide sequence comprises the amino acid sequences of tetanus toxoid epitopes P2, P16, and PX; or, the adjuvant polypeptide sequence comprises the amino acid sequences of tetanus toxoid epitopes P2, P16, P30, and PX. In some specific embodiments, the adjuvant polypeptide sequence further comprises an amino acid sequence of an MHC class I transport domain (MITD). In some specific embodiments, the amino acid sequences contained in the adjuvant polypeptide sequence can be fused in any manner. The fusion method is not limited, including but not limited to the fusion order between epitopes, direct fusion, or indirect fusion, as long as any fusion method can achieve the technical solution of this invention. Exemplary fusion sequences are as follows: P2P16PX; P2P16P30; P2P16P30PX; P2P16P30PXMITD; P16P2P30PX; P16P2PXP30; P2P30P16PXMITD; P2P30PXP16MITD. (Where P2P16PX represents the sequential fusion of the P2 sequence with the P16 sequence with the P30 sequence, and P16P2P30PX represents the sequential fusion of the P16 sequence with the P2 sequence with the P30 sequence with the PX sequence; other fusion methods are explained similarly). Since fusion proteins need to be enzymatically cleaved into small peptides of about 10-15 amino acids in length before they can be presented to effector cells by the MHC, the adjuvant polypeptide in the sequence will be presented in the form of peptide segments after enzymatic cleavage. Therefore, its order in the fusion protein does not affect its adjuvant function. In some specific embodiments, the epitope amino acid sequences are linked by linker peptides. The type of linker is not specifically limited, and includes, but is not limited to, rigid linkers and / or flexible linkers, as long as they can link the epitopes and do not necessarily have a specific function. An exemplary rigid linker is (EAAAK)*n; flexible linkers are selected from (GGGGS)*n, (GSGGG)*n, (GGGSG)*n, or (GGSGG)*n, where n is an integer from 1 to 5. In some specific embodiments, the linker is selected from one or more of GGSGGGGSGGKK, GGEAAAK, PAPAPG, GSGAKE, and GSGSS. A second aspect of the present invention provides an adjuvant nucleic acid molecule comprising a nucleotide fragment encoding the aforementioned epitope amino acid sequence. In some specific embodiments, the nucleotide fragment encoding the aforementioned epitope amino acid sequence is located within a single nucleic acid molecule in the adjuvant nucleic acid molecule. For example, the adjuvant nucleic acid molecule may contain a nucleotide fragment encoding P2P16P30; or a nucleotide fragment encoding P2P16PX; or a nucleotide fragment encoding P2P16P30PX; or a nucleotide fragment encoding P2P16P30PXMITD; or a nucleotide fragment encoding P16P2P30PX; or a nucleotide fragment encoding P16P2PXP30; or a nucleotide fragment encoding P2P30P16PXMITD; or a nucleotide fragment encoding P2P30PXP16MITD. In some specific embodiments, the nucleotide fragment encoding the aforementioned epitope amino acid sequence in the adjuvant nucleic acid molecule is located in multiple nucleic acid molecules. For example, the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2, a nucleic acid molecule encoding P16, and a nucleotide fragment encoding P30; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2, a nucleotide fragment encoding P16, a nucleotide fragment encoding P30, and a nucleotide fragment encoding PX; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2P16, a nucleic acid molecule encoding P30, and a nucleotide fragment encoding PX; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2, a nucleotide fragment encoding P16, a nucleotide fragment encoding P30, a nucleotide fragment encoding PX, and a nucleotide fragment encoding MITD; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2P16P30. The adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2P16P30, a nucleic acid molecule encoding PX, and a nucleotide fragment encoding MITD; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2P16PX, a nucleic acid molecule encoding P30, and a nucleotide fragment encoding MITD; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2P16P30PX and a nucleotide fragment encoding MITD; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P16P2P30PX and a nucleotide fragment encoding MITD; or the adjuvant nucleic acid molecule comprises a nucleotide fragment encoding P2P16PXP30 and a nucleotide fragment encoding MITD. In some specific embodiments, the amino acid sequence of the tetanus toxoid epitope P2 is as shown in SEQ ID No. 1, or is an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 1. In some specific embodiments, the amino acid sequence of the tetanus toxoid epitope P16 is as shown in SEQ ID No. 2, or is an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 2. In some specific embodiments, the amino acid sequence of the tetanus toxoid epitope P30 is as shown in SEQ ID No. 3, or is an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 3. In some specific embodiments, the amino acid sequence of the tetanus toxoid epitope PX is as shown in SEQ ID No. 4, or is an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 4. In some specific embodiments, the amino acid sequence of the MHC class I transport domain is as shown in SEQ ID No. 5, or is an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 5. A third aspect of the present invention provides a nucleic acid vaccine comprising mRNA and lipid nanoparticles, wherein the mRNA comprises one or more of the following: I) Multiple mRNAs, each having one or more open reading frames encoding 1-500 peptide epitopes and the aforementioned adjuvant nucleic acid molecule, wherein the open reading frames encoding 1-500 peptide epitopes and the aforementioned adjuvant nucleic acid molecule are not on the same mRNA; II) One or more mRNAs, each having one or more open reading frames encoding 1-500 peptide epitopes, wherein the mRNAs further comprise the aforementioned adjuvant nucleic acid molecule. In some specific embodiments, the peptide epitope is an HPV antigen. Specifically, the peptide epitope is selected from one or more of the following antigens: HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV35, HPV39, HPV45, HPV51, HPV56, HPV59, HPV66, HPV26, HPV53, HPV68, or HPV73 antigens. Preferably, the peptide epitope is an HPV16 antigen. Further, the antigen of HPV16 type is HPV16 E6 and / or E7 protein, wherein the amino acid sequence of the E6 protein is as shown in SEQ ID No. 6, or is an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 6; or, the amino acid sequence of the E7 protein is as shown in SEQ ID No. 7, or is an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 7. In some specific embodiments, the various mRNAs described in I) include, but are not limited to: mRNA encoding E6 and mRNA encoding P2P16P30; mRNA encoding E7 and mRNA encoding P2P16P30PX; mRNA encoding E6 and mRNA encoding P2P16P30PXMITD; mRNA encoding E7, mRNA encoding P2P16 and mRNA encoding P30; mRNA encoding E6, mRNA encoding P2P16 and mRNA encoding P30PX; and one or more of the following: mRNA encoding E6E7, mRNA encoding P2P16, mRNA encoding P30 and mRNA encoding PX. In some specific embodiments, the one or more mRNAs mentioned in II) include, but are not limited to: one mRNA encoding E6P2P16P30; an mRNA encoding E7P2P16P30; an mRNA encoding E6E7P2P16P30PX; an mRNA encoding E6E7P2P16P30PXMITD; an mRNA encoding E6P2P16PX; and multiple mRNAs encoding both E6P2P16P30PX and E7P2P16P30PX. In some specific embodiments, the aforementioned mRNA comprises a cap structure, a 5'UTR, a signal peptide, a 3'UTR, and / or poly A; the types are not limited. Preferably, the cap structure comprises m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(3′OMeA)pG linked to the 5'UTR; In some specific embodiments, the aforementioned mRNA cap structure, 5'UTR, signal peptide, 3'UTR, and / or poly A are as described in Chinese Patent CN116234568A.

[0039] ,

[0048] -

[0056] The signal peptide is described above. Specifically, the signal peptide can promote the expression and localization of the encoded antigenic peptide or protein. In some specific embodiments, the signal peptide is selected from: MRVMAPRTLILLLSGALALTETWA, MDWTWILFLVAAATRVHS, or MDMRVPAQLLGLLLLWLPGARC. In some specific implementations, the aforementioned mRNA contains unmodified bases, nucleosides, or nucleotides. In some specific embodiments, the aforementioned mRNA further comprises one or more modified bases, nucleosides, or nucleotides (referred to as "modified mRNA," "mmRNA," or "modified mRNA"). More specifically, compared to unmodified mRNA, modified mRNA can possess useful properties, including enhanced mRNA stability, reduced degradation, improved translation efficiency, and / or reduced immunogenicity resulting from mRNA introduction. Therefore, using modified mRNA can not only enhance protein expression efficiency but also reduce immunogenicity. In some embodiments, the aforementioned mRNA comprises one or more (e.g., 1, 2, 3, or 4) different modified bases, nucleosides, or nucleotides. In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified bases, nucleosides, or nucleotides. In some embodiments, the modified mRNA can slow down the degradation trend in cells compared to unmodified mRNA. In some specific embodiments, the modified base is modified uracil. Exemplary bases and nucleosides having modified uracil include, but are not limited to, pseudouridine (ψ), 2-thio-pseudouridine, 5-methoxy-uridine (mo5U), N1-methylpseudouridine (N1Mψ), 2-thiouridine, and 4'-thiouridine. In some specific embodiments, the modified base is a modified cytosine. Exemplary bases and nucleosides having modified cytosine include, but are not limited to, pseudocytidine, 3-methylcytidine (m3C), 5-methylcytidine (m5C), 5-hydroxymethylcytidine (hm5C), and 5-methylcytosine. In some specific embodiments, the modified base is adenine. Exemplary bases and nucleosides having modified adenine include, but are not limited to, 1-methyl-adenosine (m1A), N6-methyl-adenosine (m6A), N6,N6-dimethyl-adenosine (m62A), N6,2'-O-dimethyl-adenosine (m6Am), and α-thio-adenosine. In some specific embodiments, the modified base is modified guanine. Exemplary bases and nucleosides having modified guanine include, but are not limited to, inosine (I), α-thio-guanine, 6-thio-7-deazo-guanine, 7-methyl-guanine (m7G), and 7-methyl-8-oxo-guanine. In some specific embodiments, the aforementioned mRNA comprises pseudouridine (ψ); or, the aforementioned mRNA comprises pseudouridine and 5-methylcytidine (m5C); or, the aforementioned mRNA comprises N1-methyl-pseudouridine (N1Mψ); or, the aforementioned mRNA comprises N1-methyl-pseudouridine (N1Mψ) and 5-methylcytidine (m5C); or, the aforementioned mRNA comprises 2-thiouridine (s2U); or, the aforementioned mRNA comprises 2-thiouridine and 5-methylcytidine (m5C); or, the aforementioned mRNA comprises... The mRNA contains 5-methoxyuridine (mo5U); or, the mRNA contains 5-methoxyuridine (mo5U) and 5-methylcytidine (m5C); or, the mRNA contains 2'-O-methyluridine; or, the mRNA contains 2'-O-methyluridine and 5-methylcytidine (m5C); or, the mRNA contains N6-methyladenosine (m6A); or, the mRNA contains N6-methyladenosine (m6A) and 5-methylcytidine (m5C). In some specific implementations, the aforementioned mRNA is uniformly modified (i.e., completely modified, modified throughout the entire sequence) for a particular modification. For example, the mRNA can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, the aforementioned mRNA can be uniformly modified by replacing any type of base present in the sequence with modified base substitutions such as those shown above. In some embodiments, the aforementioned mRNA may be modified in a coding region (e.g., an open reading frame encoding a polypeptide). In some embodiments, the aforementioned mRNA may be modified in a region outside the coding region. For example, in some embodiments, a 5'UTR and / or a 5'UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region. In some specific embodiments, the lipid nanoparticles further comprise one or more combinations of the following ionizable lipid compounds, their stereoisomers, their tautomers, or pharmaceutically acceptable salts: Where n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G1 and G2 are each independently C1-C10 alkylene groups; R1, R2, R3, and R4 are each independently H, a straight-chain or branched alkane group of C1-C20, or a straight-chain or branched olefin group of C2-C20. G3 is a C1-C10 alkylene group; or G3 is (CH2). a -O-(CH2)b Where a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10; L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-; L2 is either -NH(C=O)O- or -O(C=O)NH-. The “C1-C20 straight-chain or branched alkane group”, “C2-C20 straight-chain or branched olefin group”, and “C1-C10 alkylene group” mentioned in this invention are respectively as described in Chinese patent application CN115947671A.

[0047] ,

[0048] and

[0049] As stated above. In some specific embodiments of the present invention, the ionizable lipids -CH(R1)R2 and -CH(R3)R4 are as described in Chinese patent application CN115947671A.

[0019] ,

[0020] as well as

[0021] As stated above. In some specific embodiments of the present invention, the ionizable lipid is described in Chinese patent application CN115947671A.

[0022] ,

[0023] ,

[0024] and

[0025] As stated above. The term "stereoisomer" refers to isomers that have the same atomic connection order but different spatial arrangements of atoms. The term "tautomer" refers to the phenomenon where the structure of a compound undergoes an equilibrium interconversion between two functional group isomers, and the corresponding isomers are called tautomers. The term "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention existing as free bases or free acids can be converted into pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of the compounds of the present invention can be converted into their free base or acid forms using standard techniques. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange, and containing an amino group. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Other pharmaceutically acceptable salts include those formed by the quaternization of amines using suitable electrophilic agents (e.g., alkyl halides) to form quaternized alkylated amino salts. In some specific embodiments, the aforementioned lipid nanoparticles further comprise any one or more combinations of structural lipids, auxiliary lipids, PEG-lipids, and polymers. The term "structural lipid" refers to a structure that can stabilize the composition, including but not limited to one or more combinations of sterols and their derivatives and non-sterols and their derivatives. In some specific embodiments, the structural lipids include, but are not limited to, one or more combinations of sterols and their derivatives, nonsterols, sitosterol, ergosterol, cholesterol, cholesterolenone, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, coprosterol, α-tocopherol, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholesterol, 5α-coprosterol, cholesteryl-(2′-hydroxy)ethyl ether, cholesteryl-(4′-hydroxy)butyl ether, and 6-ketocholesterol; non-polar analogs such as 5α-cholesterol, cholesterolenone, 5α-cholesterone, and decanoic acid cholesterol ester; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4′-hydroxy)butyl ether. This is not an exhaustive list; the choice of structural lipids is not limited, and any structural lipid can be used in this invention. In some specific embodiments, the structural lipid is one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives. In some specific implementations, the structural lipid is cholesterol. There is no limitation on the type of "auxiliary lipids", but phospholipid lipids are preferred, including but not limited to: one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and myristoyl phosphatidylglycerol. In some specific embodiments, the auxiliary lipid may be selected from: 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). ), 1,2-Coctodecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), dimyristoyl phosphate ethanolamine (DMPE), distearate-phosphatidyl-ethanolamine (DSPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1- Oleoyl-2-cholestyl-sn-glycerol-3-phosphate choline (OchemsPC), 1-O-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docohexanoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearateyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinyl Acyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docohexanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and sphingomyelin are one or more combinations thereof. In some specific implementations, phosphatidylcholine is one or a combination of DSPC, DPPC, DMPC, DOPC, and POPC. In some specific implementations, the auxiliary lipid is phosphatidylcholine, specifically DSPC. In some specific embodiments, the auxiliary lipid is phosphatidylethanolamine, specifically DOPE. The "PEG-lipid" mentioned in this invention generally refers to a conjugate formed by linking PEG (polyethylene glycol) and lipid molecules through chemical bonds. This includes, but is not limited to, PEG-modified phospholipids and derived lipids, exemplified by one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy polyethylene glycol bis(tetradecyl)acetamide. In some specific embodiments, the PEG-lipids include, but are not limited to, PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearate phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, and polyethylene glycol. The following are combinations of one or more of the following: PEG-DMA, PEG distearate glycerol, PEG dipalmitoyl, PEG dioleyl, PEG distearate, PEG diacyl glycamide, PEG dipalmitoyl phosphatidylethanolamine, PEG phosphatidylethanol, PEG phosphatidyl ethylenedimyristyloxypropyl-3-amine, PEG oxypropyl alcoholamine, 1,2-distearate oxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol bis(tetradecyl acetamide) (ALC0159). In some specific implementations, the PEG-lipid is PEG-DMG. In some specific embodiments of the present invention, the weight-average molecular weight of PEG in the PEG-lipid is 1000 to 10000, for example, 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, preferably 2000. In some specific embodiments, the lipid nanoparticles comprise 20-65% of the compound shown in formula (I), 20-60% of the structured lipid, 3-40% of the auxiliary lipid, and 0.1-10% of the PEG-lipid, where % refers to molar percentage. In some specific embodiments, the lipid nanoparticles comprise 35-49% of the compound represented by formula (I), 35-50% of the structured lipid, 5-20% of the auxiliary lipid, and 1-2% of the PEG-lipid, where % refers to molar percentage. In some specific embodiments, one or more different mRNAs encoding antigens or TT epitopes may be formulated in the same lipid nanoparticle (e.g., mRNAs of two HPV antigens and four TT epitopes are formulated in one lipid nanoparticle). In some specific embodiments, one or more different mRNAs encoding antigens or TT epitopes can be formulated separately in individual lipid nanoparticles (each mRNA formulated in a separate lipid nanoparticle). These lipid nanoparticles can then be combined and administered as a single vaccine composition (e.g., containing multiple mRNAs encoding multiple antigens), or they can be administered separately. In some specific embodiments, the aforementioned nucleic acid vaccine also includes pharmaceutically acceptable excipients. These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, intracavitary injection, inhalation administration, implantation administration, etc. Furthermore, the nucleic acid vaccine of the present invention can be formulated as an inhaled nebulizer formulation (e.g., dry powder formulation, aerosol formulation, inhaled mist droplet formulation, etc.), an implantable gel formulation, a microneedle formulation, and can also be formulated as an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is the therapeutically effective amount, for example, about 10 micrograms / kg body weight per day to about 50 milligrams / kg body weight per day. "Pharmaceutical acceptable" means that when a drug is properly administered to animals or humans, it will not produce adverse, allergic, or other adverse reactions. "Pharmaceutical-acceptable excipients" should be compatible with the active ingredient, meaning they can be mixed with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable excipients include sugars such as glucose, mannitol, sucrose, lactose, trehalose, and maltose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; alcohols such as ethanol, propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyophilization protectants; colorants; flavoring agents; tableting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffer solutions, and combinations thereof. These substances are used as needed to improve the stability of the formulation or to help improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally. Furthermore, the nucleic acid vaccine of the present invention can also be used in combination with other therapeutic agents, including but not limited to anti-CTLA4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PD-L1 antibody or its antigen-binding fragment, and combinations thereof. The fourth aspect of this invention provides the use of the aforementioned adjuvant polypeptide sequence or adjuvant nucleic acid molecule or nucleic acid vaccine in the preparation of preventive or therapeutic vaccines / medications. In some specific embodiments, the preventive or therapeutic vaccine / drug may be a vaccine / drug for the prevention / treatment of any one or more of the following diseases: infectious diseases, inflammatory diseases, immune diseases, or tumors. The infectious or inflammatory diseases include, but are not limited to, one or more combinations of enteritis, mesenteric infarction, sepsis, viral hepatitis or hepatitis caused by other infectious agents, acute kidney injury, renal ischemia, glomerulonephritis, gastric ulcer, acute and chronic pancreatitis, dermatitis, eczema, and psoriasis; the infectious diseases include, but are not limited to, one or more combinations of bacterial infection, fungal infection, and viral infection. In some specific embodiments, the infectious diseases include, but are not limited to, diseases caused by one or more combinations of influenza virus, parainfluenza virus, measles virus, mumps virus, herpes virus, adenovirus, respiratory syncytial virus, poliovirus, Coxsackie virus, or echovirus. The immune system diseases mentioned include, but are not limited to, one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, systemic lupus erythematosus nephritis, AIDS, rheumatoid arthritis, inflammatory bowel disease, psoriasis, vasculitis, bronchial asthma, chronic obstructive pulmonary disease, and eosinophilic sinusitis. The tumors include, but are not limited to, one or more of the following: skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma), head and neck cancer, lung cancer (e.g., squamous cell carcinoma of the lung), liver cancer, stomach cancer, esophageal cancer (e.g., squamous cell carcinoma of the esophagus), breast cancer, uterine cancer (e.g., endometrial cancer, uterine sarcoma, and cervical cancer), ovarian cancer, vaginal cancer, vulvar cancer, prostate cancer, testicular cancer, penile cancer, pancreatic cancer, peritoneal cancer, intestinal cancer (e.g., rectal cancer, colon cancer, and small bowel cancer), kidney cancer, urinary tract cancer (e.g., bladder cancer, ureteral cancer, renal pelvis cancer, and urethral cancer), pharyngeal squamous cell carcinoma, tongue cancer, malignant lymphoma, laryngeal squamous cell carcinoma, small cell carcinoma, and brain tumor; preferably, the tumor is an HPV-positive tumor. In some specific embodiments, the preventive or therapeutic vaccine / drug is a product for the prevention or treatment of HPV-related diseases. In some specific embodiments, the HPV virus may be selected from one or more of the following subtypes: HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV35, HPV39, HPV45, HPV51, HPV56, HPV59, HPV66, HPV26, HPV53, HPV68, or HPV73. Preferably, the HPV virus is HPV16. In some specific embodiments, the HPV-related disease is an HPV-positive tumor. The tumor includes, but is not limited to, one or more of the following: head and neck squamous cell carcinoma (HNSCC), cervical cancer, vaginal cancer (such as vaginal squamous cell carcinoma and adenocarcinoma), vulvar squamous cell carcinoma, penile cancer, anal squamous cell carcinoma (ASCC), oral squamous cell carcinoma (OPSCC), nasopharyngeal carcinoma (NPC), oropharyngeal squamous cell carcinoma (OPSCC), hypopharyngeal squamous cell carcinoma (HPSCC), and laryngeal squamous cell carcinoma (LSCC). In some specific embodiments, the nucleic acid vaccine is an mRNA vaccine. The present invention also provides a method for treating a disease, wherein the method comprises administering an effective amount of the aforementioned adjuvant polypeptide sequence, the aforementioned adjuvant nucleic acid molecule, or the aforementioned nucleic acid vaccine to a subject. In some specific embodiments, the route of administration can vary and includes, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional, percutaneous, tracheal, intraperitoneal, intraarterial, intravesical, intraocular, intratumoral, intraocular, intratumoral, inhalation, infusion, lavage, and oral administration and formulation. Treatment regimens may also vary and are generally dependent on the type of disease, the site of disease, the progression of disease, and the patient's health condition and age. In some specific embodiments, the object can be a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, or humans. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, felines such as domestic cats, canines such as dogs, foxes, wolves, and bird species. Preferably, the object is a human. In this invention, the term "peptide epitope" generally refers to a short protein sequence that can be recognized by the immune system and trigger an immune response. In molecular immunology, a peptide epitope is part of an antigen that can specifically bind to receptors on the surface of immune cells, such as receptors on B cells or T cells. This binding can activate immune cells, thereby initiating an immune response. In this invention, the term "open reading frame" or "ORF" generally refers to a segment or region of an mRNA molecule encoding a polypeptide. An ORF comprises a continuous, non-overlapping in-frame codon that begins with a start codon and ends with a stop codon, and is translated by the ribosome. In this invention, the term "identity" generally refers to the relationship between sequences of two or more polypeptides or polynucleotides, as determined by sequence comparison. In the art, identity also refers to the degree of sequence relevance between them, as determined by the number of matches between segments having two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between two or more sequences relative to a smaller sequence, employing vacancy alignment processed by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be readily calculated by known methods. "Identity %" is defined, when applied to polypeptide or polynucleotide sequences, as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to residues in the amino acid or nucleic acid sequence of a second sequence after sequence alignment and, if necessary, the introduction of vacancy to achieve the maximum identity percentage. Methods and computer programs used for alignment are well known in the art. It should be understood that identity depends on the calculation of the identity percentage but can differ numerically due to vacancy and penalty introduced in the calculation. Typically, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity with that particular reference polynucleotide or polypeptide, as determined by sequence alignment procedures and parameters described herein and known to those skilled in the art. In this invention, the term "MHC class I transport domain" generally refers to the transmembrane and cytoplasmic domains of MHC class I molecules, which can be recognized by CD8+ T cells (also known as cytotoxic T cells) and bind to MHC class I molecules through their T cell receptors (TCRs). The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention. The sequence information used in this application is as follows: P2 amino acid sequence: P16 amino acid sequence: P30 amino acid sequence: PX amino acid sequence: MITD amino acid sequence: E6 amino acid sequence: E7 amino acid sequence: T4S amino acid sequence: (E6E7P2P16P30PX) E6-7T4M amino acid sequence: (E6E7P2P16P30PXMITD) E6 T2M amino acid sequence: (E6P2P16MITD) E7 T2M amino acid sequence: (E7P2P16MITD) E6-7T4M Mutant amino acid sequence: (E6E7MutantP2P16P30PXMITD) mE6E7P21630 amino acid sequence: mE6E7P216X amino acid sequence: SEQ ID No. 14: E6P21630 amino acid sequence: E7P21630 amino acid sequence: T4S mRNA sequence: E6-7T4M mRNA sequence: E6 T2M mRNA sequence: E7 T2M mRNA sequence: E6-7T4M Mutant mRNA sequence: mE6E7P21630 mRNA sequence: mE6E7P216X mRNA sequence: E6P21630 mRNA sequence: E7P21630 mRNA sequence: Example 1: Preparation of HPV16 vaccine antigen mRNA-LNP The mRNA of this invention can be prepared using methods known in the art, including but not limited to chemical synthesis or in vitro transcription, and the sequence correctness is confirmed by sequencing. Then, the mRNA undergoes transformation, E. coli fermentation culture, plasmid extraction, and digestion with the restriction endonuclease BsaI to obtain a linearized plasmid template. Subsequently, under the action of T7 RNA polymerase, an in vitro transcription synthesis reaction was completed using a GAG cap analog and 100% N1M-pseudouridine-5′-triphosphate. 100 μL of the prepared IVT reaction solution (GTP 5 mM, CTP 5 mM, ATP 5 mM, N1M MTP 5 mM, GAG 4 mM, 1X transcription buffer, 1 μg plasmid template, T7 RNA polymerase 2.5 U / μL) was placed in a PCR instrument at 37°C for 3 h. After the IVT reaction, 5 μL of DNase I was added, and the instrument was placed back in the PCR instrument at 37°C for 15 min. After the DNase I digestion reaction, the mRNA was recovered using an RNA purification kit and eluted with 500 μL of DNase / RNase-free purified water. The concentration of the purified mRNA was determined using Nano Drop assay to confirm the IVT yield, ultimately yielding expressible mRNA. The synthesized mRNA was stored at -80°C for future use. Preparation of mRNA-LNP: Step 1: Dissolve the ionizable lipid compound (Lipid), DSPC, cholesterol, and PEG-lipid in ethanol at a molar percentage of 46.3 / 9.4 / 42.7 / 1.6 to prepare a lipid-ethanol solution (Lipid concentration 20 mg / mL). Step 2: Prepare mRNA at a lipid nanoparticle to mRNA mass ratio of 10:1 to 30:1, and dilute the mRNA to 0.2 mg / mL using citrate or sodium acetate buffer (pH = 3 or 5). Step 3: Thoroughly mix the lipid-ethanol solution obtained in Step 1 with the mRNA solution at a volume ratio of 1:5 to 1:1. The obtained nanoparticles are purified by ultrafiltration and dialysis. After filtration and sterilization, the average particle size and PDI of the mRNA-LNP are characterized, and the encapsulation efficiency of the mRNA is determined using an RNA quantification kit. The lipid nanoparticles in this example can form stable nanostructures with a narrow size distribution. The size varies with the structure of different lipid nanoparticles, ranging from 60 to 120 nm. Example 2: Validation of HPV16 vaccine antigen mRNA transfection expression The mRNA prepared in Example 1 was transfected into cells to detect the expression of the target protein. HEK293 cells were seeded at an appropriate density in 24-well plates, and after 24 hours, the cell monolayer density reached approximately 80%. The mRNA was transfected at a concentration of 1 μg / ml using Lipofectamine 3000. The culture medium was replaced with normal medium 5 hours after transfection. Cells were harvested and lysed after 24-48 hours to obtain the cell-derived protein. After verifying the expression of the target product, subsequent experiments were performed. Example 3: Efficacy validation of E6-7T4M mRNA-LNP and Control E6 / E7 T2M vaccines Female C57BL / 6 mice aged 4–6 weeks were subcutaneously injected with 0.1 ml of a solution containing 5 × 10⁻⁶ mg / L of iodine. 5 TC-1 tumor cells were randomly divided into groups (n=6) after the tumors grew to a suitable size. Immunization was performed via intramuscular injection of E6-7T4M mRNA-LNP (containing nucleic acids with nucleotide sequences as shown in SEQ ID NO.18, which translate to the polypeptide shown in SEQ ID NO.9), Control E6 / E7 T2M mRNA-LNP (i.e., a combination of E6 T2M mRNA-LNP and E7 T2M mRNA-LNP, containing nucleic acids with nucleotide sequences as shown in SEQ ID NO.19 and 20 respectively, which translate to the polypeptides shown in SEQ ID NO.10 and 11), or a saline blank control. The immunization dose was 1 μg / mouse / dose, administered three times at 7-day intervals, with 6 mice per group. Tumor volume was measured twice weekly after administration, and the values ​​were recorded. Animals were euthanized within 28 days of the first administration. Spleen, tumor tissue, and serum were collected and stored in pre-cooled RPMI 1640 for subsequent analysis. In all experimental groups of mice, tumor inhibition was observed. As shown in Figure 1, E6-7T4MmRNA-LNP and Control E6 / E7 T2M vaccine showed better tumor inhibition, while the saline group showed normal tumor growth. Example 4: Humoral and cellular immunity detection after E6-7T4M mRNA-LNP immunization The E7 antigen was coated onto an ELISA plate, blocked, washed three times, and patted dry. Then, serially diluted E6-7T4MmRNA-LNP vaccine or mouse serum immunized three times with saline was added. After 1 hour of incubation, the plate was washed three times, patted dry, and HRP-labeled Goat anti-mouse IgG antibody was added. After 45 minutes of incubation, the plate was washed three times, patted dry, and chromogenic solution was added. Data were recorded after 6-10 minutes. As shown in Figure 2a, this vaccine induced significantly better specific antibodies against the E7 antigen than the Model group. Splenic lymphocytes were isolated by grinding the spleen and suspended in 1640 medium. HPV16 antigen peptide library was added to a final concentration of 1 μg / ml, and the cells were seeded at a concentration of 5 × 10⁶ cells / ml. 5 Cells were cultured in 96-well U-shaped plates at 37°C for 24 hours in a 5% CO2 incubator. Afterward, cells were incubated with antibodies using flow cytometry to detect CD45 levels. + / CD3 + / CD8 + Cells account for CD45 + Cell specific gravity, and CD45 were also detected. + / CD3 + / CD8 + / IFN-γ + The proportion of CD45+ cells among CD8+ cells is shown in Figures 2b and 2c. This vaccine can induce an increase in CD45+ cells. + / CD3 + / CD8 + and CD45 + / CD3 + / CD8 + / IFN-γ + The cells were significantly better than the Model group. Example 5: Pharmacological validation of E6-7T4M mRNA-LNP and T4S mRNA-LNP Female C57BL / 6 mice aged 4–6 weeks were subcutaneously injected with 0.1 ml of a solution containing 5 × 10⁻⁶ mg / L of iodine. 5 TC-1 tumor cells were randomly divided into groups (n=6) after the tumors grew to a suitable size. Mice were immunized intramuscularly with E6-7T4M mRNA-LNP, T4S mRNA-LNP (containing the nucleotide sequence shown in SEQ ID NO.17, which translates to the polypeptide shown in SEQ ID NO.8), or a saline control. The immunization dose was 1 μg / mouse / dose, administered three times at 7-day intervals, with 6 mice in each group. Tumor volume was measured twice weekly after administration, and the values ​​were recorded. Animals were euthanized within 28 days of the first administration. Spleen, tumor tissue, and serum were collected and stored in pre-cooled RPMI 1640 for subsequent analysis. As shown in Figure 3, tumor inhibition was observed in all experimental groups. E6-7T4M mRNA-LNP and E6-7T4S vaccines showed superior tumor inhibition, while the saline group showed normal tumor growth. Example 6: Detection of humoral and cellular immunity after E6-7T4M mRNA-LNP and T4S mRNA-LNP immunization The E7 antigen was coated onto an ELISA plate, blocked, washed three times, and patted dry. Then, serially diluted E6-7T4M mRNA-LNP, T4S mRNA-LNP vaccines, or serum from mice immunized three times with saline were added. After 1 hour of incubation, the plates were washed three times, patted dry, and HRP-labeled Goat anti-mouse IgG antibody was added. After 45 minutes of incubation, the plates were washed three times, patted dry, and chromogenic buffer was added. Data were recorded after 6-10 minutes. As shown in Figure 4a, the E6-7T4M mRNA-LNP and E6-7T4S mRNA-LNP vaccines induced significantly better specific antibodies against the E7 antigen than the Model group. Splenic lymphocytes were isolated by grinding the spleen and suspended in 1640 medium. HPV16 antigen peptide library was added to a final concentration of 1 μg / ml, and the cells were seeded at a concentration of 5 × 10⁶ cells / ml. 5 Cells were cultured in 96-well U-shaped plates at 37°C for 24 hours in a 5% CO2 incubator. Afterward, cells were incubated with antibodies using flow cytometry to detect CD45 levels. + / CD3 + / CD8 + Cells account for CD45 + Cell specific gravity, and CD45 were also detected. + / CD3 + / CD8 + / IFN-γ + Cells account for CD8 + Cellular specific gravity, as shown in Figures 4b and 4c, indicates that E6-7T4M mRNA-LNP and T4S mRNA-LNP can induce elevated CD45 levels. + / CD3 + / CD8 + and CD45 + / CD3 + / CD8 + / IFN-γ + The cells were significantly better than the Model group. Example 7: Antigen-specific cellular immunoassay after E6-7T4M mRNA-LNP immunization Splenic lymphocytes were isolated by grinding the spleen and suspended in 1640 medium. HPV16 antigen peptide library was added to a final concentration of 1 μg / ml, and the cells were seeded at a concentration of 5 × 10⁶ cells / ml. 5 Cells were cultured in 96-well U-shaped plates at 37°C for 24 hours in a 5% CO2 incubator. Afterward, cells were incubated with antibodies using flow cytometry to detect CD45 levels. + / CD3 + / CD8 + / H-2Db(RAHYNIVTF) positive cells account for a significant proportion of CD8 + As shown in Figure 5, E6-7T4M mRNA-LNP can induce elevated CD45 levels. + / CD3 + / CD8 + and CD45 + / CD3 + / CD8 + / H-2Db(RAHYNIVTF) + The cells were significantly better than the Model group. Example 8: Validation of the efficacy of E6-7T4M Mutant mRNA-LNP after antigen mutation Female C57BL / 6 mice aged 4–6 weeks were subcutaneously injected with 0.1 ml of a solution containing 5 × 10⁻⁶ mg / L of iodine. 5 TC-1 tumor cells were randomly divided into groups (n=15) after the tumors grew to a suitable size. Mice were immunized intramuscularly with either E6-7T4M mutant mRNA-LNP (containing the nucleotide sequence shown in SEQ ID NO.21, which translates to the polypeptide shown in SEQ ID NO.12) or a saline control, with a dose of 1 μg / mouse / dose, administered three times at 7-day intervals, with 6 mice per group. Tumor volume was measured twice weekly after administration and the values ​​were recorded. Tumor measurements were stopped within 28 days after the first administration, and mouse survival was observed. Mice reaching the ethical endpoint were euthanized; those not reaching the endpoint were observed until day 60 of model initiation. As shown in Figure 6a, tumor inhibition was observed in all experimental groups. The E6-7T4M mutant mRNA-LNP vaccine group showed significant tumor inhibition, while the saline group showed normal tumor growth. As shown in Figure 6b, the mice treated with E6-7T4M mutant mRNA-LNP had significantly better survival at 60 days than the model group. Example 9: Validation of other mRNA-LNP efficacy Female C57BL / 6 mice aged 4–6 weeks were subcutaneously injected with 0.1 ml of a solution containing 5 × 10⁻⁶ mg / L of iodine. 5TC-1 tumor cells were randomly divided into groups (n=15) after the tumors grew to a suitable size. The mice were immunized via intramuscular injection with mE6E7P21630 mRNA-LNP (containing the nucleotide sequence shown in SEQ ID NO.22, which translates to the polypeptide shown in SEQ ID NO.13), mE6E7P216X mRNA-LNP (containing the nucleotide sequence shown in SEQ ID NO.23, which translates to the polypeptide shown in SEQ ID NO.14), E6P21630 / E7P21630 mRNA-LNP (containing the nucleotide sequences shown in SEQ ID NO.24 and 25, which translates to the polypeptides shown in SEQ ID NO.15 and 16), or a saline blank control. The immunization dose was 1 μg / mouse / dose, administered three times at 7-day intervals, with 6 mice per group. Tumor volume was measured twice weekly after administration, and the values ​​were recorded. Tumor measurements were completed within 28 days after the first administration, and mouse survival was observed. Mice that reached the ethical endpoint were euthanized, while those that did not were observed until day 50 of the modeling process. As shown in Figure 7, tumor inhibition was observed in all experimental groups. The mE6E7P21630 mRNA-LNP and mE6E7P216X mRNA-LNP vaccine groups showed significant tumor inhibition, while the saline group showed normal tumor growth. As shown in Figure 8, the mice treated with mE6E7P21630 mRNA-LNP, mE6E7P216X mRNA-LNP, and E6P21630 / E7P21630 mRNA-LNP had significantly better survival at 50 days than the Model group. The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An adjuvant polypeptide sequence, characterized in that, The adjuvant polypeptide sequence contains the amino acid sequences of tetanus toxoid epitopes P2 and P16, and also contains the amino acid sequences of P30 and / or PX.

2. The adjuvant polypeptide sequence according to claim 1, characterized in that, The adjuvant polypeptide sequence also includes an amino acid sequence containing an MHC class I transport domain.

3. The adjuvant polypeptide sequence according to claim 1, characterized in that, The adjuvant polypeptide sequence comprises one or more of the following features: 1) The amino acid sequence of the tetanus toxoid epitope P2 is as shown in SEQ ID No. 1, or is an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the amino acid sequence shown in SEQ ID No. 1; 2) The amino acid sequence of the tetanus toxoid epitope P16 is as shown in SEQ ID No. 2, or is an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the amino acid sequence shown in SEQ ID No. 2; 3) The amino acid sequence of the tetanus toxoid epitope P30 is as shown in SEQ ID No. 3, or is an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the amino acid sequence shown in SEQ ID No. 3; 4) The amino acid sequence of the tetanus toxoid epitope PX is as shown in SEQ ID No. 4, or is an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the amino acid sequence shown in SEQ ID No.

4. 5) The amino acid sequence of the MHC class I transport domain is as shown in SEQ ID No. 5, or is an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the amino acid sequence shown in SEQ ID No.

5.

4. An adjuvant nucleic acid molecule, characterized in that, The adjuvant nucleic acid molecule comprises a nucleotide fragment encoding a polypeptide sequence as described in claims 1-3, the nucleotide fragment being located in one or more nucleic acid molecules.

5. A nucleic acid vaccine, characterized in that, The nucleic acid vaccine comprises mRNA and lipid nanoparticles, wherein the mRNA is selected from one of the following: I) Multiple mRNAs, each having one or more open reading frames encoding 1-500 peptide epitopes and the adjuvant nucleic acid molecule of claim 4, wherein the open reading frames encoding 1-500 peptide epitopes and the adjuvant nucleic acid molecule of claim 4 are not on the same mRNA; II) One or more mRNAs, each having one or more open reading frames encoding 1-500 peptide epitopes, wherein the mRNA further comprises the adjuvant nucleic acid molecule of claim 4.

6. The peptide epitope according to claim 5 is a human papillomavirus antigen; preferably, the peptide epitope is selected from one or more of the following antigens: HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV35, HPV39, HPV45, HPV51, HPV56, HPV59, HPV66, HPV26, HPV53, HPV68, or HPV73 antigens; more preferably, the HPV16 antigen is HPV16 E6 and / or E7 protein; more preferably, the amino acid sequence of the E6 protein is as shown in SEQ ID No. 6, or is an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No. 6; more preferably, the amino acid sequence of the E7 protein is as shown in SEQ ID No.

6. The amino acid sequence shown in No. 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence shown in SEQ ID No.

7.

7. The nucleic acid vaccine according to claim 5, characterized in that, The lipid nanoparticles also comprise one or more combinations of compounds of formula (I), pharmaceutically acceptable salts thereof, or isomers thereof: Where n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G1 and G2 are each independently C1-C10 alkylene groups; R1, R2, R3, and R4 are each independently H, a straight-chain or branched alkane group of C1-C20, or a straight-chain or branched olefin group of C2-C20. G3 is a C1-C10 alkylene group; or G3 is (CH2). a -O-(CH2) b Where a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10; L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-; L2 is either -NH(C=O)O- or -O(C=O)NH-.

8. The use of the adjuvant polypeptide sequence according to any one of claims 1-3, the adjuvant nucleic acid molecule according to claim 4, or the nucleic acid vaccine according to any one of claims 5-7 in the preparation of preventive or therapeutic drugs.

9. The application according to claim 8, characterized in that, The preventive or therapeutic drug is a vaccine / drug for the prevention / treatment of one or more of the following diseases: infectious diseases, inflammatory diseases, immune system diseases, or tumors; preferably, the drug is an HPV-related disease prevention or treatment product; preferably, the HPV virus is selected from one or more of the following subtypes: HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV35, HPV39, HPV45, HPV51, HPV56, HPV59, HPV66, HPV26, HPV53, HPV68, or HPV73; more preferably, the HPV virus is HPV16.

10. The application according to claim 8, characterized in that, The tumor is selected from one or more of the following: skin cancer, head and neck cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, breast cancer, uterine cancer, ovarian cancer, vaginal cancer, vulvar cancer, prostate cancer, testicular cancer, penile cancer, pancreatic cancer, peritoneal cancer, intestinal cancer, kidney cancer, urinary tract cancer, pharyngeal squamous cell carcinoma, tongue cancer, malignant lymphoma, laryngeal squamous cell carcinoma, small cell carcinoma, and brain tumor; preferably, the tumor is an HPV-positive tumor.

11. A method for treating a disease, characterized in that, The method involves administering to a subject the commercially available adjuvant polypeptide sequence of any one of claims 1-3, the adjuvant nucleic acid molecule of claim 4, or the nucleic acid vaccine of any one of claims 5-7.

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