Polynucleotide molecules for preventing or treating diseases associated with HPV infection

Abstract

The present invention relates to polynucleotides that can be used for the prevention or treatment of diseases associated with HPV infection, as well as mRNA vaccines, pharmaceutical compositions or pharmaceutical products containing such polynucleotides.

Description

[Technical Field]

[0001] The present application relates to the field of biotechnology, and in particular to mRNA vaccines for treating diseases associated with HPV infection by inducing HPV antigen-specific immune responses. [Background technology]

[0002] Diseases caused by high-risk human papillomavirus (HPV) infection account for 5% of all diseases worldwide, and 70% of cervical cancers are caused by persistent infection with HPV types 16 and 18. The HPV genome contains up to seven early genes (E1-E7) and two late genes (L1 and L2). The E6 and E7 proteins are expressed in almost all cervical cancer cells and are required for maintaining the disease phenotype, making them ideal target proteins for therapeutic vaccines.

[0003] HPV therapeutic vaccines currently under research mainly include DNA vaccines, subunit vaccines, and recombinant vector vaccines. mRNA vaccines have the advantages of being free from the risk of integration, having a short half-life, and being highly safe. They can induce antigen-specific immune responses by expressing viral antigens, killing infected cells (e.g., tumor cells), and thus achieving the goal of treating associated tumors. The purpose of this application is to prepare an mRNA vaccine for the treatment of HPV infection-related diseases. Summary of the Invention [Problem to be solved by the invention]

[0004] The present application provides nucleic acids and fusion polypeptides for the prevention or treatment of diseases associated with HPV infection, pharmaceutical compositions or products comprising such therapeutic nucleic acids or fusion polypeptides, and uses of such nucleic acids and fusion polypeptides. [Means for solving the problem]

[0005] Specifically, on the one hand, the present application provides a polynucleotide molecule comprising a coding sequence for at least an HPV antigenic polypeptide, said antigenic polypeptide comprising, in order from N-terminus to C-terminus, at least the following: 1) amino acid sequence A and amino acid sequence B; 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B; 3) amino acid sequence B and amino acid sequence A; 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A; 5) amino acid sequence A, amino acid sequence B, and amino acid sequence C; 6) amino acid sequence B, amino acid sequence A, and amino acid sequence C; 7) amino acid sequence A, amino acid sequence C, and amino acid sequence B; or 8) amino acid sequence B, amino acid sequence C, and amino acid sequence A; the amino acid sequence A comprises, in order from the N-terminus to the C-terminus, SEQ ID NOs: 1, 2, 3, 4, or variants thereof, and each of the amino acid sequences represented by such SEQ ID NOs: are linked directly or via a linker; amino acid sequence B comprises, in order from the N-terminus to the C-terminus, SEQ ID NOs: 5, 6, 7, 8 or variants thereof, and each of the amino acid sequences represented by such SEQ ID NOs: are linked directly or via a linker; Amino acid sequence C provides a polynucleotide molecule that includes the HPV E2 antigen sequence.

[0006] In some embodiments, the variants are conservative substitution variants. In some embodiments, each amino acid sequence of the variants of SEQ ID NOS: 1-4 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5% or more sequence identity to one of the amino acid sequences of SEQ ID NOS: 1-4, respectively. In some embodiments, each amino acid sequence of the variants of SEQ ID NOS: 5-8 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5% or more sequence identity to one of the amino acid sequences of SEQ ID NOS: 5-8, respectively.

[0007] In some embodiments, the amino acid sequence C is an HPV E2 antigen sequence.

[0008] In some embodiments, the HPV E2 antigen sequence is SEQ ID NO:9 or a variant thereof. In some embodiments, the variant of SEQ ID NO:9 is a conservative substitution variant. In some embodiments, the variant of SEQ ID NO:9 has an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% sequence identity to SEQ ID NO:9.

[0009] In some embodiments, the linker peptide comprises one, two, or more amino acid residues. In some embodiments, the linker peptide is a flexible linker peptide, a rigid linker peptide, or a combination thereof. In some embodiments, each of the amino acid sequences set forth in each SEQ ID NO: is linked via a different linker peptide. In some embodiments, each of the amino acid sequences set forth in each SEQ ID NO: is linked via the same linker peptide. In some embodiments, the linker peptide consists of 2 to 10 amino acid residues. In some embodiments, the amino acid residues are glycine residues, serine residues, and / or alanine residues. In some embodiments, the linker peptide is selected from a GS linker, (Gly)8, an alpha helix peptide fragment, (XP)n, etc. In some embodiments, each of the amino acid sequences set forth in a SEQ ID NO: is linked via two alanine residues.

[0010] In some embodiments, the HPV antigenic polypeptide comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or comprises an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the HPV antigenic polypeptide comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or comprises an amino acid sequence having 98%, 98.5%, 99%, and 99.5% or greater sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the HPV antigenic polypeptide is SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0011] In some embodiments, the polynucleotide molecule further comprises a coding sequence for an immune activator or a functional domain thereof. In some embodiments, the coding sequence for the immune activator or a functional domain thereof is located at the 3' or 5' end of the coding sequence for the HPV antigenic polypeptide. In some embodiments, the immune activator is selected from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, pre-IFNα-2β, MIP-α, STING, HSP70, and an immune checkpoint inhibitor. In some embodiments, the immune activator is an antibody or antigen-binding fragment thereof that targets any one or more of the following checkpoint molecules: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, PD-1, or VSIG8. In some embodiments, the immune activator is Flt3L. In some embodiments, the immune activator peptide sequence comprises at least the amino acid sequence of SEQ ID NO: 10, a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 10. In some embodiments, the immune activator peptide sequence is the amino acid sequence of SEQ ID NO: 10, a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 10.In some embodiments, the coding sequence of the immune activator comprises or is the polynucleotide sequence of SEQ ID NO: 29. In some embodiments, the coding sequence of the immune activator is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 29.

[0012] In some embodiments, the coding sequence for the immune activator comprises or is a synonymous variant of SEQ ID NO:29.

[0013] In some embodiments, the polynucleotide molecule further comprises a signal peptide coding sequence. In some embodiments, the signal peptide coding sequence is located at the 5' end of the coding sequence for the HPV antigenic polypeptide. In some embodiments, the signal peptide is a secretory signal peptide. In some embodiments, the secretory signal peptide is selected from signal peptides of mammalian secretory proteins. In some embodiments, the mammal is a human. In some embodiments, the secretory signal peptide is tPA-SP. In some embodiments, the secretory signal peptide comprises the amino acid sequence of SEQ ID NO: 11, or a conservative substitution variant of SEQ ID NO: 11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 11. In some embodiments, the secretory signal peptide is the amino acid sequence set forth in SEQ ID NO: 11, or a conservative substitution variant of SEQ ID NO: 11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 11. In some embodiments, the coding sequence for the secretory signal peptide comprises or is the polynucleotide sequence of SEQ ID NO: 28. In some embodiments, the coding sequence for the secretory signal peptide is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 28.

[0014] In some embodiments, the coding sequence for the secretory signal peptide comprises or is a synonymous variant of SEQ ID NO:28.

[0015] In some embodiments, the polynucleotide molecule comprises the coding sequences for the signal peptide, the immunoactivator, and the HPV antigen polypeptide, linked in order from the 5' end to the 3' end. In some embodiments, the coding sequence for the signal peptide, the immunoactivator, and the HPV antigen polypeptide are linked directly or via a polynucleotide chain. In some embodiments, the polynucleotide chain comprises three or a multiple of three nucleotides. Alternatively, in some embodiments, the polynucleotide molecule consists of the coding sequence for the signal peptide, the immunoactivator, and the HPV antigen polypeptide, linked in order from the 5' end to the 3' end. In some embodiments, the coding sequence for the signal peptide, the immunoactivator, and the HPV antigen polypeptide are located in the same reading frame. In some embodiments, the coding sequence of the reading frame is any one of the polynucleotide sequences set forth in SEQ ID NOs: 47-54, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to any one of the nucleotide sequences set forth in SEQ ID NOs: 47-54. In some embodiments, the reading frame encodes any one of the proteins set forth in SEQ ID NOs: 17-21, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to any one of the proteins set forth in SEQ ID NOs: 17-21.

[0016] In some embodiments, the polynucleotide molecule is DNA, RNA, or a hybrid of DNA and RNA. In some embodiments, the polynucleotide molecule is extracted from a cell. In some embodiments, the polynucleotide molecule is chemically synthesized. In some embodiments, the polynucleotide molecule is not chemically modified in vitro. In some embodiments, the polynucleotide molecule is chemically modified in vitro. In some embodiments, the chemical modification is selected from one or more of the following: m6A, m1A, m5C, m7G, ac4C, 2'-O-methylation, and pseudouracil substitution.

[0017] In some embodiments, the polynucleotide molecule comprises any one of the polynucleotide sequences selected from SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of any one of the polynucleotide sequences selected from SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of any one of the polynucleotide sequences selected from SEQ ID NOs: 39-54. In some embodiments, the polynucleotide molecule comprises a nucleic acid fragment encoded by any one of the polynucleotide sequences selected from SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule is encoded by any one of the polynucleotide sequences selected from SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule is encoded by any one of the polynucleotide sequences selected from SEQ ID NOs: 39-54. In some embodiments, the polynucleotide molecule comprises a sequence complementary to any one of the polynucleotide sequences selected from SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of a sequence complementary to any one of the polynucleotide sequences selected from SEQ ID NOs: 28-54. In some embodiments, the polynucleotide molecule consists of a sequence complementary to any one of the polynucleotide sequences selected from SEQ ID NOs: 39-54.

[0018] In some embodiments, the polynucleotide molecule further comprises a 5'UTR structure. In some embodiments, the polynucleotide molecule comprises a 3'UTR structure. In some embodiments, the polynucleotide molecule further comprises a 5'UTR structure and a 3'UTR structure. In some embodiments, the 5'UTR structure comprises at least the polynucleotide sequence set forth in SEQ ID NO:22 or SEQ ID NO:25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85%, or 80% sequence identity to SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 5'UTR structure is the polynucleotide sequence set forth in SEQ ID NO:22 or SEQ ID NO:25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85%, or 80% sequence identity to SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 3'UTR structure comprises at least the polynucleotide sequence set forth in SEQ ID NO:23 or SEQ ID NO:26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85%, or 80% sequence identity to SEQ ID NO:23 or SEQ ID NO:26. In some embodiments, the 3'UTR structure is the polynucleotide sequence set forth in SEQ ID NO:23 or SEQ ID NO:26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 85%, or 80% sequence identity to SEQ ID NO:23 or SEQ ID NO:26.

[0019] In some embodiments, the polynucleotide molecule is an mRNA molecule. In some embodiments, some or all of the uridines in the mRNA are pseudouridine or 1-methyl-pseudouridine. In some embodiments, the mRNA further comprises a 5' cap structure. In some embodiments, the types of 5' cap structures are cap0, cap1, and cap2. In some embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the mRNA further comprises a poly(A) tail. In some embodiments, the poly(A) tail sequence comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 adenylic acid residues. In some embodiments, the poly(A) tail sequence comprises up to 500, up to 400, up to 300, up to 200, up to 150, up to 140, up to 130, up to 120, up to 110, up to 100, up to 90, up to 80, up to 70, and up to 60 adenylic acids (As), particularly about 120 As. In some embodiments, the poly(A) tail comprises at least the polynucleotide sequence set forth in SEQ ID NO:24 or SEQ ID NO:27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:24 or SEQ ID NO:27. In some embodiments, the poly(A) tail is the polynucleotide sequence set forth in SEQ ID NO:24 or SEQ ID NO:27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:24 or SEQ ID NO:27.

[0020] Additionally, the present application also provides polynucleotide molecules comprising a polynucleotide sequence complementary to the polynucleotide sequences of the aforementioned polynucleotide molecules.

[0021] In some embodiments, the polynucleotide molecules provided herein can be single-stranded, double-stranded, or circular. In some embodiments, the polynucleotide molecules provided herein include single-stranded and double-stranded structures.

[0022] In a second aspect, the present application also provides a fusion polypeptide. In some embodiments, the fusion polypeptide is encoded by a polynucleotide molecule described in the first aspect. In some embodiments, the fusion polypeptide comprises, in order from N-terminus to C-terminus, at least the following: 1) amino acid sequence A and amino acid sequence B; 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B; 3) amino acid sequence B and amino acid sequence A; 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A; 5) amino acid sequence A, amino acid sequence B, and amino acid sequence C; 6) amino acid sequence B, amino acid sequence A, and amino acid sequence C; 7) amino acid sequence A, amino acid sequence C, and amino acid sequence B; or 8) Amino acid sequence B, amino acid sequence C, and amino acid sequence A. The amino acid sequence A comprises, from the N-terminus to the C-terminus, SEQ ID NOs: 1, 2, 3, 4 or variants thereof, and the amino acid sequences represented by the SEQ ID NOs are linked in order either directly or via a linker peptide; The amino acid sequence B comprises SEQ ID NO: 5, 6, 7, 8 or a variant thereof, and each amino acid sequence shown in the SEQ ID NO: is directly linked or linked via a linker peptide; Such amino acid sequence C includes the HPV E2 antigen sequence.

[0023] Preferably, the variants are conservative substitution variants.

[0024] In some embodiments, the amino acid sequence C is an HPV E2 antigen sequence.

[0025] In some embodiments, the HPV E2 antigen sequence is SEQ ID NO:9.

[0026] In some embodiments, the linker peptide comprises one, two, or more amino acid residues. In some embodiments, the linker peptide is a flexible linker peptide, a rigid linker peptide, or a combination thereof. In some embodiments, the amino acid sequences set forth in each SEQ ID NO are linked via different linker peptides. In some embodiments, the amino acid sequences set forth in each SEQ ID NO are linked via the same linker peptide. In some embodiments, the amino acid sequences set forth in each SEQ ID NO are linked via two alanine residues.

[0027] In some embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or comprises a conservatively substituted variant of the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or comprises an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In some embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or a conservatively substituted variant of, or an amino acid sequence having 98%, 98.5%, 99%, 99.5% or more sequence identity to, the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the fusion polypeptide is the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0028] In some embodiments, the fusion polypeptide further comprises a full-length or functional domain of an immune activator peptide. In some embodiments, the immune activator protein or functional domain thereof is located at the C-terminus or N-terminus of the fusion polypeptide. In some embodiments, the immune activator is chosen from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, pre-IFNα-2β, MIP-α, STING, MHSP70, and an immune checkpoint inhibitor. In some embodiments, the immune activator is an antibody or antigen-binding fragment thereof that targets any one or more of the following checkpoint molecules: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, PD-1, or VSIG8. In some embodiments, the immune activator is Flt3L. In some embodiments, the polypeptide sequence of the immune activator comprises at least the amino acid sequence set forth in SEQ ID NO: 10, or a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 10. In some embodiments, the polypeptide sequence of the immune activator is the amino acid sequence of SEQ ID NO: 10, or a conservatively substituted variant of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 10.

[0029] In some embodiments, the fusion polypeptide further comprises a signal peptide. In some embodiments, the signal peptide is located at the C-terminus or N-terminus of the fusion peptide. In some embodiments, the signal peptide is a secretory signal peptide. In some embodiments, the secretory signal peptide is selected from signal peptides of mammalian secreted proteins. In some embodiments, the mammal is human. In some embodiments, the secretory signal peptide is tPA-SP. In some embodiments, the secretory signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 11, or a conservative substitution variant of SEQ ID NO: 11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 11. In some embodiments, the secretory signal peptide is the amino acid sequence set forth in SEQ ID NO: 11, or a conservative substitution variant of SEQ ID NO: 11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 11.

[0030] In some embodiments, the fusion polypeptide comprises, linked in N- to C-terminal order, the coding sequences for the signal peptide, the immunoactivator, and the fusion polypeptide.

[0031] The present invention also provides HPV E2 antigen polypeptides, polynucleotide molecules encoding such HPV E2 antigen polypeptides, and uses thereof. In some embodiments, the HPV E2 antigen polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the amino acid sequence of the HPV E2 antigen polypeptide is set forth in SEQ ID NO:9. In some embodiments, the HPV E2 antigen polypeptide comprises or consists of a conservative substitution variant of SEQ ID NO:9. Uses of HPV E2 antigen polypeptides include administering them to an individual in need thereof in combination with other HPV antigen polypeptides, or fusion with other HPV antigen polypeptides and then administering them to an individual in need thereof, in order to obtain a stronger immune response against HPV. The stronger immune response refers to an immune response that is stronger than the immune response obtained by an individual when the other HPV antigen polypeptide is administered alone. In some embodiments, the polynucleotide molecule encoding the HPV E2 antigen polypeptide comprises or consists of the polynucleotide sequence set forth in SEQ ID NO:38. In some embodiments, the polynucleotide molecule encoding the HPV E2 antigenic polypeptide comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 38, or a conservatively substituted variant of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO: 38. Uses of the polynucleotide molecules encoding the HPV E2 antigenic polypeptides include combining them with polynucleotide molecules encoding other HPV antigenic polypeptides and administering them to an individual in need thereof, or linking the polynucleotide sequences with sequences of polynucleotide molecules encoding other HPV antigenic polypeptides to form new polynucleotide molecules which are then administered to an individual in need thereof to express the HPV E2 antigenic polypeptide and the other HPV antigenic polypeptide, or a fusion protein of the HPV E2 antigenic polypeptide and the other HPV antigenic polypeptide. In some embodiments, the individual in need thereof is afflicted with cervical cancer.

[0032] A third aspect of the present application provides a delivery vehicle comprising the polynucleotide molecule of the first aspect or the fusion polypeptide of the second aspect. In some embodiments, the delivery vehicle may be a liposome, a viral particle, or a quantum dot. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, an ionizable lipid, or any combination thereof. In some embodiments, the LNP consists of an ionizable lipid, a phospholipid, cholesterol, a polyethylene glycol (PEG)-lipid, and the polynucleotide molecule of the first aspect.

[0033] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, cholesterol, and a PEG lipid, wherein the ionizable lipid content is 35 mol% to 65 mol%, the combined phospholipid and cholesterol content is 35 mol% to 65 mol%, and the PEG lipid content is 0.5 mol% to 5 mol%. In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, cholesterol, and a PEG lipid. In the LNP, the ionizable lipid content is 40 mol% to 50 mol%, the phospholipid content is 10 mol% to 15 mol%, the cholesterol content is 35 mol% to 45 mol%, and the PEG lipid content is 1.5 mol% to 2.5 mol%.

[0034] A fourth aspect of the present application provides a cell comprising the polynucleotide molecule of the first aspect or the fusion polypeptide of the second aspect. In some embodiments, the cell is a bacterial, fungal, or mammalian cell.

[0035] A fifth aspect of the present application provides a pharmaceutical composition, pharmaceutical product, or kit comprising the polynucleotide molecule of the first aspect, the fusion polypeptide of the second aspect, the delivery vehicle of the third aspect, and / or the cell of the fourth aspect. In some embodiments, the pharmaceutical composition or pharmaceutical product is an mRNA vaccine and comprises mRNA from the polynucleotide molecule of the first aspect. In some embodiments, the mRNA is encoded by a polynucleotide selected from SEQ ID NOs: 28-54 or any one of nucleotide sequences having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NOs: 28-54. In some embodiments, the mRNA is encoded by a polynucleotide selected from SEQ ID NOs: 39-54 or any one of nucleotide sequences having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NOs: 39-54.

[0036] In some embodiments, the mRNA further comprises a 5' UTR structure. In some embodiments, the mRNA comprises a 3' UTR structure. In some embodiments, the mRNA further comprises a 5' UTR structure and a 3' UTR structure. In some embodiments, the 5' UTR structure comprises at least the polynucleotide sequence set forth in SEQ ID NO:22 or SEQ ID NO:25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 5' UTR structure is the polynucleotide sequence set forth in SEQ ID NO:22 or SEQ ID NO:25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 3'UTR structure comprises at least the polynucleotide sequence set forth in SEQ ID NO:23 or SEQ ID NO:26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:23 or SEQ ID NO:26. In some embodiments, the 3'UTR structure is the polynucleotide sequence set forth in SEQ ID NO:23 or SEQ ID NO:26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:23 or SEQ ID NO:26.

[0037] In some embodiments, the mRNA molecule of the present application is a mature mRNA molecule and comprises, from the 5' end to the 3' end, a 5' cap, a 5' UTR, a coding sequence for the secretory signal peptide, a coding sequence for the immune activator, a coding sequence for the HPV antigen polypeptide, a 3' UTR, and a polyA tail, wherein the 5' UTR, the coding sequence for the secretory signal peptide, the coding sequence for the immune activator, the coding sequence for the HPV antigen polypeptide, the 3' UTR, and the polyA tail are operably linked to each other. In some embodiments, the 5' cap has the structure m7G(5')ppp(5')(2'-OMeA)pG.

[0038] In some embodiments, the 5' end of the 5' UTR also contains an AGG, AUG, or other nucleotide triplet, or one or two bases on the 5' UTR and its 5' end together form an AGG, AUG, or other nucleotide triplet for use with different capping systems. The start sites required for different capping systems (e.g., Clean Cap AG, Clean Cap AU, etc.) are known in the art and can be routinely selected by one of ordinary skill in the art.

[0039] In some embodiments, the mRNA molecules of the present application comprise a Kozak sequence, hi some particular embodiments, the Kozak sequence comprises GCCACC located 5' to the coding sequence of the secretory signaling peptide.

[0040] In some embodiments, some or all of the uridines in the mRNA are chemically modified uridines, hi some embodiments, some or all of the uridines in the mRNA are pseudouridine or 1-methyl-pseudouridine.

[0041] In some embodiments, some or all of the uracil nucleotides in the mRNA are substituted with pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.

[0042] In some embodiments, the mRNA further comprises a 5' cap structure. In some embodiments, the 5' cap structure is cap0, cap1, and cap2. In some embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the mRNA further comprises a poly(A) tail. In some embodiments, the poly(A) tail sequence comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 adenylate amino acids. In some embodiments, the poly(A) tail sequence comprises up to 500, up to 400, up to 300, up to 200, up to 150, up to 140, up to 130, up to 120, up to 110, up to 100, up to 90, up to 80, up to 70, or up to 60 adenylic acids (As), particularly about 120 As. In some embodiments, the poly(A) tail comprises at least the polynucleotide sequence set forth in SEQ ID NO:24 or SEQ ID NO:27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:24 or SEQ ID NO:27. In some embodiments, the poly(A) tail is the polynucleotide sequence set forth in SEQ ID NO:24 or SEQ ID NO:27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:24 or SEQ ID NO:27.

[0043] In some embodiments, the pharmaceutical composition, pharmaceutical product, or kit further comprises an immune activator and / or adjuvant. In some embodiments, the immune activator is selected from one or more of the following: IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, pre-IFNα-2β, MIP-α, STING, HSP70, immune checkpoint inhibitors, or coding polynucleotides encoding the same. In some embodiments, the STING is a STING V155M In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the encoding polynucleotide is mRNA.

[0044] A sixth aspect of the present application provides a method for treating or preventing HPV infection and diseases associated with HPV infection, comprising administering to an individual the polynucleotide molecule of the first aspect, the fusion polypeptide of the second aspect, the delivery vehicle of the third aspect, the cell of the fourth aspect, or the pharmaceutical composition or pharmaceutical product of the fifth aspect. In some embodiments, the disease associated with HPV infection is cervical cancer. In some embodiments, the administration is by intratumoral or peri-lymph node injection, or intramuscular injection. In some embodiments, the method further comprises administering to the individual an immune activator, chemotherapy, radiation therapy, and / or targeted therapy. In some embodiments, the targeted therapy refers to an antibody or functional domain thereof that targets a specific tumor target in cervical cancer.

[0045] It should be understood that the aspects and embodiments described herein include aspects and embodiments that "comprising," "consisting of," and "substantially consisting of." Preferred embodiments of the present invention have been described above in detail. However, the present invention is not limited thereto. Within the scope of the technical concept of this application, various simple modifications of the technical solutions may be made, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application, and all of them are encompassed within the scope of protection of this application. [Brief explanation of the drawings]

[0046] [Figure 1A] 1 shows the cellular immune responses induced by different doses of HPV-M mRNA vaccine in normal mice. [Figure 1B] 1 shows the cellular immune responses induced by different doses of HPV-M mRNA vaccine in normal mice. [Figure 2A] Antitumor effect of HPV-M mRNA vaccine after intramuscular or intratumoral injection in TC-1 tumor model mice. [Figure 2B] Antitumor effect of HPV-M mRNA vaccine after intramuscular or intratumoral injection in TC-1 tumor model mice. [Figure 3A] Antitumor effect of HPV-M mRNA vaccine after intramuscular or perilymph node injection near the tumor in TC-1 tumor model mice. [Figure 3B] Antitumor effect of HPV-M mRNA vaccine after intramuscular or perilymph node injection near the tumor in TC-1 tumor model mice. [Figure 4] The levels of HPV16 and HPV18 E6- and E7-specific IFNγ and IL-2 induced by two immunizations with five different mRNA vaccines (5 μg), as detected by ELISpot. [Figure 5]Flow cytometric detection of HPV16 and HPV18 E6- and E7-specific T cell responses induced by two immunizations with five different mRNA vaccines (5 μg). [Figure 6] The levels of HPV16 and HPV18 E6- and E7-specific IFNγ and IL-2 induced by three immunizations with four different mRNA vaccines (12.5 μg) were detected by ELISpot. [Figure 7] Flow cytometric detection of HPV16 and HPV18 E6- and E7-specific T cell responses induced by three immunizations with four different mRNA vaccines (12.5 μg). [Figure 8] This shows the antitumor effect of three immunizations (injections) of four different mRNA vaccines (12.5 μg) on ​​TC-1 tumor model mice. [Figure 9A] Antitumor effects of three immunizations of four different mRNA vaccines (12.5 μg) on ​​TC-1 tumor model mice. [Figure 9B] Antitumor effects of three immunizations of four different mRNA vaccines (12.5 μg) on ​​TC-1 tumor model mice. [Figure 10A] This study evaluates the antitumor effect of a combination of HPV-5 mRNA vaccine and PD-L1 antibody. [Figure 10B] This study evaluates the antitumor effect of a combination of HPV-5 mRNA vaccine and PD-L1 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0047] The present application provides novel nucleotide sequences that can be used to prepare prophylactic or therapeutic nucleic acids and fusion polypeptides for HPV infection-related diseases, while the present application also provides pharmaceutical compositions or pharmaceutical products comprising said therapeutic nucleic acids or fusion polypeptides, e.g., mRNA vaccines, and their use for treating diseases.

[0048] definition As used herein, a "coding sequence" may refer to a ribonucleotide sequence that can be translated into a protein in mature mRNA, as well as the complementary sequence of a deoxyribonucleotide (DNA) sequence used as a template for transcribing the ribonucleotide (RNA) sequence. In addition, the "coding sequence" of this application may further include a polynucleotide sequence that encodes a functional nucleic acid, such as miRNA, shRNA, dsRNA, etc.

[0049] As used herein, the term "HPV E2 antigenic sequence" refers to an amino acid sequence that has immunogenicity in the E2 protein derived from HPV. In some embodiments, the HPV E2 antigenic sequence is derived from the E2 protein sequence of any wild-type or artificially mutated HPV subtype, or is a fusion protein of the E2 protein sequences of multiple wild-type or artificially mutated HPV subtypes. In some embodiments, the HPV E2 antigenic sequence is derived from a conserved peptide sequence of the HPV E2 protein, or is a combination of two or more conserved peptide sequences. In some embodiments, the HPV E2 antigenic sequence is a fusion protein of one or more conserved peptide sequences of the HPV E2 protein with one or more specific E2 protein sequences of a wild-type and / or artificially mutated HPV subtype. The conserved peptide sequence may be a peptide sequence conserved within a genotype, i.e., an amino acid sequence conserved in different variants of the E2 protein of a certain HPV subtype. It may also be a peptide sequence conserved between genotypes, i.e., an amino acid sequence conserved in the E2 proteins of multiple HPV subtypes. Methods for obtaining conserved peptide sequences (or conservation evaluation methods) are known in the art. Illustratively, for example, available full-length sequences of E2 proteins from different HPV genotypes can be collected from protein databases such as NCBI and used as raw data input. All available full-length sequences are used to ensure that the selected conserved peptide sequences equally represent the entire environmental population. For example, before conservation evaluation, all genotypes are aligned, and the sequences within each genotype are weighted to ensure equal representation of genetic polymorphisms, thereby ensuring that the HPV E2 antigen sequence candidates represent the entire environmental population.Next, a sliding window of 15 amino acids is used to evaluate intra-genotype conservation (intra-genotype conservation), determining a conservation value for each window based on the commonality of amino acids within the combined window and a weighting of the values ​​for each sequence. This identifies fragments that are conserved within each genotype and within the intra-genotype conserved peptide sequences generated for each window. "Intra-genotype conserved peptide sequences" refers to amino acid sequences that represent a weighted set of genotype sequences, rather than the most common amino acid at each position. To be classified as conserved, a window must have a conservation value within the first quartile of the conservation values ​​of all windows in the protein. Subsequently, conserved intra-genotype windows that are located at the same position across all genotypes are identified (inter-genotype conservation), regardless of the percentage identity of the intra-genotype standardized consensus sequence shared within the genotype. A phylogeny of the resulting regions is then created, and intra-tree group sequences are combined to generate conserved peptide sequences across genotypes with a high level of shared common identity.

[0050] As used herein, a "linker peptide" refers to an amino acid residue or a peptide chain containing two or more amino acid residues that connects two peptide fragments in a fusion protein. In some embodiments, the linker peptide is a flexible linker peptide that allows some flexibility for the two linked amino acid fragments. The addition of Ser and Thr allows hydrogen bonds to form between the linker peptide and water molecules, conferring stability to the linker peptide in aqueous solution and thereby reducing interactions between the linker peptide and the two proteins before and after the linker peptide. A common flexible linker peptide consists of Gly and Ser residues (GS linker). In addition to the GS flexible linker peptide, several other flexible linker peptides are known in the art, such as (Gly)8. In some embodiments, the linker peptide is a rigid linker peptide that can be used to completely separate the two linked proteins and maintain their independent functions. Commonly used rigid linker peptides include α-helical peptide fragments, such as (XP)n, where P represents proline, X can be any amino acid, preferably Ala, Lys, or Glu, and n represents the number of XP repeats. Those skilled in the art can independently tailor and select different linker peptides based on the specific application scenario and the 3D structural requirements of the fusion protein.

[0051] In the present application, the term "5'-end" is used to describe the relative positional relationship between two fragments of the same nucleotide sequence. Here, the "5'-end" refers to the end of the polynucleotide sequence containing a free 5'-hydroxyl group. For example, "further comprising a coding sequence for an immune activator or a functional domain thereof at the 5'-end of the coding sequence for an HPV antigen polypeptide" means that the "coding sequence for the immune activator or a functional domain thereof" is closer to the 5'-end of the common nucleotide sequence than the "coding sequence for the HPV antigen polypeptide."

[0052] The term "signal peptide" refers to a short peptide chain that directs the localization or translocation of a newly synthesized protein. Here, a signal peptide that directs the translocation of a newly synthesized protein into the secretory pathway is also known as a "secretory signal peptide." In most cases, a signal peptide is located at the N-terminus of an amino acid sequence. In mRNA, the coding sequence for the signal peptide is usually located after the start codon and is an RNA region that encodes a hydrophobic amino acid sequence. After directing the protein to complete its localization, the signal peptide is usually cleaved under the action of a signal peptidase. The term "tPA-SP" refers to the tissue plasminogen activator signaling peptide, a secretory signal peptide.

[0053] As used herein, a "hybrid of a DNA sequence and an RNA sequence" is a polynucleotide sequence in which some of the nucleotides that make up the polynucleotide sequence are DNA and some are RNA.

[0054] The term "5' cap" refers to a cap located at the 5' end of an mRNA and containing a methylated guanylate. The methylated guanylate is added to the 5' end of an mRNA via pyrophosphate and forms a 5',5'-triphosphate bond with the adjacent nucleotide. There are three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNp), commonly known as Cap0, Cap1, and CapII, respectively. Cap0 refers to a cap in which the ribose of the terminal nucleotide is unmethylated, Cap1 refers to a cap in which the ribose of one terminal nucleotide is methylated, and CapII refers to a cap in which the ribose of both terminal nucleotides is methylated. Herein, "CleanCap AG" refers to the m7G(5')ppp(5')(2'-OMeA)pG cap.

[0055] As used herein, the term "poly(A) tail" or "poly(A) sequence" refers to an uninterrupted or interrupted sequence of adenylic acid residues typically located at the 3'-end of an RNA molecule. Poly(A) tails or poly(A) sequences are known to those skilled in the art and can be selected according to actual needs. In mRNA, if a 3'-UTR is present, the poly(A) sequence is linked to the 3'-end of the 3'-UTR. An uninterrupted poly(A) tail is characterized by the presence of consecutive adenylic acid residues. The poly(A) tail can be of any length. In some embodiments, the poly(A) tail comprises or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 adenylic acids (As), particularly about 120 As. Typically, the majority of the nucleotides in the poly(A) tail are adenosines, where majority refers to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides, while the remaining nucleotides can be nucleotides other than A, such as U (uridylic acid), G (guanylic acid), or C (cytidylic acid).

[0056] As used herein, a percentage of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity, refers to a degree of similarity between amino acid or nucleotide sequences determined by sequence alignment of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, by introducing gaps and other methods, two sequences can have the same residue at as many positions as possible, and the percentage of positions with the same base or amino acid residue relative to the total number of positions can be determined. The percentage of "identity" can be determined using software programs known in the art. A preferred method is to use default parameters for alignment. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgibin / BLAST.

[0057] As used herein, "complementarity" of a nucleic acid refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. The percentage of complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, and 10 out of 10 are about 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Full complementarity" refers to the formation of hydrogen bonds between all consecutive residues of a nucleic acid sequence and the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to two nucleic acids that hybridize under stringent conditions or with a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% within a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides. With respect to a single base or nucleotide, when A pairs with T or U and C pairs with G or I according to the Watson-Crick base pairing rules, it is referred to as complementary or matching, and vice versa. Other base pairings are referred to as non-complementary. A "complementary polynucleotide sequence" of a polynucleotide sequence herein refers to a polynucleotide sequence that is completely complementary to that particular polynucleotide sequence.

[0058] As used herein, the term "delivery vehicle" refers to a structure that packages or encapsulates large biomolecules, such as polynucleotides and polypeptides, to form a structure that has a high affinity for cell membranes and facilitates their transmembrane transport from the outside to the inside of the cell, thereby helping the biomolecules enter the cell. Delivery vehicles, including but not limited to liposomes (e.g., lipid nanoparticles (LNPs)), viruses (e.g., AAV, lentivirus), and quantum dots, and their preparation methods are known in the art. Methods for preparing LNPs are known in the art and are disclosed, for example, in Chinese Patent Application Publication No. 114901360(A) and Chinese Patent Application Publication No. 113941011(A). In some embodiments, the LNPs contain PEG-modified lipids, non-cationic lipids, sterols, ionizable lipids, or any combination thereof.

[0059] As used herein, the term "immune activator" refers specifically to a protein, peptide, or nucleic acid molecule that can be produced in a mammal and that can enhance the antigen response of the immune system, including, but not limited to, cytokines that enhance immune cell processing and / or antigen presentation capabilities (e.g., dendritic cell growth factor (e.g., Flt3L, etc.)); molecules that subvert immunosuppression, including, but not limited to, immune checkpoint inhibitors; pro-inflammatory cytokines (e.g., granulocyte-macrophage colony-stimulating factor, IFNα-2a, IFNα-2β, Pre-IFNα-2β, IL-2, etc.); and the like.

[0060] As used herein, the term "adjuvant" means an exogenous substance that can be added to a pharmaceutical composition or formulation to enhance the response of an individual's immune system to an antigen, including, but not limited to, chemical adjuvants and bacterial antigens.

[0061] As used herein, "Flt3L" refers to FMS-like tyrosine kinase 3 ligand. In some embodiments of the present application, Flt3L is human Flt3L, for example, Flt3L registered in the NCBI database under gene ID: 2323.

[0062] The term "immune checkpoint" refers to molecules in the immune system that can turn on signals (costimulatory molecules) or turn off signals. Many cancers protect themselves from damage by the immune system by inhibiting T cell signaling. As used herein, the term "immune checkpoint inhibitor" can serve as a protective mechanism to prevent cancer by acting on immune checkpoints. For example, an immune checkpoint inhibitor can be an antibody or antigen-binding fragment thereof that targets any one or more of the following checkpoint molecules: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, or VSIG8, where "PD-1" (Programmed T Cell Death Receptor) is a transmembrane protein found on the surface of T cells. PD-1 binds to PD-L1 (Programmed T Cell Death Ligand 1) on tumor cells, resulting in inhibition of T cell activity and a decrease in T cell-mediated cytotoxicity. PD-1 and PD-L1 are thus immune down-regulators or "off switches" for immune checkpoint signaling.

[0063] As used herein, "immune checkpoint inhibitors" also include agonists of costimulatory molecules, such as CD28, CD122, and CD137. CD28 is constitutively expressed on almost all human CD4+ T cells and approximately half of CD8+ T cells and promotes T cell proliferation. CD122 can increase the proliferation of CD8+ effector T cells. 4-1BB (also known as CD137) is involved in T cell proliferation and can protect T cells, particularly CD8+ T cells, from activation-induced cell death by mediating signal transduction.

[0064] As used herein, "HPV infection-associated disease" refers to any disease caused primarily or partially by human papillomavirus infection. Most HPV infections do not cause symptoms and can resolve spontaneously. However, in some cases, they may persist and lead to the appearance of common warts or precancerous lesions. In this application, "HPV infection-associated disease" includes, but is not limited to, cervical cancer caused or partially caused by HPV. Methods for diagnosing whether a disease is caused or partially caused by HPV are known in the art, such as determining by confirming a history of HPV infection, detecting HPV antigens and / or antibodies in a patient's affected tissue, blood, body fluids, or other relevant tissues or tissue fluids, etc. In this application, HPV can cover any subtype of human papillomavirus, including, but not limited to, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV54, HPV56, HPV58, HPV59, etc.

[0065] As used herein, "mRNA" (messenger RNA) is any RNA that encodes a naturally occurring, non-naturally occurring, or modified amino acid polymer of at least one protein, and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Those of skill in the art will know that unless otherwise specified, the polynucleotide sequences described herein may refer to thymine as "T" when referring to a DNA sequence, but that "T" is replaced by "U" (uridine) when the polynucleotide sequence represents RNA (e.g., mRNA). Accordingly, any DNA set forth and identified in this specification by a particular sequence number (SEQ ID NO:) also discloses the RNA (e.g., mRNA) sequence complementary to or corresponding to that DNA, in which each "T" in the DNA sequence is replaced by a "U."

[0066] As used herein, an "open reading frame (ORF)" is a contiguous DNA or RNA segment that begins with a start codon (e.g., a methionine codon (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins.

[0067] As used herein, the terms "fusion polypeptide" and "fusion protein" are used interchangeably and should be understood to refer to a polypeptide that includes a combination of sequences derived from different gene products (e.g., homologous proteins of different subtypes of HPV, different proteins of the same subtype of HPV, and heterologous proteins of different subtypes of HPV), or from the same gene product (e.g., a single HPV protein), where these sequences are derived from different / distinct regions of the wild-type gene product. For example, a fusion polypeptide can include a combination of sequences that are typically separated by other wild-type sequence fragments, as well as fusions of peptide fragments remaining after removal of one or more sequences.

[0068] As used herein, "comprises at least" means includes or is, for example "From the N-terminus to the C-terminus, at least in order, 1) amino acid sequence A and amino acid sequence B; 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B; 3) amino acid sequence B and amino acid sequence A; or 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A; "Including" means a): From the N-terminus to the C-terminus "1) amino acid sequence A and amino acid sequence B; 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B; 3) amino acid sequence B and amino acid sequence A; or 4) Amino acid sequence C, amino acid sequence B, and amino acid sequence A, and further comprising one or more amino acids between at least two adjacent amino acid sequences (e.g., sequences A and B, sequences A and C, sequences C and B); or b) From the N-terminus to the C-terminus, "1) amino acid sequence A and amino acid sequence B; 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B; 3) amino acid sequence B and amino acid sequence A; or 4) It refers to a sequence including amino acid sequence C, amino acid sequence B, and amino acid sequence A, and not including one or more amino acids between any two adjacent amino acid sequences (e.g., sequences A and B, sequences A and C, sequences C and B).

[0069] As used herein, when "direct linkage" is used to describe the relationship between two amino acid sequences, it means that there are no other amino acids between the two amino acid sequences. In some embodiments, "direct linkage" refers to linking two amino acid sequences via a chemical bond. In some embodiments, "direct linkage" refers to linking two amino acid sequences via a peptide bond (amide bond).

[0070] Polynucleotide sequence In one aspect, the present application provides a polynucleotide sequence that can be used for the prevention or treatment of diseases associated with HPV infection. Through extensive comparison and experimentation, the inventors have finally determined that the polynucleotide sequence of the present application can induce a significant specific immune response against HPV in healthy mice and exhibits significant anti-tumor activity in HPV-positive tumor model mice.

[0071] "Variant" refers to a sequence or molecule that retains the same or substantially the same biological activity as the original sequence. The variant may be from the same species or a different species (e.g., homologous proteins from different variants of the same HPV subtype), or may be a synthetic sequence based on a natural or pre-existing molecule. In this application, "variant" can be used to refer to variants of proteins, peptides, or amino acid sequences, as well as variants of nucleic acid molecules or polynucleotide sequences.

[0072] Those skilled in the art can easily determine the variants of SEQ ID NOS: 1 to 9. For example, by sequence alignment, the positional segment of each of the above amino acid sequences can be determined in the protein amino acid sequence of the corresponding HPV subtype. The sequences in this segment of all variants of HPV subtypes having mutations in the positional segment are all variants corresponding to each of the amino acid sequences.Therefore, the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof comprises the E6 protein amino acid sequence corresponding to the 1st to 85th amino acids of the E6 protein reference sequence in HPV16 subtype variants (NCBI accession number QHA94929 or AAL96630.1); the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof comprises the E7 protein amino acid sequence corresponding to the 1st to 65th amino acids of the E7 protein reference sequence in HPV-16 subtype variants (NCBI accession number ATI99837 or NP:041326.1); the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof comprises the E6 protein amino acid sequence corresponding to the 71st to 158th amino acids of the E6 protein reference sequence in HPV-16 subtype variants; and the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof comprises the E7 protein amino acid sequence corresponding to the 51st to 98th amino acids of the E7 protein reference sequence in HPV-16 subtype variants. the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof comprises the E6 protein amino acid sequence corresponding to the 1st to 85th amino acids of the E6 protein reference sequence for HPV-18 subtype variants (NCBI accession number ABP99784); the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof comprises the E7 protein reference sequence corresponding to the 1st to 65th amino acids of the E7 protein reference sequence for HPV-18 subtype variants (NCBI accession number UZQ21949 or ABP99785.1); the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof comprises the E6 protein amino acid sequence corresponding to the 71st to 158th amino acids of the E6 protein reference sequence for HPV-18 subtype variants; and the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof comprises the E7 protein amino acid sequence corresponding to the 51st to 105th amino acids of the E7 protein reference sequence for HPV-18 subtype variants. On the other hand, the amino acid sequence shown in SEQ ID NO: 9 is formed by fusing multiple E2 proteins from HPV 16, HPV 18, and HPV 31. The inventors of the present application confirmed that the HPV E2 antigen sequence has the effect of enhancing the immunogenicity of HPV proteins.

[0073] In some embodiments, a "variant" of an amino acid sequence has at least one amino acid difference with respect to the amino acid sequence, e.g., at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution may be a conservative amino acid substitution, i.e., a replacement of the original corresponding amino acid with an amino acid having similar properties. "Conservative substitutions" include polar amino acid substitutions for polar amino acids such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); nonpolar amino acid substitutions for nonpolar amino acids such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), and phenylalanine (F, Phe); acidic amino acid substitutions for acidic amino acids such as aspartic acid (D, Asp) and glutamic acid (E, Glu); The variants may be alkaline-for-alkaline amino acid substitutions such as alkaline amino acid substitutions, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys), charged-for-charge amino acid substitutions, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys), and arginine (R, Arg), or hydrophobic-for-hydrophobic amino acid substitutions, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp), and methionine (M, Met). In some other embodiments, the variants may also include non-conservative substitutions. In some embodiments, a "variant" of the amino acid sequence may have at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence.Compared to an amino acid sequence, a "variant" of that amino acid sequence may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or within a range consisting of any two of the foregoing values. As used herein, a "conservative substitution variant" of a protein, polypeptide, or amino acid sequence refers to a variant in which one or more amino acid residues are replaced by an amino acid without altering the overall conformation and function of the protein or enzyme. This includes, but is not limited to, substitutions of amino acids in the amino acid sequence of the parent protein in the manner described above under "conservative substitution." Therefore, the similarity between two proteins or amino acid sequences with similar functions may vary; for example, 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "Conservative substitution variants" also include peptides or enzymes that have 60% or more, preferably 75% or more, more preferably 85% or more, and most preferably 90% or more amino acid identity as determined by BLAST or FASTA algorithms, and which have the same or essentially similar properties or functions compared to the native or parent protein or enzyme.

[0074] Therefore, the HPV antigen polypeptides and fusion polypeptides described herein should include the aforementioned variants.

[0075] Those skilled in the art should recognize that variants of nucleic acid molecules or polynucleotide sequences that encode proteins include "synonymous variants," which refer to nucleic acid molecules or polynucleotide sequences that result from the replacement of one or more codons in the nucleic acid molecule or polynucleotide sequence with other codons that encode the same amino acids.

[0076] The term "comprises at least" means that the polynucleotide sequence may consist of the coding sequence of the above-mentioned HPV antigen polypeptide, or may contain other polynucleotide sequences other than the coding sequence comprising the above-mentioned HPV antigen polypeptide.

[0077] For example, sequences that regulate the expression of the aforementioned HPV antigen polypeptides, sequences that make the polynucleotide more stable, and other polynucleotide sequences that can promote stimulation of an immune response by the aforementioned HPV antigen polypeptides in the subject's body.

[0078] In some embodiments, the other polynucleotide sequence may be a coding sequence for any immune activator, such as Flt3L.

[0079] The polynucleotide sequence can be a DNA sequence, an RNA sequence, or a hybrid of a DNA sequence and an RNA sequence.

[0080] mRNA vaccines The present application also provides an mRNA vaccine for preventing or treating diseases associated with HPV infection, which comprises an RNA sequence selected from the aforementioned polynucleotide sequences. The mRNA vaccine achieves preventive immunity by introducing a sequence comprising a polynucleotide encoding an HPV antigen polypeptide into a subject, directly translating it to form the corresponding antigen protein, and inducing a specific immune response in the body. At the same time, the mRNA vaccine can target and kill tumor cells containing HPV antigens.

[0081] Methods for preparing mRNA vaccines are known in the art. In particular, the mRNA in such mRNA vaccines further contains, in addition to the polynucleotide sequence, coding sequences for multiple functional components necessary for expressing, regulating, or enhancing the expression level of the above-mentioned HPV antigen polypeptides. These functional components include, but are not limited to, a 5' cap, a 5' UTR, a 3' UTR, and a poly(A) tail. These functional components are known in the art, and those skilled in the art can select and combine them according to their actual needs. Both the 5' UTR and the 3' UTR are elements typically present in immature mRNA (also known as precursor mRNA or pre-mRNA) transcribed from genomic DNA. Characteristic structural features of mature mRNA (e.g., a 5' cap and a 3' poly(A) tail) are typically added to the transcribed (immature) mRNA during mRNA processing. Thus, in some embodiments, the mRNA is a precursor mRNA. In some embodiments, the mRNA is a mature mRNA.

[0082] In some embodiments, the mRNA vaccine comprises an RNA polynucleotide sequence having an open reading frame encoding at least one antigenic peptide with at least one modification and at least one 5' cap, and is formulated within a lipid nanoparticle. The following chemical RNA cap analogs can be used to simultaneously complete the 5'-capping of the polynucleotide during the in vitro transcription reaction according to the manufacturer's protocol to generate a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA Cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA), or m7G(5')ppp(5')(2'-OMeA)pG (CleanCapAG). The 5' end of modified RNA can be capped after transcription is complete using vaccinia virus capping enzyme to generate the O-type cap structure m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). CapI structures can be generated using both vaccinia virus capping enzyme and 2'-O-methyltransferase to generate m7G(5')ppp(5')(2'-OMeA)pG, and these CapI structures can also be generated by the Cleancap method. CapII structures can be generated from CapI structures by 2'-O-methylating the third-to-last nucleotide of the 5' end using 2'-O-methyltransferase. CapIII structures can be generated from CapII structures by 2'-O-methylating the fourth-to-last nucleotide of the 5' end using 2'-O-methyltransferase.

[0083] A 3'-poly(A) tail is typically added to the 3' end of a transcribed mRNA. In some embodiments, the 3'-poly(A) tail can contain up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail can be essential for the stability of an individual mRNA.

[0084] In some embodiments, the mRNA also includes a stabilizing element. The stabilizing element may include, for example, a histone stem loop. In some embodiments, the mRNA includes a coding region, at least one histone stem loop, and optionally a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal should typically enhance the expression level of the encoded protein. In some embodiments, the mRNA includes a combination of a poly(A) sequence or polyadenylation signal and at least one histone stem loop, which have alternative mechanisms in nature, but whose synergistic effect can increase protein expression to a level exceeding that observed with either element alone. The synergistic effect of the combination of poly(A) and at least one histone stem loop is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem-loop is typically derived from a histone gene and comprises a loop formed by intramolecular base pairing of two adjacent or perfectly reverse-complementary sequences separated by a spacer (consisting of a short sequence). The unpaired loop region typically cannot form base pairs with any one of the stem-loop elements. The stability of the stem-loop structure typically depends on its length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) can be generated. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15-45 nucleotides. In some embodiments, the mRNA does not contain a histone downstream element (HDE). A "histone downstream element (HDE)" comprises a purine-rich polynucleotide fragment of approximately 15-20 nucleotides located at the 3' end of a naturally occurring stem-loop and represents a binding site for U7 snRNA, which is involved in processing histone pre-mRNA into mature histone mRNA.

[0085] In some embodiments, one or more AU-rich sequences of the mRNA can be removed. AU-rich sequences are destabilizing sequences found in the 3'UTR, sometimes referred to as AURES. AURES can be removed from the mRNA. Alternatively, AURES can be retained in the mRNA.

[0086] In some embodiments, the mRNA is disposed within a lipid nanoparticle (LNP). In some embodiments, lipids are mixed with the mRNA to form the lipid nanoparticle. In some embodiments, RNA is formulated in the lipid nanoparticle. In some embodiments, the lipid nanoparticles are initially formed as empty lipid nanoparticles, which are combined with or encapsulate the vaccine mRNA immediately prior to administration (e.g., within minutes to an hour).

[0087] The lipid nanoparticles typically contain ionizable lipids, non-cationic lipids, sterols, and PEG-lipid components, and a nucleic acid of interest, such as the aforementioned mRNA. The lipid nanoparticles disclosed herein can be produced using components, compositions, and methods generally known in the art, such as those described in International Application Nos. PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US201600129, and PCT / US2016 / 014 280, International Application No. PCT / US2016 / 014280, International Application No. PCT / US2017 / 038426, International Application No. PCT / US2014 / 027077, International Application No. PCT / US2014 / 055394, International Application No. PCT / US2016 / 52117, International Application No. PCT / US2012 / 069610, International Application No. PCT / US2017 / 027492, International Application No. PCT / US2016 / 059575, and International Application No. PCT / US2016 / 069491, all of which are incorporated herein by reference in their entireties.

[0088] In some embodiments, the mRNA vaccine may further comprise one or more adjuvants. Adjuvants are known in the art and can be selected based on the specific antigen and disease state. Exemplary adjuvants include aluminum salt adjuvants (such as aluminum hydroxide or aluminum phosphate solutions), nucleic acid-based adjuvants (such as CpG-ODN), lipid-containing adjuvants (such as LPS), mixed adjuvants (such as MF59 and Freund's adjuvant), and aggregate structure adjuvants (such as RAM1, RAM2, RAM3).

[0089] Pharmaceutical composition or product The pharmaceutical compositions or pharmaceutical products provided herein include the polynucleotide sequences, nucleic acid molecules containing the polynucleotide sequences, delivery vehicles or cells, fusion polypeptides encoded by the polynucleotide sequences, or mRNA vaccines, and the nucleic acid molecules, delivery vehicles, cells, fusion polypeptides, and mRNA vaccines have a purity that meets clinical needs.

[0090] Alternatively, the pharmaceutical composition or pharmaceutical product of the present application may further contain one or more other active compounds depending on the needs of the particular indication being treated. Preferably, the compounds have a complementary, auxiliary, or enhancing effect on the aforementioned nucleic acid molecule, delivery vehicle, cell, or fusion polypeptide, e.g., an effect of enhancing the ability of the nucleic acid molecule, delivery vehicle, cell, or fusion polypeptide to induce an immune response, or a compound that enhances the immune system's immune response to the nucleic acid molecule, delivery vehicle, cell, or fusion polypeptide without adversely affecting each other. Such compounds can be present in the pharmaceutical composition or pharmaceutical product in a desired amount for a desired purpose. For example, in some embodiments, the other active compounds may include one or more immune activators, such as Flt3L, granulocyte-macrophage colony-stimulating factor, IFNα-2a, IFNα-2β, pre-IFNα-2β, IL-2, and an immune checkpoint inhibitor such as a PD-1 or PD-L1 antibody.

[0091] The above-mentioned active ingredients, such as nucleic acid molecules, carriers, cells, fusion polypeptides, etc., can be sandwiched or encapsulated in carriers or colloid drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, or nanocapsules. When the pharmaceutical composition or pharmaceutical product contains two or more active ingredients, the active ingredients can be mixed with each other or separated from each other, for example, coexisting in the same carrier, colloid particle, or microcapsule, or separately existing in different carriers, colloid particle, or microcapsule.

[0092] Alternatively, if desired, the pharmaceutical composition or product may further comprise one or more pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition (2000)) in the form of an aqueous solution, lyophilized agent, or other dry formulation. The pharmaceutically acceptable carrier, excipient, or stabilizer is nontoxic to the recipient at the dosage and concentration employed, and includes buffers (such as phosphate, citrate, histidine, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives, low molecular weight (fewer than about 10 amino acid residues) peptides, proteins (such as serum albumin, gelatin, or immunoglobulins), hydrophilic polymers such as polyvinylpyrrolidone, amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin), chelating agents (such as EDTA), polysaccharides (such as sucrose, mannose, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes, and nonionic surfactants (e.g., TWEEN™, PLURONICS™, or polyethylene glycol). Sustained-release formulations can be prepared. Suitable examples of sustained-release preparations are semipermeable matrices of solid hydrophobic polymers containing the immunoglobulins of the present application, which matrices are in the form of shaped articles such as films or microcapsules.

[0093] It should be understood that the present application includes various aspects, embodiments, and combinations of such aspects and / or embodiments described herein. The above description and the examples that follow are intended to illustrate, rather than limit, the scope of the present application. Other aspects, improvements, and modifications within the scope of the present application will be apparent to those skilled in the art. Accordingly, those skilled in the art should recognize that the scope of the present application also includes improvements and modifications to the described aspects and embodiments. [Example]

[0094] Example 1. Construction and preparation of HPV vaccine sequences 1.1 Synthesis of HPV vaccine sequences and construction of recombinant vectors In this example, the antigen sequences of the mRNA vaccine for HPV-associated tumors were the E6 and E7 proteins of HPV types 16 and 18, and their coding fragments were tandemly linked to obtain a polynucleotide sequence. The coding nucleotide sequence, including, in order from the 5' end, a T7 promoter with an XbaI restriction site at its 5' end, the 5' UTR, tPA-SP, Flt3L, HPV E2 (if present), the E6 / E7 protein or a variant thereof, the 3' UTR, and / or the poly(A) tail, was digested with restriction endonucleases XbaI and NotI and ligated with a pUC57-GW-Kan (GENEWIZ) vector backbone fragment digested with XbaI and NotI to generate a recombinant plasmid. In Examples 1 to 6, nucleic acid sequence component 1 was used, and its ORF coding sequences are SEQ ID NOs: 47-48, 50-52, and 54. In Examples 7-8, nucleic acid sequence component 2 was used, and its ORF coding sequences are SEQ ID NO:49, SEQ ID NO:53, and SEQ ID NO:54.

[0095] 1.2 Preparation of mRNA 1.2.1 Plasmid Linearization The recombinant plasmid prepared in step 1 contains a SapI restriction site after the last A in the poly(A) tail sequence. The plasmid containing the target gene was linearized using the restriction endonuclease SapI. The reaction system is shown in Table 1, and the digestion was carried out at 37°C for 3 hours.

[0096] [Table 1]

[0097] 2 μL of the digestion product was collected and subjected to 1% agarose gel electrophoresis to detect linearization of the plasmid. The linearized plasmid was purified using a PCR product recovery kit (CWBIO).

[0098] 1.2.2 In vitro transcription and purification The linearized recombinant plasmid obtained in step (1) was used as a template for in vitro transcription, using a high-yield T7 RNA transcription kit. The High-Yield T7 RNA Transcription Kit (product name: High Yield T7 RNA Synthesis Kit, Shanghai Hongene Bioengineering Co., Ltd., product catalog number: ON-040), 5x reaction buffer, 100 mM ATP solution, 100 mM CTP solution, 100 mM GTP solution, enzyme mix, DNase I, ammonium acetate stop solution, and lithium chloride (LiCl) precipitation solution are all components of the High-Yield T7 RNA Transcription Kit. 100 mM ΨUTP solution (pseudouridine triphosphate), officially named N1-Me-pUTP, 100 mM, is manufactured by Shanghai Hongene Bioengineering Co., Ltd., product catalog number: R5-027. Each component (using a 20 μL reaction system as an example) was added according to the following system (Table 2), mixed well, and reacted at 37° C. for 3 hours.

[0099] [Table 2]

[0100] Of these, CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG, product number ON-134, manufactured by Shanghai Hongene Bioengineering.

[0101] After the transcription reaction was completed, 1 μL of DNase I was added and incubated at 37°C for 15 minutes. 15 μL of ammonium acetate stop solution was added and mixed thoroughly. Then, 1 / 3 volume of 7.5 M lithium chloride (LiCl) precipitation solution was added (to a final concentration of 2.5 M), and the mixture was maintained at -20°C for 30 minutes. The mixture was then centrifuged at 12,000 g for 15 minutes to precipitate the RNA, and the supernatant was discarded. The RNA was washed by adding 1 mL of 70% ethanol, and the mixture was centrifuged at 12,000 g for 5 minutes. The supernatant was discarded. After air-drying, 50 μL of RNase-free water was added to dissolve the precipitate. The mRNA was quantified using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

[0102] 1.3 Lipid Nanoparticle (LNP) Encapsulation The mRNA stock solution obtained in step 1.2 was dispersed in a 20 mM acetic acid solution (pH 5.0) to obtain an RNA solution with an mRNA concentration of 200 μg / mL. A lipid mixture was obtained by mixing the ionizable lipid:cholesterol:DSPC:DMG-PEG2000 solution in a molar ratio of 50:38.5:10:1.5. The flow rates of the aqueous and oil phases were controlled to mix the mRNA and lipid mixture using a T-type mixing flow. The infusion pump was started to mix the mRNA solution with the lipid mixture to form LNPs. This solution was then diluted 10-fold with diluent and centrifuged in an ultrafiltration tube, followed by three solution exchanges. The resulting solution was added to a Tris aqueous solution and adjusted to pH 7.0-8.0 to obtain an mRNA solution encapsulated in LNPs. LNP refers to lipid nanoparticles.

[0103] The concentration and particle size of the mRNA encapsulated in the LNPs were measured using a Ribogreen RNA quantification kit (Invitrogen, R11490) and a Darwin ZetaSizer particle size analyzer, respectively. LNPs without any encapsulated material were used as a control in the experiment.

[0104] Example 2. Evaluation of the dose-dependent cellular immunological effects of HPV-M mRNA vaccines Twenty-five SPF-grade female C57BL / 6 mice, 6-8 weeks old, were randomly assigned to five groups of five mice per group. According to the groupings shown in Table 4, the mice were immunized with the mRNA vaccine every two weeks for a total of three times. Seven days after immunization, the mice were sacrificed, and the spleens were removed. They were placed on a 70 μm nylon cell filter and thoroughly dispersed in 2 mL of RPMI-1640 complete culture medium to form a cell suspension, and then the cells were counted.

[0105] [Table 3]

[0106] 2.1 Specific IFNγ and IL-2 levels against HPV16 and HPV18 E6 and E7 detected by ELISpot 1.5 x 10 per well 5Cells were seeded onto ELISpot plates, and then an overlapping peptide library (synthesized by SBS Company) of HPV16 and HPV18 E6 and E7 proteins was added. The peptides contained 15 amino acids, with eight amino acid residues overlapping each consecutive peptide. The positive stimulators, phorbol ester PMA and ionomycin, were added to positive control wells, while no stimulators were added to negative control wells. The plates were then placed in a 5% CO2 incubator and incubated at 37°C for 20 hours. The cells in the plates were incubated with antibodies and developed according to the protocol of the ELISpot assay kit (Dacron, 2210001 and Mabtech, 2210001). After air-drying, the plates were read using a Mabtech IRIS ELISpot / FluoroSpot reader equipped with Mabtech Apex software (version 1.1.45.114) to detect spot-forming units (SFU) on the plates.

[0107] 2.2 Flow cytometric detection of specific T cell responses to E6 and E7 of HPV16 and HPV18 To comprehensively evaluate the cellular immune response induced by the HPV vaccine, an intracellular cytokine staining assay was also performed. The obtained single cells were disrupted, filtered, and cultured in a 96-well U-bottom cell culture plate at 1 × 10 per well. 6The plates were seeded at 1000 x 1000 cells / well. An overlapping peptide library of HPV16 and HPV18 E6 and E7 proteins (synthesized by SBS) or PMA and ionomycin were used as stimulators, and BFA and monensin (Biolegend, 420601 and 420701) were used as blockers. After overnight incubation in a 37°C, 5% CO2 incubator, extracellular staining (CD3-cy5.5, CD4-APC or FITC, and CD8-APC or FITC) was performed first, followed by membrane disruption and fixation, followed by staining for the intracellular cytokines IFN-γ-PE or TNF-α-PE. The resulting cells were detected using a Cytoflex flow cytometer (Beckman Coulter). The levels of IFN-γ and TNF-α in CD4+ and CD8+ cells were obtained by gating.

[0108] One-way ANOVA statistical method was used to analyze the significance of each experimental group and control group ( *** p<0.001 vs. LNP group; ###p<0.001 vs. HPV-M-2.5μg; ##p<0.01 vs. HPV-M-2.5μg, &&p<0.01 vs. HPV-M-12.5μg; &&p<0.05 vs. HPV-M-12.5μg). As shown in Figure 1, both the Elispot and flow cytometry results indicated that the cellular immune response produced by HPV-M exhibited a dose-dependent effect, reaching a plateau period after 25μg.

[0109] Example 3. Pharmacodynamic evaluation of HPV-M mRNA vaccines 3.1 Antitumor effects of HPV-M vaccines after intramuscular or intratumoral injection This study used the TC-1 mouse tumor model to evaluate the pharmacodynamic effects of the HPV-M vaccine. TC-1 cells in logarithmic growth phase were subcutaneously implanted into 6-8 week-old female C57BL / 6 mice with SPF grade. Tumors were 100 mm in volume. 3When tumor volume reached 1000, the mice were randomly divided into groups with 10 mice per group based on tumor volume. Mice were immunized with mRNA vaccines once a week for three doses according to the groupings shown in Table 4. Tumor size was measured twice a week, and the survival status of the mice was recorded. Tumor volume was calculated as follows: Volume of implanted tumor = major axis × minor axis × minor axis / 2, i.e., V = ab 2 Statistical analysis was performed on tumor volume and survival rate between groups using one-way ANOVA and log-rank, respectively.

[0110] [Table 4]

[0111] As shown in Figure 2, in the TC-1 mouse tumor model, the mean tumor volume of mice treated with each dose of HPV-M, whether intramuscularly or intratumorally, was significantly reduced compared to the control group of mice treated with LNP, and the survival time of the mice was also significantly prolonged.

[0112] 3.2 Antitumor efficacy of HPV-M vaccine injected intramuscularly or into lymph nodes near tumors According to the above-mentioned method for evaluating antitumor effects, mice were immunized with mRNA vaccines according to the groups shown in Table 5.

[0113] [Table 5]

[0114] As shown in Figure 3, in the TC-1 mouse tumor model, each dose of HPV-M, whether injected intramuscularly or perilymph node, significantly inhibited tumor growth and significantly prolonged mouse survival compared with the control group of mice treated with LNP.

[0115] Example 4. Evaluation of cellular immunity of various HPV mRNA vaccines in mice after vaccination Thirty-five SPF-grade female C57BL / 6 mice, 6 to 8 weeks old, were randomly assigned to seven groups of five mice per group. According to the groupings shown in Table 4, the mice were immunized twice with the mRNA vaccine, once every two weeks. Seven days after immunization, the mice were sacrificed, and the spleens were removed and placed on a 70 μm nylon cell filter. The spleens were thoroughly ground in 2 mL of RPMI-1640 complete medium to obtain a cell suspension, and the cell numbers were counted.

[0116] [Table 6]

[0117] 4.1 HPV16 and HPV18 E6- and E7-specific IFNγ and IL-2 levels detected by ELISpot The levels of antigen-specific IFN-γ and IL-2 induced by the above five HPV vaccines were detected using the same ELISpot method as in Example 2.

[0118] As shown in Figure 4, when immunized with a low dose of 5 μg in two injections, the SFU / 10 derived from mice in the five experimental groups 6 The mean number of IFN-γ and IL-2 spots in splenocytes was higher than that in the negative control group. Compared with the HPV-M vaccine group, the HPV-1, HPV-2, HPV-3, and HPV-4 vaccine groups all significantly increased the levels of IFN-γ and IL-2 expressed by mouse splenocytes stimulated with specific peptides, effectively inducing antigen-specific cellular immune responses. One-way ANOVA statistical analysis of the significance between each group was performed using GraphPad Prism 8 software ( *** p<0.001 vs. LNP control; ** p<0.01 vs. LNP control; * p<0.05 vs. LNP control; ###p<0.001 vs. HPV-M; ##p<0.01 vs. HPV-M; #p<0.05 vs. HPV-M).

[0119] 4.2 Flow cytometric detection of HPV16 and HPV18 E6- and E7-specific T cell responses At the same time, the antigen-specific T cell responses induced by immunization with the above five HPV vaccines were detected using the same flow cytometry method as in Example 2. Cells were gated to obtain the levels of IFN-γ and TNF-α in CD4+ cells and CD8+ cells, and significance analysis was performed for each experimental group and control group using one-way ANOVA statistical method. Pairwise comparisons were made between the experimental group and the HPV-M original sequence group using Student's t-test. As shown in Figure 5, when immunized with two injections of a low dose of 5 μg, the CD4 and TNF-α levels of mice spleen cells in the four experimental groups, HPV-1, HPV-2, HPV-3, and HPV-4, were significantly higher than those in the control group. 4+ Cells and CD 8+ The percentages of IFN-γ and TNF-α in the cells were significantly higher than those in the HPV-M vaccine group, indicating that vaccination can successfully induce an effective immune response. One-way ANOVA statistical analysis of the significance between each group was performed using GraphPad Prism 8 software ( *** p<0.001 vs. LNP control; ** p<0.01 vs. LNP control; * p<0.05 vs. LNP control; ###p<0.001 vs. HPV-M; ##p<0.01 vs. HPV-M; #p<0.05 vs. HPV-M).

[0120] Example 5. Evaluation of cellular immunity to various HPV mRNA vaccines in mice after vaccination Twenty-five SPF-grade female C57BL / 6 mice, 6-8 weeks old, were randomly assigned to seven groups of five mice per group. According to the groupings shown in Table 4, the mice were immunized with the mRNA vaccine three times, once every two weeks. Seven days after immunization, the mice were sacrificed, and the spleens were removed. They were placed on a 70 μm nylon cell filter and thoroughly ground into a cell suspension in 2 mL of RPMI-1640 complete medium, followed by cell counting.

[0121] [Table 7]

[0122] 5.1 HPV16 and HPV18 E6- and E7-specific IFNγ and IL-2 levels detected by ELISpot The levels of antigen-specific IFN-γ and IL-2 induced by the above four HPV vaccines were detected using the same ELISpot method as in Example 2.

[0123] As shown in Figure 6, when immunized with three injections at a dose of 12.5 μg, the SFU / 10 mice in the four experimental groups were 6 The mean number of IFN-γ and IL-2 spots in splenocytes was higher than that in the negative control group. The HPV-1, HPV-4, and HPV-5 vaccine groups all significantly increased the levels of IFN-γ and IL-2 expressed by mouse splenocytes stimulated with specific peptides compared to the HPV-M vaccine group, indicating that these three vaccines significantly enhanced antigen-specific cellular immune responses. One-way ANOVA statistical analysis of the significance between each group was performed using GraphPad Prism 8 software ( *** p<0.001 vs. LNP control; ** p<0.01 vs. LNP control; * p<0.05 vs. LNP control; ###p<0.001 vs. HPV-M; ##p<0.01 vs. HPV-M; #p<0.05 vs. HPV-M).

[0124] 5.2 Flow cytometric detection of HPV16 and HPV18 E6- and E7-specific T cell responses Meanwhile, the antigen-specific T cell responses induced by immunization with the above four HPV vaccines were detected using the same flow cytometry method as in Example 2. Cells were gated to obtain the levels of IFN-γ and TNF-α in CD4+ and CD8+ cells, and significance analysis was performed between each experimental group using one-way ANOVA statistical method. As shown in Figure 7, when immunized with three injections at a dose of 12.5 μg, the percentages of IFN-γ and TNF-α in CD4+ and CD8+ cells of mouse splenocytes in the four experimental groups were all significantly higher than those in the negative control group. Compared with the HPV-M vaccine group, HPV-1, HPV-4, and HPV-5 all significantly increased the percentages of IFN-γ and TNF-α in CD8+ cells, further indicating that these three vaccines can induce stronger antigen-specific cellular immune responses than the HPV-M vaccine. One-way ANOVA statistical analysis for significance between each group was performed using GraphPad Prism 8 software ( *** p<0.001 vs. LNP control; ** p<0.01 vs. LNP control; * p<0.05 vs. LNP control; ###p<0.001 vs. HPV-M; ##p<0.01 vs. HPV-M; #p<0.05 vs. HPV-M).

[0125] Example 6. Antitumor effects of different HPV mRNA vaccines in the TC-1 mouse tumor model In this study, the pharmacodynamic effects of different HPV mRNA vaccines in the TC-1 mouse tumor model were evaluated using the same methods as in Example 3. Mice were administered the mRNA vaccines according to the groupings shown in Table 8.

[0126] [Table 8]

[0127] As shown in Figure 8, in the TC-1 mouse tumor model, the mean tumor volume of the mice treated with each vaccine was significantly reduced compared to the control group of mice treated with LNP, and the survival time of the mice was also significantly extended. At 22 days after administration, the percentage of tumor-free mice in the HPV-M, HPV-1, HPV-4, and HPV-5 vaccine groups was 30% (3 / 10), 50% (5 / 10), 60% (6 / 10), and 70% (7 / 10), respectively. Therefore, overall, the antitumor effects of HPV-1, HPV-4, and HPV-5 at the same dose were better than those of the HPV-M vaccine, with HPV-5 being the most favorable.

[0128] Example 7. Pharmacodynamic evaluation of different HPV mRNA vaccines 6.1 Antitumor effect of intramuscular injection of HPV mRNA vaccine In this study, the pharmacodynamic effects of different HPV mRNA vaccines on the TC-1 mouse tumor model were evaluated using the same method as in Example 3. Mice were immunized with the mRNA vaccines three times weekly according to the groupings shown in Table 9 (Figure 9). Tumor size was measured three times weekly, and the survival status of the mice was recorded.

[0129] [Table 9]

[0130] As shown in Figure 9, in the TC-1 mouse tumor model, the mean tumor volume of the mouse groups treated with each vaccine was significantly reduced compared to the control group of mice treated with LNP. At 22 days post-administration, the percentage of tumor-free mice in the HPV-M, HPV-1, HPV-4, and HPV-5 vaccine groups was 40% (4 / 10), 70% (7 / 10), 70% (7 / 10), and 90% (9 / 10), respectively. The survival time of the mouse groups treated with each vaccine was significantly extended, with the HPV-5 treatment group showing significantly better results than the HPV-M treatment group. Therefore, overall, the antitumor effects of HPV-1, HPV-4, and HPV-5 at the same dose were better than those of the HPV-M vaccine, with HPV-5 showing the best results.

[0131] 6.2 Studies on long-term antitumor effects and immune memory of different HPV mRNA vaccines Mice that showed complete tumor regression were selected, and 68 days after the initial administration, an equal amount of TC-1 tumor cells was inoculated on the opposite side of the back of the mice. Normal untreated mice were inoculated with tumor cells and used as a control group. Tumor growth and mouse survival were recorded, and the cellular immunity levels of the mice were detected at the end of the experiment using the same ELISpot and flow cytometry methods as in Example 2.

[0132] As shown in Figure 9, all mice treated with the HPV mRNA vaccine (except for 14.3% (1 / 7) of deaths in the HPV-4 group) had no tumor recurrence compared to the rapid tumor growth in the control group and remained completely tumor-free for at least 42 days. As shown in Figure 9, the SFU / 10 of mice in the four experimental groups 6 The mean number of spots of IFN-γ and IL-2 in the spleen cells was higher than that of the negative control group, indicating that CD4+ 4+ Cells and CD 8+ The percentage of cells and their IFN-γ and TNF-α were higher than those of the negative control group. 8+ The percentage of IFN-γ and TNF-α in the cells was significantly increased. All these results indicated that these four vaccines, especially HPV-5, can induce long-term antigen-specific immune memory and provide strong protection against tumor recurrence. One-way ANOVA statistical analysis for significance between each group was performed using GraphPad Prism 8 software ( *** p<0.001 vs. LNP control; ** p<0.01 vs. LNP control; * p<0.05 vs. LNP control; ns, not significant).

[0133] Example 8. Evaluation of antitumor effect of combined use of HPV-5 mRNA vaccine and PD-L1 antibody This study evaluated the antitumor effect of the combination of HPV-5 mRNA vaccine and PD-L1 in a TC-1 mouse tumor model using the same method as in Example 3. Mice were administered the mRNA vaccine and PD-L1 antibody according to the groupings shown in Table 10. The PD-L1 antibody (10F.9G2) and rabbit isotype IgG2b (LTF-2) were both purchased from BioXcell and intraperitoneally injected at a dose of 10 mg / kg. The specific dosing regimen is shown in Figure 10. Tumor size was measured three times a week, and the survival status of the mice was recorded. Tumor growth and survival curves were plotted based on the measurement results, and the relative tumor growth rate (T / C (%)) and synergistic efficiency of the drug combination were calculated using the relative tumor volume (RTV). The specific calculation formula is as follows: RTV = V t / V0, where V t and V0 are the tumor volumes measured at a certain time point (dt) and at the time when the mice were separated into different cages and treatment was initiated (d0), respectively. T / C(%)=(T RTV / C RTV ) × 100%, where T RTV and C RTV are the RTV values ​​of the experimental group and the control group, respectively. Synergistic efficiency of drugs in groups A and B (%) = (A T / C ×B T / C / AB T / C ) × 100%, where A T / C , B T / C and AB T / C are the T / C values ​​for drug A group, drug B group, and AB drug combination group, respectively.

[0134] [Table 10]

[0135] As a result, the PBS control group and the PD-L1 antibody monotherapy group in the TC-1 mouse tumor model showed rapid tumor growth and shorter survival times, as shown in Figure 10. Both the 3 μg HPV-5 monotherapy group and the HPV-5 and PD-L1 antibody combination group significantly inhibited tumor growth and significantly extended mouse survival times compared to PBS.

[0136] [Table 11]

[0137] Tumor data on day 16 after administration were calculated, and the results are shown in Table 11. The synergistic effects of the combination of HPV-5 (0.3 μg) or HPV-5 (3 μg) with PD-L1 antibody were 2.91 and 19.60, respectively (when the value was greater than 1, the two had synergistic effects), indicating good anti-tumor synergy between the HPV-5 mRNA vaccine and PD-L1 antibody.

[0138] The sequences used in the above examples of the present application are shown in the sequence listing below. It should be understood that the following sequences are merely exemplary sequences for the embodiments of the present application and do not constitute any limitation on the embodiments of the present application. The nucleic acid sequences in the sequence listing below can represent DNA sequences or RNA sequences, and when they represent RNA sequences, "T" therein represents uridine.

[0139] [Table 12]

[0140] [Table 13]

[0141] [Table 14]

[0142] [Table 15]

[0143] [Table 16]

[0144] Table 17

[0145] Table 18

[0146] Table 19

[0147] Table 20

[0148] Table 21

[0149] Table 22

[0150] Table 23

[0151] Table 24

[0152] Table 25

[0153] Table 26

[0154] Table 27

[0155] Table 28

[0156] Table 29

[0157] Table 30

[0158] Table 31

[0159] Table 32

[0160] Table 33

Claims

1. A polynucleotide molecule comprising at least a coding sequence for an HPV antigenic polypeptide, the antigenic polypeptide comprising, in order from N-terminus to C-terminus, at least: 1) amino acid sequence A and amino acid sequence B; 2) amino acid sequence C, amino acid sequence A, and amino acid sequence B; 3) amino acid sequence B and amino acid sequence A; or 4) amino acid sequence C, amino acid sequence B, and amino acid sequence A; Including, the amino acid sequence A comprises, in order from the N-terminus to the C-terminus, at least SEQ ID NOs: 1 to 4 or variants thereof, and each of the amino acid sequences represented by SEQ ID NOs: is linked directly or via a linker; the amino acid sequence B comprises, in order from the N-terminus to the C-terminus, at least SEQ ID NOs: 5 to 8 or variants thereof, and each of the amino acid sequences represented by SEQ ID NOs: are linked directly or via a linker; the amino acid sequence C comprises an HPV E2 antigen sequence; Preferably, the variant is a conservative substitution variant.

2. 2. The polynucleotide molecule of claim 1, wherein the HPV E2 antigen sequence is SEQ ID NO: 9 or a variant thereof.

3. The polynucleotide molecule of claim 1 or 2, wherein the linker peptide comprises one, two or more amino acid residues.

4. The polynucleotide molecule according to claim 1 or 2, wherein the linker peptide consists of 2 to 10 amino acid residues, preferably the amino acid residues are glycine residues, serine residues and / or alanine residues, more preferably the linker peptide consists of 2 alanine residues.

5. 2. The polynucleotide molecule of claim 1, wherein the HPV antigen polypeptide comprises the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:

16.

6. The polynucleotide molecule according to any one of claims 1 to 5, further comprising a coding sequence for an immunoactivator or a functional domain thereof at the 5'-terminal side of the coding sequence for the HPV antigen polypeptide.

7. The polynucleotide molecule of claim 6, wherein the immunoactivator is Flt3L.

8. The polynucleotide molecule of claim 7, wherein the polypeptide sequence of the immune activator comprises at least the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:

10.

9. The polynucleotide molecule according to any one of claims 1 to 8, further comprising a coding sequence for a secretory signal peptide, preferably wherein the coding sequence for the secretory signal peptide is located at the 5'-terminal side of the coding sequence for the HPV antigen polypeptide.

10. The polynucleotide molecule of claim 9, wherein the secretory signal peptide is tPA-SP.

11. 11. The polynucleotide molecule of claim 10, wherein the secretory signal peptide comprises an amino acid sequence represented by SEQ ID NO:11 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:

11.

12. The polynucleotide molecule according to any one of claims 9 to 11, comprising coding sequences for the secretory signal peptide, the immunoactivator and the HPV antigen polypeptide, which are linked in order from the 5' end to the 3' end.

13. The polynucleotide molecule of any one of claims 1 to 12, which is DNA, RNA, or a hybrid of DNA and RNA.

14. The polynucleotide molecule according to any one of claims 1 to 13, comprising any one polynucleotide sequence selected from SEQ ID NOs: 28 to 54, consisting of any one polynucleotide sequence selected from SEQ ID NOs: 28 to 54, or encoded by any one polynucleotide sequence selected from SEQ ID NOs: 28 to 54.

15. 15. The polynucleotide molecule of any one of claims 1 to 14, further comprising a 5'UTR structure, wherein preferably said 5'UTR structure comprises at least a polynucleotide sequence represented by SEQ ID NO:22 or SEQ ID NO:25, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:22 or SEQ ID NO:

25.

16. 16. The polynucleotide molecule of any one of claims 1 to 15, further comprising a 3'UTR structure, preferably wherein said 3'UTR structure comprises at least a polynucleotide sequence represented by SEQ ID NO:23 or SEQ ID NO:26, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:23 or SEQ ID NO:

26.

17. The polynucleotide molecule of any one of claims 1 to 16, which is an mRNA molecule.

18. The polynucleotide molecule according to claim 17, wherein some or all of the uridines in the mRNA molecule are chemically modified uridines, and preferably the chemically modified uridines are pseudouridine or N1-methyl-pseudouridine.

19. 19. The polynucleotide molecule of claim 17 or 18, wherein the mRNA further comprises a 5' cap structure, preferably the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

20. 20. The polynucleotide molecule of any one of claims 17 to 19, wherein the mRNA further comprises a poly(A) tail, preferably the sequence of the poly(A) tail comprises at least 50, at least 60, or at least 100 A nucleotides, and preferably the poly(A) tail comprises at least the polynucleotide sequence represented by SEQ ID NO:24 or SEQ ID NO:27, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:24 or SEQ ID NO:

27.

21. A polynucleotide molecule complementary to the polynucleotide molecule of any one of claims 1 to 20.

22. A fusion polypeptide encoded by the polynucleotide molecule of any one of claims 1 to 20, or having the same amino acid sequence as a polypeptide encoded by the polynucleotide molecule of any one of claims 1 to 20.

23. 23. The fusion polypeptide of claim 22, comprising an amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to the amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:

21.

24. A delivery vehicle comprising the polynucleotide molecule of any one of claims 1 to 21 or the fusion polypeptide of claim 22 or 23.

25. The delivery vehicle according to claim 24, wherein the delivery vehicle is a lipid nanoparticle (LNP).

26. The delivery vehicle of claim 25, wherein the LNP comprises an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG)-lipid.

27. A cell comprising a polynucleotide molecule according to any one of claims 1 to 21 or a fusion polypeptide according to claim 22 or 23.

28. A pharmaceutical composition or pharmaceutical product comprising the polynucleotide molecule of any one of claims 1 to 21, the fusion polypeptide of claim 22 or 23, the delivery vehicle of any one of claims 24 to 26, or the cell of claim 27.

29. 29. The pharmaceutical composition or product of claim 28, further comprising an immunostimulator and / or an adjuvant.

30. 30. The pharmaceutical composition or pharmaceutical product according to claim 29, wherein the immune activator is any one or more selected from the group consisting of IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, Flt3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, Pre-IFNα-2β, MIP-α, STING, HSP70, and an immune checkpoint inhibitor (preferably a PD-1 inhibitor or a PD-L1 inhibitor).

31. The STING is Ｖ１５５Ｍ 31. The pharmaceutical composition or product of claim 30, wherein

32. 31. The pharmaceutical composition or product of claim 30, wherein said immune activator is a protein or a nucleic acid molecule encoding said protein, preferably said nucleic acid molecule is mRNA.

33. The pharmaceutical composition or product of any one of claims 28 to 32, which is an mRNA vaccine.

34. A method for treating or preventing HPV infection or a disease associated with HPV infection, the method comprising administering to an individual the polynucleotide molecule of any one of claims 1 to 21, the fusion polypeptide of claim 22 or 23, the delivery vehicle of any one of claims 24 to 26, the cell of claim 27, or the pharmaceutical composition or pharmaceutical product of any one of claims 28 to 33.

35. 35. The method of claim 34, wherein the HPV infection-related disease is cervical cancer.

36. 36. The method of claim 35, wherein the administration is intratumoral, perilymphatic, or intramuscular injection.

37. 37. The method of claim 36, further comprising administering to the individual an immune activator, chemotherapy, radiation therapy, and / or targeted therapy.

38. 38. The method of claim 37, wherein the immune activator is one or more selected from the group consisting of IL-3, IL-7, IL-2, IL-4, IL-5, IL-12, IL-13, FI3L, G-CSF, M-CSF, GM-CSF, EPO, TPO, SCF, IFNα-2α, IFNα-2β, Pre-IFNα-2β, MIP-α, STING, HSP70, and an immune checkpoint inhibitor (preferably a PD-1 inhibitor or a PD-L1 inhibitor).

39. 39. The method of claim 38, wherein the immunoactivator is a protein or a nucleic acid molecule encoding the protein, preferably the nucleic acid molecule is mRNA.

40. Use of the polynucleotide molecule of any one of claims 1 to 21, the fusion polypeptide of claim 22 or 23, the delivery vehicle of any one of claims 24 to 26, the cell of claim 27, or the pharmaceutical composition or pharmaceutical product of any one of claims 28 to 33 in the preparation of a medicament for treating or preventing HPV infection or a disease associated with HPV infection.

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