HK40108427A - Polynucleotide molecule for preventing or treating hpv infection-related diseases - Google Patents

Description

Polynucleotide molecules used for the prevention or treatment of HPV infection-related diseases

Technical Field

This application relates to the field of biotechnology, specifically to an mRNA vaccine that treats HPV infection-related diseases by inducing an HPV antigen-specific immune response.

Background Technology

High-risk human papillomavirus (HPV) infection accounts for 5% of all diseases worldwide, with 70% of cervical cancers 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). Among them, E6 and E7 proteins are expressed in almost all cervical cancer cells and are essential for maintaining the disease phenotype, making them ideal targets for therapeutic vaccines.

Currently, HPV therapeutic vaccines under development mainly include DNA vaccines, subunit vaccines, and recombinant vector vaccines. mRNA vaccines have advantages such as no integration risk, short half-life, and high safety. They can induce antigen-specific immune responses by expressing viral antigens, killing infected cells, such as tumor cells, thereby achieving the goal of treating related tumors. The purpose of this application is to prepare an mRNA vaccine for the treatment of HPV infection-related diseases.

Invention Overview

This application provides for preventive or therapeutic nucleic acids and fusion peptides for HPV infection-related diseases, pharmaceutical compositions or pharmaceutical articles comprising said therapeutic nucleic acids or fusion peptides, and the use of said nucleic acids and fusion peptides.

Specifically, this application provides a polynucleotide molecule that at least contains the coding sequence of an HPV antigenic polypeptide, wherein the antigenic polypeptide comprises at least the following 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;

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.

Among them, amino acid sequence A contains SEQ ID NO: 1, 2, 3, 4 or variants thereof from N-terminus to C-terminus, and each amino acid sequence shown in SEQ ID NO is directly linked or linked by a linking peptide in sequence.

The amino acid sequence B contains SEQ ID NO:5, 6, 7, 8 or variants thereof from the N-terminus to the C-terminus, and the amino acid sequences shown in SEQ ID NO are directly linked or linked by linking peptides in sequence.

The amino acid sequence C contains the HPV E2 antigen sequence.

In some embodiments, the variants are conserved substitution variants. In some embodiments, the amino acid sequences of each variant of SEQ ID NO: 1-4 have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5% sequence identity with the amino acid sequences of one of SEQ ID NO: 1-4. In some embodiments, the amino acid sequences of each variant of SEQ ID NO: 5-8 have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5% sequence identity with the amino acid sequences of one of SEQ ID NO: 5-8.

In some embodiments, the amino acid sequence C is the HPV E2 antigen sequence.

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 conserved substitution variant. In some embodiments, the variant of SEQ ID NO:9 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% sequence identity with SEQ ID NO:9.

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 shown in each SEQ ID NO are linked by different linker peptides. In some embodiments, the amino acid sequences shown in each SEQ ID NO are linked by the same linker peptide. In some embodiments, the linker peptide consists of 2-10 amino acid residues. In some embodiments, the amino acid residues are glycine, serine, and/or alanine residues. In some embodiments, the linker peptide is selected from GS linker, (Gly) ₈ , α-helical peptides, (XP)n, etc. In some embodiments, the amino acid sequences shown in each SEQ ID NO are formed by two alanine residues linked together.

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 with it. 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 more than 98%, 98.5%, 99%, or 99.5% sequence identity with 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.

In some embodiments, the polynucleotide molecule further comprises a coding sequence for an immunostimulatory factor or its functional domain. In some embodiments, the coding sequence for the immunostimulatory factor or its functional domain is located on one side of the 3' or 5' end of the coding sequence of the HPV antigenic polypeptide. In some embodiments, the immunostimulatory factor 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 immune checkpoint inhibitors. In some embodiments, the immunostimulatory factor is an antibody or antigen-binding fragment thereof against 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, galactagogue-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, VSIG8. In some embodiments, the immunostimulatory factor is Flt3L. In some embodiments, the polypeptide sequence of the immunostimulatory factor comprises at least the amino acid sequence shown in SEQ ID NO:10, or a conserved substitution 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 with SEQ ID NO:10. In some embodiments, the polypeptide sequence of the immunostimulatory factor is the amino acid sequence shown in SEQ ID NO:10, or a conserved substitution 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 with SEQ ID NO:10. In some embodiments, the coding sequence of the immunostimulatory factor comprises or is the polynucleotide sequence shown in SEQ ID NO:29. In some embodiments, the coding sequence of the immunostimulatory factor is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 29.

In some embodiments, the coding sequence of the immunostimulatory factor contains or is a synonymous mutant of SEQ ID NO: 29.

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 of the HPV antigen polypeptide. In some embodiments, the signal peptide is a secreted signal peptide. In some embodiments, the secreted signal peptide is selected from signal peptides of mammalian secreted proteins. In some embodiments, the mammal is human. In some embodiments, the secreted signal peptide is tPA-SP. In some embodiments, the secreted signal peptide comprises the amino acid sequence shown in SEQ ID NO:11, or a conserved 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 with SEQ ID NO:11. In some embodiments, the secretory signal peptide is an amino acid sequence as shown in SEQ ID NO:11, or a conserved 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 with SEQ ID NO:11. In some embodiments, the coding sequence of the secretory signal peptide comprises or is a polynucleotide sequence as shown in SEQ ID NO:28. In some embodiments, the coding sequence of the secretory signal peptide is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:28.

In some embodiments, the coding sequence of the secreted signal peptide includes or is a synonymous mutant of SEQ ID NO: 28.

In some embodiments, the polynucleotide molecule comprises, from its 5' end to its 3' end, the coding sequences of the aforementioned signal peptide, the aforementioned immunostimulatory factor, and the aforementioned HPV antigenic peptide, linked sequentially. In some embodiments, the coding sequences of the signal peptide, the immunostimulatory factor, and the HPV antigenic peptide are directly linked or linked via a polynucleotide chain. In some embodiments, the polynucleotide chain comprises three or multiples of three nucleotides. Alternatively, in some embodiments, the polynucleotide molecule consists of the coding sequences of the signal peptide, the immunostimulatory factor, and the HPV antigenic peptide linked sequentially from its 5' end to its 3' end. In some embodiments, the coding sequences of the signal peptide, the immunostimulatory factor, and the HPV antigenic peptide are located within the same reading frame. In some embodiments, the reading frame encodes any polynucleotide sequence shown in SEQ ID NO:47-54 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with any polynucleotide sequence shown in SEQ ID NO:47-54; in some embodiments, the reading frame encodes a protein as shown in any of SEQ ID NO:17-21 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with any protein shown in SEQ ID NO:17-21.

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 cells. 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.

In some embodiments, the polynucleotide molecule comprises any polynucleotide sequence selected from SEQ ID NO: 28-54. In some embodiments, the polynucleotide molecule consists of any polynucleotide sequence selected from SEQ ID NO: 28-54. In some embodiments, the polynucleotide molecule consists of any polynucleotide sequence selected from SEQ ID NO: 39-54. In some embodiments, the polynucleotide molecule comprises a nucleic acid fragment encoded by any polynucleotide sequence selected from SEQ ID NO: 28-54. In some embodiments, the polynucleotide molecule is encoded by a nucleic acid encoded by any polynucleotide sequence selected from SEQ ID NO: 28-54. In some embodiments, the polynucleotide molecule is encoded by a nucleic acid encoded by any polynucleotide sequence selected from SEQ ID NO: 39-54. In some embodiments, the polynucleotide molecule comprises a sequence complementary to any polynucleotide sequence selected from SEQ ID NO: 28-54. In some embodiments, the polynucleotide molecule consists of a sequence complementary to any polynucleotide sequence selected from SEQ ID NO: 28-54. In some embodiments, the polynucleotide molecule consists of a sequence complementary to any polynucleotide sequence selected from SEQ ID NO: 39-54.

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 both a 5'UTR structure and a 3'UTR structure. In some embodiments, the 5'UTR structure comprises at least the polynucleotide sequence shown 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 with SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 5'UTR structure is the polynucleotide sequence shown 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 with SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 3'UTR structure comprises at least the polynucleotide sequence shown 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 with SEQ ID NO:23 or SEQ ID NO:26. In some embodiments, the 3'UTR structure is the polynucleotide sequence shown 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 with SEQ ID NO:23 or SEQ ID NO:26.

In some embodiments, the polynucleotide molecule is an mRNA molecule. In some embodiments, some or all of the uridine in the mRNA molecule is pseudouridine or 1-methyl-pseudouridine. In some embodiments, the mRNA further comprises a 5' cap structure. In some embodiments, the 5' cap structure is type O, type I, or type II. In some embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the mRNA further comprises a polyA tail. In some embodiments, the polyA 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 adenosine nucleotides. In some embodiments, the polyA tail comprises up to 500, 400, 300, 200, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 adenosine nucleotides (A), particularly about 120 A's. In some embodiments, the polyA tail comprises at least the polynucleotide sequence shown 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 with SEQ ID NO:24 or SEQ ID NO:27. In some embodiments, the polyA tail is a polynucleotide sequence as shown 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 with SEQ ID NO:24 or SEQ ID NO:27.

In addition, this application also provides a polynucleotide molecule containing a sequence complementary to the polynucleotide sequence of the above-mentioned polynucleotide molecule.

The polynucleotide molecules provided in this application are, in some embodiments, single-stranded, double-stranded, or cyclic molecules. In some embodiments, the polynucleotide molecules provided in this application include single-stranded and double-stranded structures.

Secondly, this application also provides a fusion polypeptide. In some embodiments, the fusion polypeptide is encoded by the polynucleotide molecule described in the first aspect. In some embodiments, the fusion polypeptide comprises at least the following 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;

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.

Among them, amino acid sequence A contains SEQ ID NO: 1, 2, 3, 4 or variants thereof from the N-terminus to the C-terminus, and each amino acid sequence shown in SEQ ID NO is directly linked or linked sequentially through a linker peptide.

Amino acid sequence B contains SEQ ID NO:5, 6, 7, 8 or variants thereof, and the amino acid sequences shown in SEQ ID NO are directly linked or linked sequentially via linking peptides.

The amino acid sequence C contains the HPV E2 antigen sequence.

Preferably, the variant is a conservative substitution variant.

In some implementations, amino acid sequence C is the HPV E2 antigen sequence.

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

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 shown in SEQ ID NO are linked by different linker peptides. In some embodiments, the amino acid sequences shown in SEQ ID NO are linked by the same linker peptide. In some embodiments, the amino acid sequences shown in SEQ ID NO are linked by two alanine residues.

In some embodiments, the fusion polypeptide comprises the amino acid sequence shown 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 conserved substitution variant of the amino acid sequence shown 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 with it. In some embodiments, the fusion polypeptide comprises the amino acid sequence shown 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 conserved substitution variant of the amino acid sequence shown 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 an amino acid sequence having 98%, 98.5%, 99%, or 99.5% or more sequence identity with it. In some embodiments, the fusion polypeptide is an amino acid sequence shown 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 further comprises the full-length immunostimulatory factor polypeptide or its functional domain. In some embodiments, the immunostimulatory factor protein or its functional domain is located at the C-terminus or N-terminus of the fusion polypeptide. In some embodiments, the immunostimulatory factor 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, MHSP70, and immune checkpoint inhibitors. In some embodiments, the immunostimulatory factor is an antibody or antigen-binding fragment thereof against 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, galactagogue-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, VSIG8. In some embodiments, the immunostimulatory factor is Flt3L. In some embodiments, the polypeptide sequence of the immunostimulatory factor comprises at least the amino acid sequence shown in SEQ ID NO:10, or a conserved substitution 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 with SEQ ID NO:10. In some embodiments, the polypeptide sequence of the immunostimulatory factor is the amino acid sequence shown in SEQ ID NO:10, or a conserved substitution 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 with SEQ ID NO:10.

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 polypeptide. In some embodiments, the signal peptide is a secreted signal peptide. In some embodiments, the secreted signal peptide is selected from signal peptides of mammalian secreted proteins. In some embodiments, the mammal is human. In some embodiments, the secreted signal peptide is tPA-SP. In some embodiments, the secreted signal peptide comprises the amino acid sequence shown in SEQ ID NO:11, or a conserved 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 with SEQ ID NO:11. In some embodiments, the secreted signal peptide is the amino acid sequence shown in SEQ ID NO:11, or a conserved 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 with SEQ ID NO:11.

In some embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus, the coding sequence of the signal peptide, the immunostimulatory factor, and the fusion polypeptide connected in sequence.

In addition, this application also provides an HPV E2 antigenic peptide, a polynucleotide molecule encoding the HPV E2 antigenic peptide, and uses thereof. In some embodiments, the HPV E2 antigenic peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:9. In some embodiments, the amino acid sequence of the HPV E2 antigenic peptide is shown in SEQ ID NO:9. In some embodiments, the HPV E2 antigenic peptide comprises or is composed of a conserved substitution variant of SEQ ID NO:9. Use of the HPV E2 antigenic peptide includes administering it in combination with other HPV antigenic peptides to an individual in need, or fusing it with other HPV antigenic peptides and administering it to an individual in need, to induce a stronger immune response against HPV in the individual, wherein a stronger immune response is defined as a stronger immune response than the immune response induced by the individual upon administration of the other HPV antigenic peptides alone. In some embodiments, the polynucleotide molecule encoding the HPV E2 antigenic peptide comprises or is composed of the polynucleotide sequence shown in SEQ ID NO:38. In some embodiments, the polynucleotide molecule encoding the HPV E2 antigenic polypeptide comprises or is composed of a conserved substitution variant of the polynucleotide sequence of SEQ ID NO: 38, or is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 38. Use of the polynucleotide molecule encoding the HPV E2 antigenic polypeptide includes administering it in combination with a polynucleotide molecule encoding other HPV antigenic polypeptides to an individual in need, or administering it to an individual in need after linking its polynucleotide sequence with the sequence of a polynucleotide molecule encoding other HPV antigenic polypeptides to form a new polynucleotide molecule, to express the HPV E2 antigenic polypeptide and other HPV antigenic polypeptides, or a fusion protein of the HPV E2 antigenic polypeptide and other HPV antigenic polypeptides. In some embodiments, the individual in need has cervical cancer.

A third aspect of this application provides a delivery system comprising the polynucleotide molecule of the first aspect or the fusion polypeptide of the second aspect. In some embodiments, the delivery system is a liposome, a viral particle, or a quantum dot. In some embodiments, the delivery system is an LNP (lipid nanoparticle). In some embodiments, the LNP comprises PEG-modified lipids, non-cationic lipids, sterols, ionizable lipids, or any combination thereof. In some embodiments, the LNP is composed of ionizable lipids, phospholipids, cholesterol, polyethylene glycol (PEG)-lipids, and the polynucleotide molecule of the first aspect.

In some embodiments, the LNP comprises ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 35 mol%-65 mol%, the sum of phospholipids and cholesterol is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%. In some embodiments, the LNP comprises ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 40 mol%-50 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%.

A fourth aspect of this application provides a cell comprising the polynucleotide molecule of the first aspect or the fusion polypeptide of the second aspect described above. In some embodiments, the cell is a bacterial, fungal, or mammalian cell.

A fifth aspect of this 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 body 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 the mRNA from the polynucleotide molecule of the first aspect. In some embodiments, the mRNA is encoded by any polynucleotide selected from SEQ ID NO: 28-54 or by a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 28-54. In some embodiments, the mRNA is encoded by any polynucleotide selected from SEQ ID NO: 39-54 or by a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 39-54.

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 both a 5'UTR structure and a 3'UTR structure. In some embodiments, the 5'UTR structure comprises at least the polynucleotide sequence shown 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 with SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 5'UTR structure is the polynucleotide sequence shown 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 with SEQ ID NO:22 or SEQ ID NO:25. In some embodiments, the 3'UTR structure comprises at least the polynucleotide sequence shown 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 with SEQ ID NO:23 or SEQ ID NO:26. In some embodiments, the 3'UTR structure is the polynucleotide sequence shown 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 with SEQ ID NO:23 or SEQ ID NO:26.

In some embodiments, the mRNA molecule of the present invention is a mature mRNA molecule, which, from its 5' to 3' ends, sequentially comprises a 5' cap, a 5' UTR, a coding sequence for the secretory signal peptide, a coding sequence for the immunostimulatory factor, 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 immunostimulatory factor, the coding sequence for the HPV antigen polypeptide, the 3' UTR, and the polyA tail are operatively linked to each other. In some embodiments, the 5' cap is an m7G(5')ppp(5')(2'-OMeA)pG structure.

In some embodiments, the 5' end of the 5' UTR also includes an AGG or AUG or other nucleotide triplet; or the 5' UTR together with one or two bases on its 5' end constitutes an AGG or AUG or other nucleotide triplet; for use in different capping systems. The starting sites required for different capping systems, such as Clean CapAG, Clean Cap AU, etc., are known in the art and can be conventionally selected by those skilled in the art.

In some embodiments, the mRNA molecule of the present invention comprises a Kozak sequence. In some specific embodiments, the Kozak sequence comprises a GCCACC located to one side of the 5' end of the coding sequence of the secreted signal peptide.

In some embodiments, some or all of the uridine in the mRNA is chemically modified uridine. In some embodiments, some or all of the uridine in the mRNA is pseudouridine or 1-methyl-pseudouridine.

In some embodiments, some or all of the uracil nucleotides of the mRNA are replaced by pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.

In some embodiments, the mRNA further comprises a 5' cap structure. In some embodiments, the 5' cap structure is type O, type I, and type II. In some embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the mRNA further comprises a polyA tail. In some embodiments, the polyA 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 adenosine nucleotides. In some embodiments, the polyA 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 adenosine nucleotides (A), particularly about 120 A's. In some embodiments, the polyA tail comprises at least the polynucleotide sequence shown 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 with SEQ ID NO:24 or SEQ ID NO:27. In some embodiments, the polyA tail is the polynucleotide sequence shown 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 with SEQ ID NO:24 or SEQ ID NO:27.

In some embodiments, the pharmaceutical composition, pharmaceutical product, or kit further comprises an immunostimulatory factor and/or adjuvant. In some embodiments, the immunostimulatory factor 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 their encoded polynucleotides. In some embodiments, the STING is 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 encoded polynucleotide is mRNA.

A sixth aspect of this application provides a method for treating or preventing HPV infection and HPV-related diseases, comprising administering to an individual the polynucleotide molecule of the first aspect, the fusion polypeptide of the second aspect, the delivery body 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 HPV-related disease is cervical cancer. In some embodiments, the administration is intratumoral, perilymphatic, or intramuscular injection. In some embodiments, the method further comprises administering to the individual an immunostimulatory factor, chemotherapy, radiotherapy, and/or targeted therapy. In some embodiments, the targeted therapy is an antibody or its functional domain targeting a cervical cancer-specific tumor target.

It should be understood that the aspects and embodiments of this application described herein include those described as "comprising," "forming," and "essentially consisting of." The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combinations of 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 are all within the protection scope of this application.

Attached Figure Description

Figure 1 shows the cellular immune response induced in normal mice after inoculation with different doses of HPV-M mRNA vaccine.

Figure 2. Antitumor effect of HPV-M mRNA vaccine after intramuscular or intratumoral injection in TC-1 tumor model mice.

Figure 3. Antitumor effect of HPV-M mRNA vaccine injected into TC-1 tumor model mice via muscle or peritumoral injection near the tumor.

Figure 4 shows the levels of specific IFNγ and IL-2 for HPV16 and 18 E6 and E7 induced by two doses of five different mRNA vaccines (5 μg) detected using ELISpot.

Figure 5 shows the flow cytometry analysis of the HPV16 and 18 E6 and E7 specific T cell responses induced by two doses of five different mRNA vaccines (5 μg).

Figure 6 shows the levels of HPV16 and 18 E6 and E7 specific IFNγ and IL-2 induced by three doses of four different mRNA vaccines (12.5 μg) using ELISpot.

Figure 7 shows the HPV16 and 18 E6 and E7 specific T cell responses induced by three doses of four different mRNA vaccines (12.5 μg) using flow cytometry.

Figure 8. Antitumor effects of three immunizations with four different mRNA vaccines (12.5 μg) in TC-1 tumor model mice.

Figure 9. Antitumor effects of three doses of four different mRNA vaccines (12.5 μg) on TC-1 tumor model mice.

Figure 10 Evaluation of the antitumor effect of HPV-5 mRNA vaccine combined with PD-L1 antibody.

Invention Details

This application provides novel polynucleotide sequences that can be used to prepare preventive or therapeutic nucleic acids and fusion peptides for HPV infection-related diseases. Furthermore, this application also provides pharmaceutical compositions or pharmaceutical products comprising the aforementioned therapeutic nucleic acids or fusion peptides, such as mRNA vaccines, and their use in treating diseases.

the term

As used herein, "coding sequence" can refer to a ribonucleotide sequence in mature mRNA that can be translated into a protein, or it can refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence used as a template for transcribing the ribonucleotide (RNA) sequence. Furthermore, the "coding sequence" of this application may further include a polynucleotide sequence encoding a functional nucleic acid, such as miRNA, shRNA, dsRNA, etc.

As used herein, the term "HPV E2 antigen sequence" refers to an immunogenic amino acid sequence derived from the E2 protein of HPV. In some embodiments, the HPV E2 antigen sequence is derived from the E2 protein sequence of any wild-type or artificially mutant HPV subtype, or is a fusion protein of multiple wild-type or artificially mutant HPV subtype E2 protein sequences. In some embodiments, the HPV E2 antigen 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 antigen sequence is a fusion protein of one or more conserved peptide sequences of the HPV E2 protein with one or more specific wild-type and/or artificially mutant HPV subtype E2 protein sequences. The conserved peptide sequence can be an intragenotypic conserved peptide sequence, i.e., a conserved amino acid sequence in different mutants of the E2 protein of a particular HPV subtype; or it can be an intergenotypic conserved peptide sequence, i.e., a conserved amino acid sequence in multiple HPV subtype E2 proteins. Methods for obtaining (or for conservatism assessment) of conserved peptide sequences are known in the art. For example, available full-length sequences of the E2 protein 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 will be equivalently representative of the entire environmental population. For example, before conservation assessment, all genotypes are aligned and sequences within each genotype are weighted to ensure equivalent representation of genotype diversity and thus ensure that HPV E2 antigen sequence candidates represent the entire environmental population. Intragenotype conservation is then assessed using, for example, a 15-amino acid sliding window (intragenotype conservation), determining the conservation value for each window based on the amino acid prevalence within the combined window and the weighted value for each sequence, to identify conserved fragments within each genotype, and the intragenotype conserved peptide sequence created for each window. 'Intragenotype conserved peptide sequence' refers to an amino acid sequence representing a weighted set of genotype sequences, rather than the most common amino acid at each position. To be classified as conserved, the window must have a conserved value within the first quartile of all window conservation values for the protein. Subsequently, conserved intragenotypic windows at the same location in all genotypes were identified, regardless of the percentage of identity of intragenotypic normalized common sequences shared between genotypes (inter-genotypic conservation). A phylogenetic study of the resulting regions was then created, and tree ingroup sequences were combined to generate inter-genotypic conserved peptide sequences with a high level of shared common identity.

As used herein, a "linker peptide" refers to an amino acid residue in a fusion protein that links two polypeptide fragments together, or a peptide chain containing two or more amino acid residues. In some embodiments, the linker peptide is a flexible linker peptide, which allows the two linked amino acid fragments to have a certain degree of mobility. The addition of Ser and Thr allows the linker peptide to form hydrogen bonds with water molecules, giving the linker peptide stability in aqueous solution, thereby reducing the interaction between the linker peptide and the two preceding and following proteins. Common flexible linkers peptides consist of Gly and Ser residues ("GS" linkers). In addition to GS flexible linkers peptides, there are other flexible linkers peptides, such as (Gly) ₈ , which are known in the art. In some embodiments, the linker peptide is a rigid linker peptide, which can be used to completely isolate two linked proteins, maintaining their independent functions. Commonly used rigid linkers peptides include α-helix peptides, (XP)n, etc., where P represents proline, X can be any amino acid, preferably Ala, Lys, Glu, and n represents the number of XP repetitions. Those skilled in the art can adjust and select different linkers peptides according to specific application scenarios and the 3D structure requirements of the fusion protein.

In this application, "5' end side" is used to describe the relative positional relationship between two sequences within the same polynucleotide sequence. "5' end" refers to the end of the polynucleotide sequence containing a free 5'-hydroxyl group. For example, "on one side of the coding sequence of the HPV antigen polypeptide, there is further a coding sequence for an immunostimulatory factor or its functional domain," means that the "coding sequence for the immunostimulatory factor or its functional domain" is closer to the 5' end of the polynucleotide sequence in which they share a common location, relative to the "coding sequence of the HPV antigen polypeptide."

The term "signal peptide" refers to a short polypeptide chain that guides the localization or translocation of newly synthesized proteins. Among them, the signal peptide that guides the translocation of newly synthesized proteins to the secretory pathway is also called a "secretory signal peptide." In most cases, the signal peptide is located at the N-terminus of the amino acid sequence. In mRNA, the coding sequence of the signal peptide is usually located after the start codon, and is an RNA region encoding a hydrophobic amino acid sequence. After the signal peptide guides the protein to complete its localization, it is usually cleaved by a signal peptidase. The term "tPA-SP," or tissue plasminogen activator signal peptide, is a type of secretory signal peptide.

As used herein, the “hybrid of DNA and RNA sequences” is a polynucleotide sequence in which the nucleotides that make up the polynucleotide sequence are partly DNA and partly RNA.

The term "5' cap" is located at the 5' end of mRNA and contains a methylated guanosine monophosphate (GMP) linked to the 5' end of the mRNA via pyrophosphate, forming a 5',5'-triphosphate linker with the adjacent nucleotide. There are generally three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as type O, type I, and type II, respectively. Type O indicates that the ribose of the terminal nucleotide is unmethylated, type I indicates that the ribose of the terminal nucleotide is methylated, and type II indicates that the ribose of both terminal nucleotides is methylated. In this article, "CleanCap AG" refers to the m7G(5')ppp(5')(2'-OMeA)pG cap.

As used herein, the term "PolyA tail" or "PolyA sequence" refers to a continuous or interrupted sequence of adenosine 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 as needed. In mRNA, in the presence of a 3'-UTR, the Poly-A sequence is attached to the 3' end of the 3'-UTR. A continuous poly-A tail is characterized by a continuous sequence of adenosine residues. A 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 adenosine residues (A), particularly about 120 A residues. Typically, the vast majority of nucleotides in the PolyA tail are adenosine, meaning 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. However, the remaining nucleotides are allowed to be nucleotides other than A, such as U (uridine monophosphate), G (guanylic acid), or C (cytidine monophosphate).

As used herein, the percentage of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5%, refers to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment. For example, it is the percentage of positions with identical bases or amino acid residues out of the total number of positions, determined by introducing vacancies or other methods to make two sequences have as many identical residues as possible. The percentage of "identity" can be determined using software programs known in the art. It is preferred to use default parameters for alignment. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these procedures can be found at the following internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

As used herein, “complementarity” of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. Percentage complementarity indicates 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., approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity out of 10, respectively). “Complete complementarity” means that all consecutive residues in a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, “substantially complementary” means the degree of complementarity of at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in a region of approximately 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or refers to two nucleic acids hybridized under stringent conditions. For a single base or nucleotide, according to the Watson-Crick base pairing rule, when A pairs with T or U, or C pairs with G or I, it is called complementary or matched, and vice versa; all other base pairings are called non-complementary. In this application, the "complementary polynucleotide sequence" of a certain polynucleotide sequence refers to a polynucleotide sequence that is completely complementary to that polynucleotide sequence.

As used herein, a "delivery body" refers to a structure that, to facilitate the entry of larger biomolecules such as polynucleotides and polypeptides into cells, is packaged or encapsulated to form a structure with higher affinity for the cell membrane, making it easier for them to be transported across the membrane from the extracellular space to the intracellular space. Delivery bodies and their preparation methods are known in the art, including but not limited to liposomes (e.g., lipid nanoparticles (LNPs)), viruses (e.g., AAV, lentiviruses), and quantum dots. Methods for preparing LNPs are known in the art, for example, those disclosed in CN114901360A and CN113941011A. In some embodiments, the LNP comprises PEG-modified lipids, non-cationic lipids, sterols, ionizable lipids, or any combination thereof.

As used herein, the term "immunostimulatory factor" specifically refers to proteins, polypeptides, or nucleic acid molecules that can be produced by mammals themselves and can enhance the immune system's response to antigens, including but not limited to: cytokines that enhance the ability of immune cells to process and/or present antigens, such as dendritic cell growth factors (e.g., Flt3L); molecules that break down immunosuppression, including but not limited to immune checkpoint inhibitors; and pro-inflammatory cytokines (e.g., granulocyte-macrophage colony-stimulating factor, IFNα-2a, IFNα-2β, Pre-IFNα-2β, IL-2).

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

As used herein, “Flt3L” refers to FMS-like tyrosine kinase 3 ligand. In some embodiments of this application, Flt3L is human Flt3L, such as Flt3L recorded in the NCBI database with gene ID: 2323.

The term "immune checkpoint" refers to molecules in the immune system that can turn a signal (a co-stimulatory molecule) on or off. Many cancers protect themselves from the immune system by suppressing T-cell signaling. As used in this article, the term "immune checkpoint inhibitor" can help block this protective mechanism of cancer by acting on immune checkpoints. For example, immune checkpoint inhibitors can be antibodies or antigen-binding fragments of 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, galactagogue-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, VSIG8. PD-1 (programmed T cell death receptor) is a transmembrane protein found on the surface of T cells. When it binds to PD-L1 (programmed T cell death ligand 1) on tumor cells, it leads to the inhibition of T cell activity and a reduction in T cell-mediated cytotoxicity. Therefore, PD-1 and PD-L1 act as "switch-off switches" for immune downregulation or immune checkpoint signaling.

In this application, "immune checkpoint inhibitors" also include agonists of co-stimulatory molecules, such as agonists of CD28, CD122, and CD137. CD28 is constitutively expressed on almost all human CD4+ T cells and about half of CD8+ T cells, promoting 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, especially CD8+ T cells, from activation-induced cell death by mediating signal transduction.

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

As used herein, “mRNA” (messenger RNA) is any RNA, naturally occurring, non-naturally occurring, or modified amino acid polymer, encoding at least one protein, and is translatable to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Those skilled in the art will appreciate that, unless otherwise stated, the polynucleotide sequence described herein may use “T” to refer to thymine when representing a DNA sequence, but when the polynucleotide sequence represents RNA (e.g., mRNA), “T” will be replaced by “U” (uracil). Therefore, any DNA disclosed and identified by a specific sequence number (SEQ ID NO) herein also discloses an RNA (e.g., mRNA) sequence complementary to or corresponding to said DNA, wherein each “T” in said DNA sequence is replaced by a “U”.

As used in this article, an "open reading frame (ORF)" is a continuous segment of DNA or RNA 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.

The terms “fusion polypeptide” and “fusion protein” used herein are used interchangeably and should be understood to mean a polypeptide comprising a combination of sequences derived from different gene products (e.g., homologous proteins of different HPV subtypes, different proteins of the same HPV subtype, and non-homologous proteins of different HPV subtypes) or a combination of sequences from the same gene product (e.g., a single HPV protein), wherein these sequences originate from different/separate regions of the wild-type gene product. For example, a fusion polypeptide may comprise a combination of sequences typically separated by other sequence segments in the wild type, as well as a fusion of the remaining peptide segments after the removal of one or more sequence segments.

As used in this article, "at least includes" means either "includes" or "is". For example,

"From N to C, at least the following must be included sequentially:"

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”

This refers to:

a):

From N-end to C-end includes:

"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", wherein at least two adjacent amino acid sequences (e.g., sequences A and B, A and C, C and B) further contain one or more amino acids between them; or

b): From N-end to C-end includes:

"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”, wherein no more amino acids are contained between any two adjacent amino acid sequences (e.g., sequences A and B, A and C, C and B).

As used herein, when “direct link” is used to describe the relationship between two amino acid sequences, it means that no other amino acids are contained between the two amino acid sequences; in some embodiments, “direct link” means that the two amino acid sequences are linked by a chemical bond; in some embodiments, “direct link” means that the two amino acid sequences are linked by a peptide bond (amide bond).

Polynucleotide sequence

This application provides a polynucleotide sequence that can be used to prevent or treat HPV infection-related diseases. Through extensive comparison and experimentation, the inventors have finally determined that the polynucleotide sequence of this application can induce a significant HPV-specific immune response in healthy mice and has significant anti-tumor activity in HPV-positive tumor model mice.

The term "variant" refers to a sequence or molecule that retains the same or substantially the same biological activity as the original sequence. The variant may originate from the same or different species (e.g., homologous proteins from different mutant strains of the same HPV subtype), or it may be a synthetic sequence based on a natural molecule or an existing molecule. In this application, "variant" can refer to variants of proteins, peptides, or amino acid sequences, or variants of nucleic acid molecules or polynucleotide sequences.

Those skilled in the art can readily identify the variants of SEQ ID NO: 1 to 9. For example, sequence alignment can determine the position of each of the aforementioned amino acid sequences within the corresponding amino acid sequence of the corresponding HPV subtype protein. The sequences of all HPV subtype mutants exhibiting mutations at that position are variants of the aforementioned amino acid sequences. Therefore, the amino acid sequence shown in SEQ ID NO:1 or its variant contains the E6 protein amino acid sequence corresponding to the 1st to 85th amino acid sites of the E6 protein reference sequence (NCBI accession number QHA94929 or AAL96630.1) in the HPV-16 subtype mutant strain; the amino acid sequence shown in SEQ ID NO:2 or its variant contains the E7 protein amino acid sequence corresponding to the 1st to 65th amino acid sites of the E7 protein reference sequence (NCBI accession number ATI99837 or NP_041326.1) in the HPV-16 subtype mutant strain; the amino acid sequence shown in SEQ ID NO:3 or its variant contains the E6 protein amino acid sequence corresponding to the 71st to 158th amino acid sites of the E6 protein reference sequence in the HPV-16 subtype mutant strain; and the amino acid sequence shown in SEQ ID NO:4 or its variant contains the E6 protein amino acid sequence corresponding to the 51st to 98th amino acid sites of the E7 protein reference sequence in the HPV-16 subtype mutant strain. 7. The amino acid sequence of the protein; the amino acid sequence shown in SEQ ID NO:5 or its variant contains the amino acid sequence of the E6 protein corresponding to amino acid sites 1 to 85 of the E6 protein reference sequence (NCBI accession number ABP99784) in the HPV-18 subtype mutant strain; the amino acid sequence shown in SEQ ID NO:6 or its variant contains the amino acid sequence of the E7 protein corresponding to amino acid sites 1 to 65 of the E7 protein reference sequence (NCBI accession number UZQ21949 or ABP99785.1) in the HPV-18 subtype mutant strain; the amino acid sequence shown in SEQ ID NO:7 or its variant contains the amino acid sequence of the E6 protein corresponding to amino acid sites 71 to 158 of the E6 protein reference sequence in the HPV-18 subtype mutant strain; the amino acid sequence shown in SEQ ID NO:8 or its variant contains the amino acid sequence of the E7 protein corresponding to amino acid sites 51 to 105 of the E7 protein reference sequence in the HPV-18 subtype mutant strain. The amino acid sequence shown in SEQ ID NO: 9 is formed by the fusion of multiple E2 protein segments of HPV16, 18 and 31. The inventors of this application have demonstrated that the HPV E2 antigen sequence has the effect of enhancing the immunogenicity of HPV proteins.

In some embodiments, the "variant" of the amino acid sequence differs from the amino acid sequence by at least one amino acid, for example, by the addition, insertion, deletion, or substitution of at least one amino acid. For example, the amino acid substitution can be a conservative amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitution" can be polar to 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 to 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); or acidic to acidic amino acids, such as aspartic acid (D, Asp) and glutamic acid (E, Gln). u); basic amino acids paired with basic amino acids, such as arginine (R, Arg), histidine (H, His), and lysine (K, Lys); charged amino acids paired with charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys), and arginine (R, Arg); hydrophobic amino acids paired with hydrophobic amino acids, 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 contain non-conservative substitutions. In some embodiments, the "variants" of the amino acid sequence may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to the amino acid sequence. Compared to this amino acid sequence, a "variant" of this amino acid sequence can have an activity ranging from 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 any combination of the foregoing values. As used herein, a "conserved substitution variant" of a protein, polypeptide, or amino acid sequence refers to one or more amino acid residues in which amino acid substitutions have been made 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 parent protein's amino acid sequence in the manner described above as "conserved substitutions." Therefore, two proteins or amino acid sequences with similar functions may have varying degrees of similarity. For example, similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" also include peptides or enzymes that, as determined by BLAST or FASTA algorithms, have more than 60% amino acid identity, preferably more than 75%, ideally more than 85%, and even better if they have more than 90%, and have the same or substantially similar properties or functions as the natural or parental protein or enzyme.

Therefore, the HPV antigen polypeptide and the fusion polypeptide described in this application should cover the above-mentioned variants.

Those skilled in the art should know that variants of nucleic acid molecules or polynucleotide sequences encoding proteins include “synonymous mutants”, which are nucleic acid molecules or polynucleotide sequences obtained by replacing one or more codons in the nucleic acid molecule or polynucleotide sequence with other codons that encode the same amino acid as the codons.

The phrase "at least includes" means that the polynucleotide sequence may consist of the coding sequence of the HPV antigen peptide, or may include other polynucleotide sequences in addition to the coding sequence of the HPV antigen peptide.

For example, sequences that regulate the expression of the aforementioned HPV antigenic peptides, sequences that make the polynucleotide more stable, and other polynucleotide sequences that can promote the stimulation of immune responses by the aforementioned HPV antigenic peptides in the subject's body.

In some embodiments, the other polynucleotide sequence may be the coding sequence of any immunostimulatory factor. Preferred immunostimulatory factors include, for example, Flt3L.

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

mRNA vaccine

This application also provides an mRNA vaccine for the prevention or treatment of HPV infection-related diseases, wherein the mRNA vaccine contains the RNA sequence from the aforementioned polynucleotide sequence. The mRNA vaccine can be administered by introducing a polynucleotide sequence containing an HPV antigen polypeptide into a subject, where it is directly translated to form the corresponding antigen protein, inducing a specific immune response in the body, thereby achieving a preventive immune effect. Simultaneously, it can target and kill tumor cells containing HPV antigens.

Methods for preparing mRNA vaccines are known in the art. Specifically, the mRNA in the mRNA vaccine, in addition to containing the aforementioned polynucleotide sequences, further contains coding sequences for several essential functional components to express, regulate, or enhance the expression level of the aforementioned HPV antigen peptides. These functional components include, but are not limited to, a 5' cap, a 5' UTR, a 3' UTR, and a polytail. These functional components are known in the art, and those skilled in the art can select and combine them according to actual needs. Both the 5' UTR and 3' UTR are typically transcribed from genomic DNA and are elements present in pre-mature mRNA (or mRNA precursor or pre-mRNA). Characteristic structural features of mature mRNA (e.g., a 5'-cap and a 3'-poly(A) tail) are typically added to the transcribed (pre-mature) mRNA during mRNA processing. Therefore, in some embodiments, the mRNA is a pre-mRNA. In some embodiments, the mRNA is mature mRNA.

In some embodiments, the mRNA vaccine comprises the aforementioned RNA polynucleotide sequence having an open reading frame encoding at least one antigenic polypeptide having at least one modification and at least one 5' cap, and is formulated within lipid nanoparticles. Depending on the manufacturer's protocol, the following chemical RNA cap analogs can be used to simultaneously perform 5' capping of the polynucleotide during in vitro transcription to produce 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 (CleanCap AG). Post-transcriptional 5' capping of modified RNA using vaccinia virus capping enzymes produces an O-type cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). A type I cap structure can be produced using both vaccinia virus capping enzymes and 2'-O methyltransferases, resulting in m7G(5')ppp(5')(2'-OMeA)pG. This type I cap structure can also be produced using the CleanCap method. A type II cap structure can be produced by 2'-O-methylation of the 5'-tertiary nucleotide using 2'-O methyltransferases. A type III cap structure can be produced by 2'-O-methylation of the 5'-quadrupole nucleotide using 2'-O methyltransferases.

The 3'-Poly(A) tail is typically added to the 3' end of the transcribed mRNA. In some embodiments, it may contain up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-Poly(A) tail may be an essential element for the stability of the individual mRNA.

In some embodiments, the mRNA further comprises a stabilizing element. The stabilizing element may include, for example, a histone stem-loop. In some embodiments, the mRNA comprises 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 generally enhance the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, whose synergistic effect, although alternative mechanisms exist in nature, can increase protein expression to levels observed by either single element. 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 an intramolecular base pairing forming a loop between two adjacent portions separated by a spacer region (consisting of a short sequence) or completely inversely complementary sequences. Unpaired loop regions are generally unable to pair with either of the stem-loop elements. The stability of stem-loop structures typically depends on length, the number of mismatches or bulges, and the base composition of the paired regions. In some embodiments, wobbly base pairing (non-Watson-Crick base pairing) may occur. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length. In some embodiments, the mRNA does not contain a histone downstream element (HDE). A “histone downstream element” (HDE) comprises a purine-rich polynucleotide segment of approximately 15 to 20 nucleotides in the 3' of a naturally occurring stem-loop, representing a binding site for U7 snRNA involved in the processing of pre-histone mRNA into mature histone mRNA.

In some implementations, one or more AU-rich sequences of the mRNA may be removed. These sequences, sometimes referred to as AURES, are destabilizing sequences found in the 3' UTR. AURES may be removed from the mRNA. Alternatively, AURES may be retained in the mRNA.

In some embodiments, the mRNA is configured within lipid nanoparticles (LNPs). In some embodiments, lipids are mixed with the mRNA to form lipid nanoparticles. In some embodiments, RNA is formulated within lipid nanoparticles. In some embodiments, the lipid nanoparticles are initially formed as empty lipid nanoparticles and then combined with or encapsulated with the vaccine's mRNA just before administration (e.g., within minutes to one hour).

The lipid nanoparticles typically comprise ionizable lipids, noncationic lipids, sterols, and PEG lipid components, as well as target nucleic acids, such as the aforementioned mRNA. The lipid nanoparticles of this disclosure can be produced using components, compositions, and methods commonly known in the art, see, for example, PCT/US2016/052352, PCT/US2016/068300, PCT/US2017/037551, PCT/US2015/027400, PCT/US2016/047406, PCT/US2016000129, PCT/US2016/014280, PCT/U The entirety of PCT/US2016/014280, PCT/US2017/038426, PCT/US2014/027077, PCT/US2014/055394, PCT/US2016/52117, PCT/US2012/069610, PCT/US2017/027492, PCT/US2016/059575 and PCT/US2016/069491 are incorporated herein by reference.

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

Pharmaceutical compositions or pharmaceutical products

The pharmaceutical composition or pharmaceutical product provided in this application comprises the above-mentioned polynucleotide sequence, a nucleic acid molecule comprising the polynucleotide sequence, a delivery body or cell, a fusion polypeptide encoded by the above-mentioned polynucleotide sequence, or an mRNA vaccine. The nucleic acid molecule, delivery body, cell, fusion polypeptide, and mRNA in the mRNA vaccine have a purity that meets clinical requirements.

Optionally, the pharmaceutical composition or pharmaceutical product of this application may further comprise one or more other active compounds as needed for a specific indication of treatment. Preferably, the compounds have complementary, auxiliary, or promoting activities with the aforementioned nucleic acid molecules, delivery bodies, cells, or fusion peptides, such as enhancing the ability of the nucleic acid molecules, delivery bodies, cells, or fusion peptides to induce an immune response, or enhancing the immune system's immune response to the nucleic acid molecules, delivery bodies, cells, or fusion peptides, without adversely affecting each other. Such compounds may be present in the pharmaceutical composition or pharmaceutical product in an amount effective for the intended purpose. For example, in some embodiments, the other active compounds may comprise one or more immunostimulatory factors, such as Flt3L, granulocyte-macrophage colony-stimulating factor, IFNα-2a, IFNα-2β, Pre-IFNα-2β, IL-2, and immune checkpoint inhibitors such as PD-1 or PD-L1 antibodies.

The aforementioned active ingredients, such as nucleic acid molecules, delivery systems, cells, and fusion peptides, can be sandwiched or encapsulated within delivery systems or colloidal drug delivery systems, for example, sandwiched or encapsulated within 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 delivery system, colloidal particle, or microcapsule, or existing separately in different delivery systems, colloidal particles, or microcapsules.

Optionally, the pharmaceutical composition or pharmaceutical article 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 or other dried formulation. These pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to subjects at the doses and concentrations used, and include buffers (such as phosphates, citrates, histidines, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives, low molecular weight (less than about 10 amino acid residues) peptides, proteins (such as serum albumin, gelatin, or immunoglobulins); and 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 (such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol). Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices containing solid hydrophobic polymers of the immunoglobulins of the present invention, in the form of heteromorphic materials such as films or microcapsules.

It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and/or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments.

Example

Example 1: Sequence construction and preparation of HPV vaccine

1.1 Synthesis of HPV vaccine sequences and construction of recombinant vectors

In this embodiment, the antigen sequence of the HPV-related tumor mRNA vaccine is the E6 and E7 proteins of HPV types 16 and 18. The coding fragments of these proteins are tandemly used to obtain a polynucleotide sequence. Starting from the 5' end, the sequence includes: a T7 promoter with XbaI at the 5' end, 5'UTR, tPA-SP, Flt3L, HPV E2 (if present), E6/E7 protein or its variants, 3'UTR, and/or the coding nucleotide sequence of a polyA tail. This sequence is then double-digested with XbaI and NotI, and ligated with the pUC57-GW-Kan (金维智) vector backbone fragment digested with XbaI and NotI to construct a recombinant plasmid. The nucleic acid sequence component 1 used in Examples 1-6 has ORF encoding sequences of SEQ ID NO: 47-48, SEQ ID NO: 50-52, and SEQ ID NO: 54; the nucleic acid sequence component 2 used in Examples 7-8 has ORF encoding sequences of SEQ ID NO: 49, SEQ ID NO: 53, and SEQ ID NO: 54.

1.2 mRNA preparation

1.2.1 Plasmid linearization

The recombinant plasmid constructed in step 1 has a SapⅠ restriction site after the last A in the polyA tail sequence. The plasmid containing the target gene was linearized with the restriction endonuclease SapⅠ, and the reaction system is shown in Table 1. The digestion was carried out at 37℃ for 3 hours.

Table 1. Plasmid linearization enzyme digestion system

Take 2 μL of the enzyme digestion product and perform 1% agarose gel electrophoresis to check the linearization of the plasmid. Purify the linearized plasmid using a PCR product recovery kit (Comway Century).

(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 Zhaowei Technology Development Co., Ltd., product catalog number: ON-040; 5×Reaction Buffer, 100mM ATP Solution, 100mM CTP Solution, 100mM 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. 100mM ΨUTP Solution (pseudouridine triphosphate), full name: N1-Me-pUTP, 100mM, Shanghai Zhaowei Technology Development Co., Ltd., product catalog number: R5-027. Add each component according to the following system (Table 2) (taking a 20 μL reaction system as an example), mix well, and react at 37°C for 3 h.

Table 2. In vitro transcription system

5×Reaction Buffer 4μL ATP (100mM) 2μL ΨTP (100mM) 2μL CTP (100mM) 2μL GTP (100mM) 2μL Enzyme Mix 1μL Linearized DNA template 500ng-1μg CleanCap AG (100mM) 1μL <![CDATA[Nuclease-free H₂O]]> Fill to 20μL

Among them, CleanCap AG is m7G(5’)ppp(5’)(2’-OMeA)pG, with the product number ON-134, manufactured by Shanghai Zhaowei.

After the transcription reaction was complete, 1 μL of DNase I was added, and the mixture was incubated at 37°C for 15 min. Then, 15 μL of Ammonium Acetate Stop Solution was added and mixed well. Next, 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 incubated at -20°C for 30 min. The mixture was centrifuged at 12000 g for 15 min, and the RNA precipitate was discarded. 1 mL of 70% ethanol was added to wash the RNA, and the mixture was centrifuged at 12000 g for 5 min, and the supernatant was discarded. After air-drying, 50 μL of RNase-free water was added to dissolve the precipitate, and mRNA quantification was performed using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

1.3 Lipid Nanoparticle (LNP) Encapsulation

The mRNA stock solution obtained in step 1.2 was dispersed in 20 mM acetic acid solution (pH 5.0) to obtain an RNA solution with an mRNA concentration of 200 μg/mL. The mixture was then prepared by mixing ionizable lipids, cholesterol, DSPC, and DMG-PEG2000 in a molar ratio of 50:38.5:10:1.5. The flow rates of the aqueous and oil phases were controlled using a T-junction method to mix the mRNA with the lipid mixture. The syringe pump was then started to further mix the mRNA solution with the lipid mixture to form LNPs. The mixture was then diluted 10-fold with diluent, concentrated by ultrafiltration centrifugation, and subjected to three solution replacements. The resulting solution was adjusted to pH 7.0–8.0 with Tris aqueous solution to obtain an LNP-encapsulated mRNA solution. LNPs are lipid nanoparticles.

The concentration and particle size of LNP-loaded mRNA were determined using a Ribogreen RNA quantification kit (Invitrogen, R11490) and a Darwin ZetaSizer particle size analyzer. An LNP sample without any loaded material was included as a control.

Example 2: Evaluation of the cellular immunological dose-response effect of HPV-M mRNA vaccine

Twenty-five 6-8 week old SPF-grade female C57BL/6 mice were randomly divided into 5 groups of 5 mice each. The mice were immunized with the mRNA vaccine according to the grouping shown in Table 4, with immunizations every two weeks for a total of three immunizations. Seven days after immunization, the mice were sacrificed, and their spleens were harvested and placed on a 70 μm nylon cell filter. The cells were thoroughly ground in 2 ml of RPMI-1640 complete culture medium to prepare a cell suspension, and cell counts were performed.

Table 3. Grouping of mice for vaccine cell immune response detection

candidate vaccines dose Immunization methods Mouse number HPV-M 2.5μg Intramuscular injection 5 HPV-M 12.5μg Intramuscular injection 5 HPV-M 25μg Intramuscular injection 5 HPV-M 50μg Intramuscular injection 5 LNP 50μg Intramuscular injection 5

2.1 ELISpot detection of HPV16 and 18 types E6 and E7 specific IFNγ and IL-2 levels

Cells were seeded at a density of 1.5 × ^10⁵ cells per well into ELISpot plates, and an overlapping peptide library of HPV16 and 18 E6 and E7 proteins (synthesized by Cyprom) was added. The peptides contain 15 amino acid sequences, with 8 amino acid residues overlapping each consecutive peptide. Positive control wells were treated with phorbol ester (PMA) and ionomycin, while negative control wells received no stimulant. The plates were then incubated at 37°C for 20 hours in a 5% CO₂ incubator. Following the ELISpot kit instructions (Dakeway, 2210001 and Mabtech, 2210001), the cells were incubated with antibodies and subjected to color development. 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 (SFUs).

2.2 Flow cytometry detection of HPV16 and 18 types E6 and E7 specific T cell responses

To comprehensively evaluate the cellular immune response induced by the HPV vaccine, intracellular cytokine staining was performed. The obtained single cells were lysed and filtered, and 1 x ^10⁶ cells were seeded into each well of a 96-well U-bottom cell culture plate. HPV16 and 18 E6 and E7 protein overlapping peptide libraries (synthesized by Cyprom) or PMA and Ionomycin were used as stimulants, and BFA and monensin (Biolegend, 420601 and 420701) were used as blocking agents. The plates were incubated overnight at 37°C in a 5% CO₂ incubator. Extracellular staining (CD3-cy5.5, CD4-APC or FITC, and CD8-APC or FITC) was performed first, followed by cell lysis and fixation, and intracellular cytokine staining (IFN-γ-PE or TNF-α-PE). The resulting cells were analyzed using a Cytoflex flow cytometer (Beckman Coulter). The levels of IFN-γ and TNF-α in CD4+ and CD8+ cells were obtained by gating cells.

One-way ANOVA was used to analyze the significance of differences between the experimental and control groups (***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 Elispot and flow cytometry results showed that the cellular immune response induced by HPV-M was dose-dependent, reaching a plateau after 25μg.

Example 3: Pharmacodynamic evaluation of HPV-M mRNA vaccine

3.1 Antitumor effect after intramuscular or intratumoral injection of HPV-M vaccine

This study evaluated the pharmacodynamics of the HPV-M vaccine using a TC-1 mouse tumor model. Six- to eight-week-old SPF-grade female C57BL/6 mice were subcutaneously inoculated with logarithmically growing TC-1 cells. When the tumor volume reached 100 ^mm³ , mice were randomly assigned to groups of 10 mice each, based on tumor volume. Mice were immunized with the mRNA vaccine weekly for three weeks, according to the groupings shown in Table 4. Tumor size was measured twice weekly, and mouse survival was recorded. Tumor volume was calculated using the following formula: Tumor volume = major axis × minor axis × minor axis / 2, i.e., V = ab²/2. One-way ANOVA and Log-rank were used to perform statistical analyses of tumor volume and survival rate among the groups.

Table 4. Mouse grouping for HPV-M vaccine efficacy evaluation

candidate vaccines dose Immunization methods Mouse number HPV-M 6.25μg Intramuscular injection 10 HPV-M 12.5μg Intramuscular injection 10 HPV-M 25μg Intramuscular injection 10 LNPCtrl 25μg Intramuscular injection 10 HPV-M 6.25μg Intratumoral injection 10 HPV-M 12.5μg Intratumoral injection 10 HPV-M 25μg Intratumoral injection 10 LNPCtrl 25μg Intratumoral injection 10

As shown in Figure 2, in the TC-1 mouse tumor model, regardless of whether the injection was intramuscular or intratumoral, compared with the control group treated with LNP, the average tumor volume of mice treated with each dose of HPV-M was significantly reduced, and the survival time of the mice was also significantly prolonged.

3.2 Antitumor effect of HPV-M vaccine after injection around muscles or lymph nodes near tumors

Following the evaluation method for anti-tumor effects described above, mice were immunized with the mRNA vaccine according to the grouping shown in Table 5.

Table 5. Mouse grouping for HPV-M vaccine efficacy evaluation

candidate vaccines dose Immunization methods Mouse number HPV-M 6.25μg Intramuscular injection 10 HPV-M 12.5μg Intramuscular injection 10 HPV-M 25μg Intramuscular injection 10 LNPCtrl 25μg Intramuscular injection 10 HPV-M 6.25μg Perilymphatic injection 10 HPV-M 12.5μg Perilymphatic injection 10 HPV-M 25μg Perilymphatic injection 10 LNPCtrl 25μg Perilymphatic injection 10

As shown in Figure 3, in the TC-1 mouse tumor model, regardless of whether it was intramuscular injection or perilymphatic injection, all doses of HPV-M significantly inhibited tumor growth and significantly prolonged the survival of mice compared with the control group treated with LNP.

Example 4: Evaluation of cellular immunity in mice after inoculation with different HPV mRNA vaccines

Thirty-five 6-8 week old SPF-grade female C57BL/6 mice were randomly divided into 7 groups of 5 mice each. Mice were immunized with the mRNA vaccine according to the grouping shown in Table 4, with immunizations every two weeks for a total of two immunizations. Seven days after immunization, the mice were sacrificed, and their spleens were harvested and placed on a 70 μm nylon cell filter. The cells were thoroughly ground in 2 ml of RPMI-1640 complete culture medium to prepare a cell suspension, and cell counts were performed.

Table 6. Grouping of Mice for Vaccine Cellular Immunoreactivity Detection

candidate vaccines dose Immunization methods Mouse number HPV-1 5μg Intramuscular injection 5 HPV-2 5μg Intramuscular injection 5 HPV-3 5μg Intramuscular injection 5 HPV-4 5μg Intramuscular injection 5 HPV-M 5μg Intramuscular injection 5 LNP 5μg Intramuscular injection 5

4.1 ELISpot detection of HPV types E6 and E7 specific IFNγ and IL-2 levels

The levels of antigen-specific IFNγ and IL-2 induced after vaccination with the five HPV vaccines were detected using the same ELISpot method as in Example 2.

As shown in Figure 4, after two injections of a low-dose 5 μg vaccine, the average number of IFN-γ and IL-2 spots in the ^spleen cells of mice in all five experimental groups was greater 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 significantly increased the levels of IFN-γ and IL-2 expressed in mouse spleen cells after stimulation with specific peptides, effectively inducing antigen-specific cellular immune responses. One-way ANOVA was performed using GraphPad Prism 8 software to analyze the significance between groups (***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).

4.2 Flow cytometry detection of HPV16 and 18 types E6 and E7 specific T cell responses

Simultaneously, the antigen-specific T-cell responses induced after vaccination with the five HPV vaccines were detected using the same flow cytometry method as in Example 2. Cell gating was used to obtain the levels of IFN-γ and TNF-α in CD4+ and CD8+ cells, and one-way ANOVA was used for significance analysis between the experimental and control groups. Student's t-test was used for pairwise comparisons between the experimental groups and the original HPV-M sequence. The results are shown in Figure 5. With two 5μg low-dose immunizations, the percentages of IFN-γ and TNF-α in CD4 ⁺ and CD8 ⁺ cells in the spleen cells of mice in the four experimental groups (HPV-1, HPV-2, HPV-3, and HPV-4) were significantly higher than those in the HPV-M vaccine group, indicating that vaccination successfully induced an effective immune response. One-way ANOVA was used to analyze the significance of the differences between groups using GraphPadPrism 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).

Example 5: Evaluation of cellular immunity in mice after inoculation with different HPV mRNA vaccines

Twenty-five 6-8 week old SPF-grade female C57BL/6 mice were randomly divided into 7 groups of 5 mice each. Mice were immunized with the mRNA vaccine according to the grouping shown in Table 4, with immunizations every two weeks for a total of three immunizations. Seven days after immunization, the mice were sacrificed, and their spleens were harvested and placed on a 70 μm nylon cell filter. The cells were thoroughly ground in 2 ml of RPMI-1640 complete culture medium to prepare a cell suspension, and cell counts were performed.

Table 7. Grouping of Mice for Vaccine Humoral Immune Response Detection

candidate vaccines dose Immunization methods Mouse number HPV-1 12.5μg Intramuscular injection 5 HPV-4 12.5μg Intramuscular injection 5 HPV-5 12.5μg Intramuscular injection 5 HPV-M 12.5μg Intramuscular injection 5 LNP 12.5μg Intramuscular injection 5

5.1 ELISpot detection of HPV types E6 and E7 specific IFNγ and IL-2 levels

The levels of antigen-specific IFNγ and IL-2 induced after the above four HPV vaccinations were detected using the same ELISpot method as in Example 2.

As shown in Figure 6, at the three-dose immunization with a 12.5 μg dose, the mean number of IFN-γ and IL-2 spots in the SFU/ ^10⁶ splenocytes of mice in all four experimental groups was greater than that in the negative control group. Compared with the HPV-M vaccine group, the HPV-1, HPV-4, and HPV-5 vaccine groups significantly increased the levels of IFN-γ and IL-2 expressed in mouse splenocytes after stimulation with specific peptides, indicating that these three vaccines significantly enhanced antigen-specific cellular immune responses. One-way ANOVA statistical analysis was performed using GraphPad Prism 8 software to analyze the significance between groups (***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).

5.2 Flow cytometry detection of HPV16 and 18 E6 and E7 specific T cell responses

Simultaneously, the antigen-specific T-cell responses induced after the four HPV vaccines were detected using the same flow cytometry method as in Example 2. The levels of IFN-γ and TNF-α in CD4+ and CD8+ cells were obtained by gating the cells, and the significance between the experimental groups was analyzed using one-way ANOVA. As shown in Figure 7, at the three-dose immunization with a 12.5 μg dose, the percentages of IFN- ^γ and TNF ^- α in the spleen cells of mice in all four experimental groups were 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 a stronger antigen-specific cellular immune response than the HPV-M vaccine. One-way ANOVA was used to analyze the significance of the differences between groups 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).

Example 6: Antitumor effects of different HPV mRNA vaccines in a TC-1 mouse tumor model

In this study, the pharmacodynamics of different HPV mRNA vaccines were evaluated in a TC-1 mouse tumor model using the same methods as in Example 3. Mice were administered the mRNA vaccines according to the groupings shown in Table 8.

Table 8. Mouse grouping for HPV-M vaccine efficacy evaluation

candidate vaccines dose Immunization methods Mouse number HPV-1 12.5μg Intramuscular administration 10 HPV-4 12.5μg Intramuscular administration 10 HPV-5 12.5μg Intramuscular administration 10 HPV-M 12.5μg Intramuscular administration 10 LNP 12.5μg Intramuscular administration 10

As shown in Figure 8, in the TC-1 mouse tumor model, compared with the control group treated with LNP, the average tumor volume of mice treated with each vaccine group was significantly reduced, and the survival time of the mice was also significantly prolonged. At 22 days post-administration, the proportions of mice with tumor disappearance in the HPV-M, HPV-1, HPV-4, and HPV-5 vaccine groups were 30% (3/10), 50% (5/10), 60% (6/10), and 70% (7/10), respectively. Therefore, overall, at the same dose, HPV-1, HPV-4, and HPV-5 showed better antitumor effects than HPV-M vaccine, with HPV-5 showing the best results.

Example 7: Pharmacodynamic evaluation of different HPV mRNA vaccines

6.1 Antitumor effect after intramuscular injection of HPV mRNA vaccine

In this study, the pharmacodynamics of different HPV mRNA vaccines were evaluated in a TC-1 mouse tumor model using the same methods as in Example 3. Mice were immunized with the mRNA vaccine according to the groupings shown in Table 9, once a week for three weeks (Figure 9). Tumor size was measured three times a week, and the survival status of the mice was recorded.

Table 9. Mouse grouping for HPV-M vaccine efficacy evaluation

candidate vaccines dose Immunization methods Mouse number LNP Ctrl 12.5μg Intramuscular injection 10 HPV-M 12.5μg Intramuscular injection 10 HPV-1 12.5μg Intramuscular injection 10 HPV-4 12.5μg Intramuscular injection 10 HPV-5 12.5μg Intramuscular injection 10

As shown in Figure 9, in the TC-1 mouse tumor model, the average tumor volume of mice treated with each vaccine group was significantly reduced compared to mice treated with LNP (as a control group). At 22 days post-administration, the proportions of mice with tumor disappearance in the HPV-M, HPV-1, HPV-4, and HPV-5 vaccine groups were 40% (4/10), 70% (7/10), 70% (7/10), and 90% (9/10), respectively. The survival time of mice treated with each vaccine group was also significantly prolonged, with the HPV-5 treatment group showing significantly better results than the HPV-M treatment group. Therefore, overall, at the same dose, HPV-1, HPV-4, and HPV-5 vaccines showed better anti-tumor effects than HPV-M vaccines, with HPV-5 being the most effective.

6.2 Study on the long-term anti-tumor effects and immune memory of different HPV mRNA vaccines

Mice whose tumors had completely regressed were selected, and on day 68 after the initial administration, TC-1 tumor cells were re-inoculated into the contralateral side of the back of the mice in an equal amount. Normal mice that had not received the drug were used as a control group. Tumor growth and mouse survival were recorded, and the cellular immune levels of the mice were detected at the experimental endpoint using the same ELISpot and flow cytometry methods as in Example 2.

As shown in Figure 9, compared to the rapid tumor growth in the control group, all mice administered the HPV mRNA vaccine (excluding the 14.3% (1/7) deaths in the HPV-4 group) showed no tumor recurrence and maintained a completely tumor-free state for at least 42 days. As shown in Figure 9, the mean number of IFN-γ and IL-2 spots in the SFU/ ^10⁶ splenocytes of the four experimental groups was greater than that in the negative control group, and the percentages of CD4 ⁺ and CD8 ⁺ cells and their IFN-γ and TNF-α in the splenocytes were also higher in the four groups. Specifically, HPV-4 and HPV-5 significantly increased the percentages of IFN-γ and TNF-α in CD8 ⁺ cells. These results indicate that these four vaccines, especially HPV-5, can induce long-term antigen-specific immune memory, providing strong protection against tumor recurrence. One-way ANOVA was performed using GraphPad Prism 8 software to analyze the significance of differences between groups (***p<0.001 vs. LNP control; **p<0.01 vs. LNP control; *p<0.05 vs. LNP control; ns, no significant difference).

Example 8 Evaluation of the antitumor effect of HPV-5 mRNA vaccine combined with PD-L1 antibody

This study evaluated the antitumor efficacy of HPV-5 mRNA vaccine combined with PD-L1 antibody 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. Both the PD-L1 antibody (10F.9G2) and rabbit isotype IgG2b (LTF-2) were purchased from BioXcell, and the dosage was 10 mg/kg via intraperitoneal injection. The specific administration regimen is shown in Figure 10. Tumor size was measured three times weekly, and mouse survival was recorded. Tumor growth curves and survival curves were plotted based on the measurement results. The relative tumor proliferation rate (T/C (%)) and the synergistic efficiency of the drug combination were calculated using relative tumor volume (RTV). The specific calculation formula is as follows: RTV = _Vt / _V0 , where _Vt and _V0 are the tumor volumes measured at a certain time point (dt) and at the time of separate administration (d0), respectively. T/C (%) = ( _TRTV / _CRTTV ) × 100%, where _TRTV / _CRTTV are the RTV values of the experimental group and the control group, respectively. Synergistic efficiency (%) of drugs A and B = ( _AT/C × _B/C / AB _/C ) × 100%, where _AT/C , B _/C , and _AB/C are the T/C values of drug A, drug B, and the combination of drugs AB, respectively.

Table 10. Mouse grouping for HPV-5 vaccine efficacy evaluation

As shown in Figure 10, in the TC-1 mouse tumor model, both the PBS control group and the PD-L1 antibody monotherapy group exhibited rapid tumor growth and shorter survival. Compared with PBS, the 3 μg HPV-5 monotherapy group, especially the HPV-5 combined with PD-L1 antibody group, significantly inhibited tumor growth and significantly prolonged mouse survival.

Table 11 Analysis of tumor growth data in a mouse TC-1 tumor model using a combination of HPV-5 and PD-L1 antibodies.

Tumor data were calculated on day 16 after administration, and the results are shown in Table 11. The synergistic efficiencies of HPV-5 (0.3 μg) and HPV-5 (3 μg) combined with PD-L1 antibody were 2.91 and 19.60, respectively (a value >1 indicates a synergistic effect). This indicates that HPV-5 mRNA vaccine and PD-L1 antibody have a good anti-tumor synergistic effect.

The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences, and when they represent RNA sequences, "T" represents uridine.

Sequence List:

Claims (40)

1. A polynucleotide molecule comprising at least the coding sequence of an HPV antigenic polypeptide, said antigenic polypeptide comprising at least the following 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; in, Amino acid sequence A contains at least SEQ ID NO: 1-4 or variants thereof from the N-terminus to the C-terminus, and the amino acid sequences shown in SEQ ID NO are directly linked or linked by linking peptides in sequence. Amino acid sequence B contains at least SEQ ID NO:5-8 or variants thereof from the N-terminus to the C-terminus, and the amino acid sequences shown in SEQ ID NO are directly linked or linked by linking peptides in sequence. The amino acid sequence C contains the HPV E2 antigen sequence. Preferably, the variant is a conservative substitution variant. 2. The polynucleotide molecule according to claim 1, wherein the HPV E2 antigen sequence is SEQ ID NO:9 or a variant thereof. 3. The polynucleotide molecule according to 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 is composed of 2-10 amino acid residues, preferably, the amino acid residues are glycine, serine and/or alanine residues, and more preferably, the linker peptide is composed of two alanine residues. 5. The polynucleotide molecule according to claim 1, wherein 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 an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with it. 6. The polynucleotide molecule according to any one of claims 1-5, further comprising, on one side of the 5' end of the coding sequence of the HPV antigen polypeptide, a coding sequence of an immunostimulatory factor or its functional domain thereof. 7. The polynucleotide molecule according to claim 6, wherein the immunostimulatory factor is Flt3L. 8. The polynucleotide molecule of claim 7, wherein the polypeptide sequence of the immunostimulatory factor comprises at least the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:10. 9. The polynucleotide molecule according to any one of claims 1-8, further comprising a secretory signal peptide coding sequence, preferably, the secretory signal peptide coding sequence being located on one side of the 5' end of the coding sequence of the HPV antigen polypeptide. 10. The polynucleotide molecule according to claim 9, wherein the secretory signal peptide is tPA-SP. 11. The polynucleotide molecule of claim 10, wherein the secretory signal peptide comprises an amino acid sequence as shown in SEQ ID NO:11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:11. 12. The polynucleotide molecule according to any one of claims 9-11, comprising, from the 5' end to the 3' end, the coding sequences of the secretory signal peptide, the immunostimulatory factor, and the HPV antigen polypeptide connected in sequence. 13. The polynucleotide molecule according to any one of claims 1-12, wherein it is DNA, RNA, or a hybrid of DNA and RNA. 14. The polynucleotide molecule according to any one of claims 1-13, comprising, being composed of, or encoded by any polynucleotide sequence selected from SEQ ID NO: 28-54. 15. The polynucleotide molecule according to any one of claims 1-14, further comprising a 5’UTR structure, preferably, the 5’UTR structure comprising at least the polynucleotide sequence shown 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 with SEQ ID NO:22 or SEQ ID NO:25. 16. The polynucleotide molecule according to any one of claims 1-15, further comprising a 3’UTR structure, preferably, the 3’UTR structure comprising at least the polynucleotide sequence shown 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 with SEQ ID NO:23 or SEQ ID NO:26. 17. The polynucleotide molecule according to any one of claims 1-16, wherein it is an mRNA molecule. 18. The polynucleotide molecule according to claim 17, wherein some or all of the uridine in the mRNA molecule is chemically modified uridine, preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine. 19. The polynucleotide molecule according to 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. The polynucleotide molecule according to any one of claims 17-19, wherein the mRNA further comprises a polyA tail, preferably, the polyA tail sequence comprising at least 50, at least 60, or at least 100 A nucleotides; preferably, the polyA tail comprises at least the polynucleotide sequence shown 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 with SEQ ID NO:24 or SEQ ID NO:27. 21. The complementary polynucleotide molecule of the polynucleotide molecule according to any one of claims 1-20. 22. A fusion polypeptide encoded by a polynucleotide molecule according to any one of claims 1-20 or having the same amino acid sequence as a polypeptide encoded by a polynucleotide molecule according to any one of claims 1-20. 23. The fusion polypeptide of claim 22, comprising the amino acid sequence shown in 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 with the amino acid sequence shown in 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 system comprising a polynucleotide molecule according to any one of claims 1-21 or a fusion polypeptide according to claim 22 or 23. 25. The delivery body according to claim 24, wherein the delivery body is a lipid nanoparticle (LNP). 26. The delivery body of claim 25, wherein the LNP comprises ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-lipids. 27. A cell comprising a polynucleotide molecule according to any one of claims 1-21 or a fusion polypeptide according to claim 22 or 23. 28. A pharmaceutical composition or pharmaceutical article comprising a polynucleotide molecule according to any one of claims 1-21, a fusion polypeptide according to claim 22 or 23, a delivery body according to claims 24-26, or a cell according to claim 27. 29. The pharmaceutical composition or pharmaceutical product according to claim 28, further comprising an immunostimulatory factor and/or an adjuvant. 30. The pharmaceutical composition or pharmaceutical product according to claim 29, wherein the immunostimulatory factor 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 immune checkpoint inhibitors (preferably PD-1 inhibitors or PD-L1 inhibitors). 31. The pharmaceutical composition or pharmaceutical product according to claim 30, wherein the STING is STING ^V155M . 32. The pharmaceutical composition or pharmaceutical product according to claim 30, wherein the immunostimulatory factor is a protein or a nucleic acid molecule encoding the protein, preferably, the nucleic acid molecule is mRNA. 33. The pharmaceutical composition or pharmaceutical product according to any one of claims 28-32, wherein it is an mRNA vaccine. 34. A method for treating or preventing HPV infection or HPV-related diseases, comprising administering to an individual a polynucleotide molecule according to any one of claims 1-21, a fusion polypeptide according to claim 22 or 23, a delivery medium according to any one of claims 24-26, a cell according to claim 27, or a pharmaceutical composition or pharmaceutical article according to any one of claims 28-33. 35. The method of claim 34, wherein the HPV infection-related disease is cervical cancer. 36. The method of claim 35, wherein the administration is intratumoral, perilymphatic, or intramuscular injection. 37. The method of claim 36, further comprising administering to an individual an immunostimulatory factor, chemotherapy, radiotherapy, and/or targeted therapy. 38. The method according to claim 37, wherein the immunostimulatory factor 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 immune checkpoint inhibitors (preferably PD-1 inhibitors or PD-L1 inhibitors). 39. The method according to claim 38, wherein the immunostimulatory factor is a protein or a nucleic acid molecule encoding the protein, preferably, the nucleic acid molecule is mRNA. 40. Use of the polynucleotide molecule according to any one of claims 1-21, the fusion polypeptide according to claim 22 or 23, the delivery body according to any one of claims 24-26, the cell according to claim 27, or the pharmaceutical composition or pharmaceutical article according to any one of claims 28-33 in the preparation of a medicament for treating or preventing HPV infection or HPV infection-related diseases.