Human papilloma virus (HPV) mRNA vaccine composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a multivalent HPV mRNA vaccine and a preparation method thereof. The present invention provides self-replicating and / or non-self-replicating mRNA molecules. The mRNA molecule provided by the invention is used for connecting E6 and E7 genes from HPV16 and HPV18, and four antigens can be expressed at the same time. After a therapeutic mRNA molecule obtained by combining the polypeptide with other elements of an mRNA vaccine is encapsulated by lipid nanoparticles, E6 and E7 four antigens of HPV16 and HPV18 can be simultaneously and successfully expressed in vivo and in vitro. When the vaccine is used for immunizing mice, higher cellular immune level and tumor curative effect can be generated, and the vaccine has a wide application prospect.

Description

[0001] This application claims priority to a Chinese patent application filed with the China Patent Office on June 20, 2023, with application number 202310735541.8 and invention name “A human papillomavirus (HPV) mRNA vaccine composition, its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biopharmaceutical technology, and in particular to an HPV mRNA vaccine and a preparation method and application thereof. Background Art

[0003] Human papillomavirus (HPV) is a DNA virus composed of nearly 8000bp double-stranded circular DNA. So far, more than 100 types of HPV viruses have been identified. Persistent infection with highly pathogenic HPV, including HPV-16, 18, 31, 45, 52, 58 and nearly 20 other types of HPV, can easily lead to the occurrence of malignant tumors including cervix, penis, vulva, vagina, anus and oropharynx. Globally, these HPV-related malignancies account for 4.5% of all human cancers. Preliminary estimates show that since 2012, the number of related cases has been between 500,000 and 600,000 per year. Cervical cancer is the second most common malignant tumor in women, and it has a particularly serious impact on developing countries. In my country, there were 111,000 new cases of cervical cancer in 2015, and as many as 34,000 deaths.

[0004] Although there are several preventive HPV vaccines on the market, including the bivalent Cervarix (targeting HPV16 and 18) launched by GlaxoSmithKline in 2006, the quadrivalent vaccine Gardasil (targeting HPV6, 11, 16 and 18) launched by Merck in 2009, and the nine-valent vaccine Gardasil (targeting HPV6, 11, 16, 18, 31, 33, 45, 52 and 58) launched in 2018. These vaccines are very effective in preventing new infections, especially against highly carcinogenic HPV-16 and -18 and low carcinogenic HPV-6 and -11, which can induce strong anti-HPV immune effects. However, millions of people with persistent HPV infection or HPV-related malignancies are unlikely to benefit from these existing HPV preventive vaccine products. Therefore, the development of new therapeutic HPV vaccines, especially those targeting important proto-oncogenes in highly pathogenic HPV, such as E6 and E7, is extremely meaningful.

[0005] Using our company's mature self-replicating mRNA vaccine and LNP formulation development platform, we have developed a number of HPV therapeutic self-replicating mRNA vaccines. In immunogenicity testing experiments, these LNP-self-replicating mRNA vaccines showed strong cellular immunity levels. In TC-1 (highly expressing HPV16 E6 and E7 antigens) and CT26 (HPV18) transgenic cell tumor-bearing mouse models, these self-replicating mRNA vaccines also showed strong tumor inhibition effects. Summary of the invention

[0006] The first object of the present invention is to provide an mRNA vaccine, and more specifically, to provide an HPV mRNA vaccine that can induce an immune response against HPV.

[0007] The second object of the present invention is to provide the mRNA construct contained in the above-mentioned vaccine, and the corresponding vectors and cells.

[0008] The third object of the present invention is to provide the pharmaceutical use of the mRNA construct, and the corresponding vectors and cells.

[0009] The fourth object of the present invention is to provide a method for inducing an immune response in an HPV-positive subject in need thereof based on the above-mentioned vaccine, or a method for treating an HPV-positive subject in need thereof.

[0010] In order to solve the above technical problems, the present invention includes the following technical solutions:

[0011] In one aspect, the present invention provides an mRNA molecule construct, characterized in that the mRNA molecule is self-replicating and / or non-self-replicating.

[0012] In some specific embodiments, the mRNA is circular RNA and / or TaRNA.

[0013] In some specific embodiments, the mRNA molecule construct includes a linked coding segment, and the protein encoded by the coding segment has HPV virus antigen-specific immunogenicity.

[0014] In some specific embodiments, the HPV virus antigen protein epitopes include proteins E6 and E7.

[0015] In some specific embodiments, the HPV virus antigen protein epitopes include E6 and E7 proteins from HPV virus subtypes 16 and 18.

[0016] In some specific embodiments, the mRNA molecule construct includes E6 and E7 proteins from HPV virus subtypes 16 and 18 connected in tandem.

[0017] In some specific embodiments, the mRNA molecule construct also includes a 5'UTR.

[0018] In some specific embodiments, the mRNA molecule construct also includes a 3'UTR.

[0019] In some specific embodiments, the mRNA molecule construct also includes a poly (A) sequence.

[0020] In some specific embodiments, the mRNA molecule construct also includes a signal peptide sequence.

[0021] In some specific embodiments, the signal peptide sequence is an IgE signal peptide.

[0022] In some specific embodiments, the self-replicating mRNA molecule construct further comprises non-structural proteins nsP1-4. In some specific embodiments, the nsP comprises the sequence shown in SEQ ID NO:9.

[0023] In some specific embodiments, the 5'UTR comprises a sequence as shown in SEQ ID NO:10 or SEQ ID NO:11.

[0024] In some specific embodiments, the 3'UTR comprises a sequence as shown in SEQ ID NO:12 or SEQ ID NO:13.

[0025] In some specific embodiments, the poly (A) sequence comprises a sequence as shown in SEQ ID NO:14 or SEQ ID NO:15.

[0026] In some specific embodiments, the IgE signal peptide comprises the sequence shown in SEQ ID NO:16.

[0027] In some specific embodiments, the protein encoded by the mRNA comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8;

[0028] In some specific embodiments, the protein encoded by the mRNA comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8;

[0029] In some specific embodiments, the protein encoded by the mRNA comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0030] In some specific embodiments, the protein encoded by the mRNA independently contains 1-12 amino acid substitutions relative to at least one of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0031] In some specific embodiments, the 1-12 amino acid substitutions are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions.

[0032] In some specific embodiments, the amino acid substitution is a conservative amino acid substitution;

[0033] In some specific embodiments, the substitutions are highly conservative amino acid substitutions.

[0034] In some specific embodiments, the protein encoded by the mRNA comprises an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0035] In some specific embodiments, the mRNA molecule construct comprises a coding nucleotide sequence corresponding to the antigen protein.

[0036] In some specific embodiments, the encoding nucleotide sequence corresponding to the antigen protein is a codon-optimized sequence.

[0037] In some specific embodiments, the encoding nucleotide sequence corresponding to the antigen protein comprises a nucleotide sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0038] In some specific embodiments, the mRNA comprises a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0039] In another aspect, the present invention provides a vaccine comprising any one of the mRNA molecule constructs described herein.

[0040] In some specific embodiments, the vaccine further comprises a delivery formulation;

[0041] In some specific embodiments, the delivery formulation is a nanoparticle.

[0042] In some specific embodiments, the delivery preparation includes lipid nanoparticles (LNP), lipopolyplex (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc.

[0043] In some specific embodiments, the nanoparticles are lipid nanoparticles (LNP).

[0044] In some specific embodiments, the vaccine uses any one of the mRNA molecule constructs described herein or a combination thereof as an immunogenic component.

[0045] In some specific embodiments, the vaccine is an HPV vaccine.

[0046] In another aspect, the present invention provides a vector comprising any one of the mRNA constructs described herein, or a combination thereof.

[0047] In another aspect, the present invention provides a cell, characterized in that it comprises any one of the mRNA constructs described herein, or a combination thereof, or any one of the vectors described herein.

[0048] In another aspect, the present invention provides a nanoparticle, characterized in that it comprises any one of the mRNA constructs described herein, or a combination thereof.

[0049] In some specific embodiments, the nanoparticles include lipid nanoparticles (LNP), lipid multipolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc.

[0050] In some specific embodiments, the nanoparticles are lipid nanoparticles (LNP).

[0051] In another aspect, the present invention provides use of any one of the mRNA constructs, vaccines, vectors, cells, or nanoparticles described herein in the preparation of a medicament for inducing an immune response in a subject in need thereof.

[0052] In another aspect, the present invention provides use of any one of the mRNA constructs, vaccines, vectors, cells, or nanoparticles described herein in the preparation of a medicament for treating or preventing an HPV-related disease in a subject in need thereof.

[0053] In another aspect, the present invention provides a method for inducing an immune response in a subject in need thereof, characterized by administering any one of the vaccines described herein to the subject;

[0054] In some specific embodiments, the methods induce an immune response against HPV by using a replicating or non-replicating virus.

[0055] In another aspect, the present invention provides a method for treating or preventing an HPV-related disease in a subject in need thereof, characterized by administering any one of the vaccines described herein to the subject.

[0056] In some specific embodiments, the methods induce an immune response against HPV by using a replicating or non-replicating mRNA vaccine.

[0057] In some specific embodiments, the vaccine composition is administered as part of a therapeutic regimen;

[0058] In some specific embodiments, the treatment regimen is radiation therapy, targeted therapy, immunotherapy, or chemotherapy.

[0059] In some specific embodiments, the individual is HPV positive.

[0060] In some specific embodiments, the HPV-related disease comprises cancer or a tumor.

[0061] In some specific embodiments, the cancer or tumor includes, for example, cervical cancer, anal cancer, penile cancer, vaginal cancer, vulvar cancer, and oropharyngeal cancer of the back of the throat, etc.

[0062] Beneficial Effects

[0063] Compared with the prior art, the technical solution of the present invention has at least one of the following beneficial effects:

[0064] 1. The HPV mRNA vaccine delivered by LNP of the present invention has good immunogenicity and can effectively stimulate the body to produce high-level immune responses, especially HPV antigen-specific cellular immunity, in different species.

[0065] 2. The HPV mRNA vaccines described in the present invention, especially the self-replicating vaccines repHPV-001 and repHPV-002, have shown strong tumor inhibition ability in different tumor models and have clinical significance for the development of tumor immunotherapy.

[0066] 3. Compared with HPV therapeutic protein vaccines or DNA vaccines, the HPV mRNA vaccine of the present invention has a simpler preparation process, higher safety, and is more suitable for clinical development.

[0067] Definitions and explanations of terms

[0068] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0069] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0070] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0071] The term "and / or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

[0072] The term "and / or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

[0073] The term "gene" herein refers to a nucleic acid fragment encoding a single protein or RNA (also referred to as a "coding sequence" or "coding region") and associated regulatory regions such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

[0074] The term "nucleic acid" herein is used in its broadest sense to include any compound and / or substance comprising a polymer of nucleotides. These polymers are called polynucleotides.

[0075] Nucleic acids (also referred to as polynucleotides) may be or may include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribose configuration, α-LNA with an α-L-ribose configuration (diastereomers of LNA), 2′-amino-LNA with 2′-amino functionalization, and 2′-amino-α-LNA with 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0076] The term "mRNA" herein means messenger RNA and refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring or modified amino acid polymer) and can be translated in vitro, in vivo, in situ or ex vivo to produce the encoded polypeptide.

[0077] The basic components of an mRNA molecule generally include at least a coding region, a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and a poly(A) tail. The polynucleotides of the present disclosure can act as mRNAs, but can be distinguished from wild-type mRNAs in their functional and / or structural design features that are used to overcome existing problems with effective polypeptide expression using nucleic acid-based therapeutics.

[0078] The mRNA described herein refers to mRNA containing a nucleic acid sequence encoding a tumor antigen, which may be 1) mRNA that only encodes and translates a certain tumor antigen, 2) a mixture of mRNAs that encode and translate multiple tumor antigens, or 3) a mixture consisting of 1), 2) and other mRNAs that do not encode tumor antigens.

[0079] The term 5'UTR (5'-untranslated region) herein refers to a specific portion of a messenger RNA (mRNA) that is located 5' to the open reading frame of the mRNA. Typically, the 5'UTR starts at the transcription start site and ends at one nucleotide before the start codon of the open reading frame. The 5'UTR may include elements for controlling gene expression, also referred to as regulatory elements. The regulatory elements may be, for example, a ribosome binding site or a 5' terminal oligopyrimidine sequence. The 5'UTR may be modified post-transcriptionally, for example by adding a 5'CAP.

[0080] The term 3'UTR (3'-untranslated region) herein is the portion of mRNA that is located between the protein coding region (i.e., open reading frame) and the poly (A) sequence of the mRNA. The 3'UTR of the mRNA is not translated into an amino acid sequence. The 3'UTR sequence is usually encoded by a gene that is transcribed into a corresponding mRNA during gene expression. The genomic sequence is first transcribed into a pre-mature mRNA, which includes optional introns. The pre-mature mRNA is then further processed into a mature mRNA during the maturation process. The maturation process includes the following steps: 5' capping, splicing of the pre-mature mRNA to remove optional introns, and modification of the 3' end, such as polyadenylation of the 3' end of the pre-mature mRNA and optional endo- or exonuclease cleavage.

[0081] The term "Poly (A)" herein refers to a (long) sequence of adenosine nucleotides added to the 3' end of RNA, which is up to about 400 adenosine nucleotides, for example, about 25 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, most preferably about 60 to about 250 adenosine nucleotides.

[0082] The term "mutation" herein includes gene mutation and amino acid mutation, wherein gene mutation refers to deletion, insertion, inversion or substitution of heterologous nucleic acid, which may lead to changes in the amino acid sequence of the corresponding protein product; amino acid mutation is also called non-synonymous single nucleotide mutation, which is caused by the change of some single bases, resulting in changes in the amino acid sequence of the protein product. Amino acid changes can affect protein stability, interaction and enzyme activity, thereby leading to the occurrence of diseases.

[0083] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein and refer to a peptide-bonded chain of any amino acids, regardless of length or co-translational or post-translational modification. This definition of a protein polypeptide or protein that is not encoded on a nucleic acid construct specifically and additionally includes chains that include one or more unnatural amino acids or amino acid-like building blocks.

[0084] The term "amino acid substitution" herein refers to those in which at least one amino acid residue in the native or starting sequence is removed and a different amino acid is inserted in its place at the same position. Substitutions may be single, in which only one amino acid in the molecule has been substituted, or they may be multiple, in which two or more amino acids in the same molecule have been substituted.

[0085] The term "conservative amino acid substitution" herein refers to replacing the amino acid that is usually present in the sequence with different amino acids having similar size, charge or polarity. The example of conservative substitution includes replacing non-polar (hydrophobic) residues such as isoleucine, valine and leucine with another non-polar residue. Similarly, the example of conservative substitution includes replacing a polar (hydrophilic) residue with another residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, replacing a basic residue such as lysine, arginine or histidine with another residue, or replacing an acidic residue such as aspartic acid or glutamic acid with another acidic residue is an additional example of conservative substitution. The example of non-conservative substitution includes replacing non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, methionine with polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid or lysine, and / or replacing polar residues with non-polar residues.

[0086] The term "mutant" herein refers to a "variant" of the protein or peptide that may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity with the amino acid sequence of the protein or peptide.

[0087] The term "vector" herein refers to a DNA molecule including single-stranded, double-stranded, circular or supercoiled DNA. Suitable vectors include retroviruses, adenoviruses, adenovirus-associated viruses, poxviruses and bacterial plasmids.

[0088] The term "antigen" herein refers to a substance that can be recognized by the immune system and can, for example, trigger an antigen-specific immune response by forming antibodies as part of an adaptive immune response. Antigens described herein include tumor antigens, preferably located on the surface of (tumor) cells. Tumor antigens can also be selected from proteins that are overexpressed in tumor cells compared to normal cells. In addition, tumor antigens also include antigens that are non-self (or initially non-self) degenerate and are associated with hypothetical tumors expressed in cells. Antigens associated with tumors also include antigens from cells or tissues that are usually embedded in tumors. In addition, some substances (such as proteins or peptides) are expressed in patients with (known or unknown) cancer, and their concentration in the body fluids of the patients increases. These substances are also referred to as "tumor antigens", but they are not antigens in the strict sense of immune response inducing substances. In addition, tumor antigens can also be present on the tumor surface in the form of, for example, mutated receptors. In this case, it can be recognized by antibodies.

[0089] The term "vaccine" herein refers to a preventive or therapeutic substance that provides at least one antigen or antigenic function that can stimulate an immune response in the body without causing disease. The antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0090] The terms "nanoparticle" and "nanoparticle" are used interchangeably herein.

[0091] The term "immune system" herein can protect an organism from infection. If a pathogen breaks through the physical barriers of an organism and enters the organism, the innate immune system provides an immediate but non-specific response. If the pathogen avoids the innate response, vertebrates have a second layer of protection, the adaptive immune system. Here, the immune system changes its response during the infection process to improve its recognition of the pathogen. Then, the improved response is retained in the form of immune memory after the pathogen is eliminated, and allows the adaptive immune system to establish a faster and stronger attack each time the pathogen is encountered. Accordingly, the immune system includes an innate and adaptive immune system. Each of these two parts contains so-called body fluids and cellular components.

[0092] The term "immune response" herein can typically be a specific response of the adaptive immune system to a specific antigen (so-called specific or adaptive immune response) or a non-specific response of the innate immune system (so-called non-specific or innate immune response). One basis of the present invention relates to the specific response of the adaptive immune system (adaptive immune response); in particular, the adaptive immune response after exposure to an antigen (such as an immunogenic polypeptide). However, this specific response can be supported by additional non-specific responses (innate immune responses). Therefore, one basis of the present invention also relates to compounds for stimulating both the innate and adaptive immune systems to stimulate an effective adaptive immune response. In the context of the present invention, an "antigenic composition" refers to a compound or mixture of compounds (such as in a solution or pharmaceutical preparation) that is capable of, used for, or used for, has the ability or can, in practice, stimulate, increase, produce or cause an immune response (preferably, an effective adaptive immune response) when administered to a subject or otherwise exposed to a subject.

[0093] The term "cellular immunity / cellular immune response" herein typically involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. In a more general manner, cellular immunity does not involve antibodies but involves the activation of immune system cells. For example, the cellular immune response is characterized by the activation of antigen-specific cytotoxic T lymphocytes, which can induce apoptosis in body cells (such as virus-infected cells, cells with intracellular bacteria) and cancer cells displaying tumor antigens that display antigenic epitopes on their surfaces; Activate macrophages and natural killer cells, allowing them to destroy pathogens; and stimulate cells to secrete a variety of cytokines that affect the functions of other cells involved in adaptive immune responses and innate immune responses.

[0094] The term "humoral immunity / humoral immune response" herein typically refers to antibody production and the auxiliary processes that may accompany it. For example, a humoral immune response can typically be characterized by Th2 activation and cytokine production, germinal center formation and allotype switching, affinity maturation and memory cell production. Humoral immunity can also typically refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation and phagocytosis and opsonization promotion of pathogen elimination. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 : Schematic diagram of mRNA construct design.

[0096] Figure 2 : Flow cytometric detection of mRNA construct stock expression in 293T cells.

[0097] Figure 3 : ELISpot detected the secretion level of IFN-γ in spleen cells after C57BL / 6 mice were immunized twice with reHPV-002 and HPV-004 vaccines delivered by LNP.

[0098] Figure 4 :Tetramer staining analysis of antigen-specific CD8 production in the spleen of C57BL / 6 mice after two immunizations with LNP-delivered reHPV-002 and HPV-004 vaccines + T cell level.

[0099] Figure 5 : ELISpot detected the secretion level of IFN-γ in spleen cells after Balb / c mice were immunized twice with LNP-delivered repHPV-002 and HPV-004.

[0100] Figure 6: Preventive efficacy of LNP-delivered repHPV-002 and HPV-004 vaccines against TC-1 tumors in C57BL / 6 mice: Tumor growth curves after the first tumor loading; the repHPV-002 and HPV-004 curves overlap.

[0101] Figure 7 : Preventive efficacy of LNP-delivered repHPV-002 and HPV-004 vaccines against TC-1 tumors in C57BL / 6 mice: Tumor growth curves after the second tumor loading; the repHPV-002 and HPV-004 curves overlap.

[0102] Figure 8 : Preventive efficacy of LNP-delivered repHPV-002 and HPV-004 vaccines against CT26 (HPV18) tumors in Balb / c mice.

[0103] Fig. 9 : Therapeutic efficacy of different doses of LNP-delivered repHPV-001, repHPV-002 vaccines and 5.0 μg of HPV-004, HPV-006 vaccines against TC-1 tumors in C57BL / 6 mice: Tumor growth curve after the first tumor loading.

[0104] Fig.10 : Therapeutic efficacy of different doses of LNP-delivered repHPV-001, repHPV-002 vaccines and 5.0 μg of HPV-004, HPV-006 vaccines against TC-1 tumors in C57BL / 6 mice: mortality within 53 days of the first tumor bearing.

[0105] Fig.11 : Therapeutic efficacy of different doses of LNP-delivered repHPV-001 and repHPV-002 vaccines against TC-1 tumors in C57BL / 6 mice: Tumor growth curve after the second tumor loading. DETAILED DESCRIPTION

[0106] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0107] The embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0108] Example 1 mRNA antigen and molecular design

[0109] By querying the Genbank database in NCBI (https: / / www.ncbi.nlm.nih.gov / ), we obtained the protein sequences of HPV16 and HPV18 E6 and E7, respectively: HPV16 E6: NP_041325.1; HPV16: E7 NP_041326.1; HPV18 E6: ATL15239.1; HPV18 E7: ATL15240.1.

[0110] Because E6 and E7 are proto-oncogenes, considering the safety of the vaccine, we have partially modified the amino acid sequences of E6 and E7, as follows: (1) The amino acid sequences of "FAF" and "CPEEK" were deleted in HPV16 E6, and "SG" was mutated to "RP"; (2) "CYE" in locus 24-26 in HPV16 E7 was mutated to "GYG"; (3) "FAF" in locus 47-49 and "NPAEK" in locus 113-117 were deleted in HPV18 E6, and "RP" in locus 10-11 was mutated to "SG"; (4) "CHE" in locus 27-29 was mutated to "GHG" in HPV18 E7. Specifically, the modification (mutation / deletion) of the E6 protein sequence is to change the p53 binding / degradation domain, while the modification (deletion or mutation) of the E7 protein is to destroy the domain that binds to the cellular retinoblastoma (Rb) protein.

[0111] In addition, we also fused the HPV16 / 18 antigen with the signal peptide IgE sequence (SEQ ID NO; 13) that can be efficiently secreted.

[0112] In order to further improve the immunogenicity of the target antigen, we also selected FMS-like tyrosine kinase 3 ligand (FLT3L) protein for co-expression to promote the proliferation, differentiation and maturation of dendritic cells and natural killer cells and improve immunogenicity. The nucleic acid sequences corresponding to the two target antigens were cloned into self-replicating or non-self-replicating vectors to construct self-replicating RNA repHPV-001 and repHPV-002, and non-self-replicating mRNA HPV-004 and HPV-006.

[0113] Schematic diagram of the construct is shown in Figure 1 ,in Figure 116E6, 18E6, 16E7 and 18E7 represent E6 protein or E7 protein of HPV subtype 16 or HP18, respectively, and the above proteins are connected in series through a GS linker.

[0114] Example 2 Preparation of self-replicating and non-self-replicating mRNA

[0115] 2.1 Construction of plasmids

[0116] 2.1.1 Construction of self-replicating vectors repHPV-001 and repHPV-002 plasmids

[0117] For the self-replicating mRNA constructs repHPV-001 and repHPV-002, the complete nucleotide sequences are shown in Table 1, SEQ ID: NO.1 and SEQ ID: NO.2. According to the amino acid sequences of the target proteins SEQ ID: NO.5 and SEQ ID: NO.6 shown in Table 1, the codon-optimized nucleotide sequences encoding the target proteins SEQ ID: NO.17 and SEQ ID: NO.18 were added with an ApaI restriction site and a subgenomic promoter sequence (nsP1-4, SEQ ID NO: 9) at the 5' end, and a SacII restriction site was added at the 3' end. After gene synthesis according to the spliced ​​nucleotide sequence, the restriction sites at both ends were cloned into the vector Vector (self-synthesized, kanamycin resistant), and the original plasmid sequences were replaced to obtain the target plasmids. The 5'UTR, 3'UTR and Poly A sequences used are shown in SEQ ID: NO.11, SEQ ID: NO.13 and SEQ ID: NO.15, respectively.

[0118] 2.1.2 Non-self-replicating HPV-004 and HPV-006 plasmid design:

[0119] For the non-self-replicating mRNA constructs HPV-004 and HPV-006, the complete nucleotide sequences are shown in Table 1, SEQ ID NO: 3 and SEQ ID: NO. 4, and the amino acid sequences are SEQ ID NO: 7 and SEQ ID NO: 8. The nucleotide sequences obtained after codon optimization according to the amino acid sequence of the target protein shown in Table 1 are SEQ ID: NO. 19 and SEQ ID: NO. 20, and XbaI restriction site, T7 promoter and 5'UTR sequence (SEQ ID NO: 10) are added to the 5' end, and 3'UTR sequence (SEQ ID NO: 12), polyA tail sequence (SEQ ID NO: 14) and NotI restriction site are added to the 3' end. After gene synthesis, the spliced ​​sequence is cloned into the vector pUC57-BsaI-BsmBI-free through the XbaI / NotI restriction sites at both ends to obtain a non-self-replicating plasmid. For the convenience of correspondence, the non-self-replicating plasmid is represented by the name of the mRNA construct, and the following self-replicating constructs and corresponding plasmids are also represented by the same name.

[0120] Table 1 Sequence information

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] Note: The nucleotide sequence in the table is represented by atgc. Those skilled in the art know that when the nucleotide is DNA, it is atgc; when transcribed into mRNA, it is augc; when used in a non-self-replicating transcription system, ψ is used in mRNA to replace u, ψ = 1-methyl-3'-pseudouridylyl.

[0134] 2.2 Strain transformation:

[0135] The non-self-replicating (HPV-004 and HPV-006) plasmids were transformed into Escherichia coli Stbl3, and different clones were verified by restriction digestion and sequencing to obtain the final clones.

[0136] The self-replicating plasmids (repHPV-001 and repHPV-002) were transformed into Escherichia coli DH5α, and different clones were verified by restriction digestion and sequencing to obtain the final clones.

[0137] The above E. coli were all added with glycerol to a final concentration of 30% and stored for a long time below -70°C.

[0138] 2.3. Plasmid extraction, template DNA amplification and identification:

[0139] Pipette 20mL of LB liquid culture medium into a sterile shake flask, add ampicillin sodium or kanamycin, inoculate glycerol bacterial solution, culture to obtain activated bacterial solution, add the activated bacterial solution to LB liquid culture medium, culture and centrifuge to discard the supernatant. Use a kit (Macherey Nagel, catalog number 740420.5) to extract the plasmid. Add restriction endonuclease BsaI-HF (NEB, #R3733L) or NotI-HF (NEB, #R3189) to obtain template DNA.

[0140] 2.4. mRNA transcription and purification:

[0141] An in vitro transcription system was prepared according to conventional methods in the art, and in vitro transcription was performed. Then, DNase I (NoviZan, catalog number: DD4104-PC-02) was added for digestion to obtain mRNA.

[0142] 2.5. mRNA capping:

[0143] The reaction system was configured and capping was performed according to conventional methods in the art. The RNA content was determined using NanoDrop, and samples were taken for RNA integrity testing. The results are shown in Table 2.

[0144] Table 2 mRNA concentration, molecular weight and base pair number

[0145] name Concentration (mg / mL) Completeness repHPV-001 1.46 71.4％ repHPV-002 1.45 68.6％ HPV-004 1.367 86.5％ HPV-006 1.89 91.6％

[0146] Based on the above parameters, mRNA has been successfully constructed, and all parameters meet the standards and can be used for subsequent experiments.

[0147] Example 3 Detection of target protein expression after cell transfection with different mRNA stock solutions

[0148] On the first day, the quantity will be 1×10 5 293T cells with an activity greater than 95% were plated in 24-well plates and cultured overnight at 37° C. The next day, mRNA stock solution transfection was performed.

[0149] 3.1 mRNA transfection

[0150] Before use, equilibrate TransIT-mRNA and mRNA Boost Reagent (Miurs, MIR-2250) to room temperature and vortex gently. Aspirate 50μL Opti-MEM I medium into a sterile tube. Add 0.5μg (0.5μL of 1μg / μL stock solution) mRNA. Gently pipette to mix thoroughly. Add 1μL mRNA Boost Reagent to the diluted mRNA mixture. Gently pipette to mix thoroughly. Add 1μL TransIT-mRNA Reagent to the diluted mRNA mixture and gently pipette to mix thoroughly. Incubate at room temperature for 2-5 minutes to allow enough time for complex formation, then gently drop the complex into the cell culture wells and gently shake the cell plate to mix.

[0151] 3.2 Processing (fixation and permeabilization) and detection of cell samples after transfection with repHPV-001 / 002 or HPV-004 / 006 stock solution or irrelevant antigen mRNA sequence (Control)

[0152] 3.2.1 Preparation of fixation and permeabilization solution: Take 1 ml of Fixation / Permeabilization Concentrate (Thermo Fisher, 00-5123) and add 3 ml of Fixation / Permeabilization Diluent (Thermo Fisher, 00-5223).

[0153] 3.2.2 Add 200 μL of prepared fixation and permeabilization solution and incubate for 40 min.

[0154] 3.2.3 Preparation of permeabilization buffer (PB): 1 ml 10× Permeabilization Buffer (ThermoFisher, 00-8333)

[0155] 3.2.4 Centrifuge and discard the supernatant, add 200 μL permeabilization buffer (PB), and after 5 minutes, add fetal bovine serum with a final concentration of 2% and block at room temperature for 15 minutes.

[0156] 3.2.5 Add Mouse anti-HPV18 E7 mAb (F-7) (Santa Cruz, sc-365035) diluted 1:200 with permeabilization buffer and incubate at room temperature for 1 hour

[0157] 3.2.6 Centrifuge and discard the supernatant, add 200 μL of permeabilization buffer (PB), and wash twice

[0158] 3.2.7 Add Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher, A32723) diluted 1:800 with permeabilization buffer and incubate at room temperature for 30 minutes

[0159] 3.2.8 Centrifuge and discard the supernatant, add 200 μL of permeabilization buffer (PB), wash twice, and add 200 μL of PBS. 24 hours after transfection, use Attune NxT acoustic focusing flow cytometer (Thermo, 2AFC236901121) for detection and analysis.

[0160] Flow cytometry (eg Figure 2 ), the expression of the target protein could be detected in all four molecules using the HPV18 E7 protein antibody, indicating that the construction and expression of repHPV-001 / 002 and HPV-004 / 006 were normal.

[0161] Example 4 LNP-mRNA Preparation

[0162] 4.1 Lipid Nanoparticle (LNP) Preparation

[0163] When preparing lipid nanoparticles, ALC0315, ALC0159, DSPC and cholesterol are used as lipid components, and the four are prepared in a conventional molar ratio in the art to form an organic phase, and the RNA stock solution and citric acid buffer are prepared into an aqueous phase. Subsequently, a conventional microfluidic method in the art is adopted to prepare lipid nanoparticles through a microfluidic device, and then the product is diluted and concentrated with a buffer, and then diluted with a buffer and packaged, and stored frozen at -80°C.

[0164] 4.2 Lipid nanoparticle size and potential detection

[0165] Malvern Zetasizer ultra was used to measure the particle size and potential of lipid nanoparticles. 10 μL of the solution containing LNP particles was diluted to 1 mL with injection water and placed in the detection cell to detect the particle size and potential of the LNP particles.

[0166] 4.3 Detection of total RNA concentration in lipid nanoparticles

[0167] The total RNA concentration of lipid nanoparticles was detected by Ribogreen method. The LNP solution was demulsified with Triton, 100 μL of the demulsified solution was added to a 96-well plate, and then 100 μL of Ribogreen dye solution was added and placed on a flat shaker for 5 minutes at 600 rpm. The SpectraMax iD3 multifunctional microplate reader was used for detection. The total RNA concentration was calculated using the standard curve.

[0168] Table 3 Physical and chemical tests of LNP preparations

[0169]

[0170]

[0171] The results in Table 3 show that the particle size, PDI, Zeta position and total RNA concentration of repHPV-001 / 002 and HPV-004 / 006 can meet the requirements of subsequent experiments.

[0172] In addition, LNP-coated repHPV-001 / 002 and HPV-004 / 006 were transfected with 293T cells in vitro and cultured overnight for 24 hours. Using flow cytometry (HPV18 E7 antibody, Santa Cruz, sc-365035), the transfection results were all positive, which can meet the needs of subsequent animal experiments.

[0173] Example 5 Immunogenicity of LNP / HPV mRNA Vaccine in Mice

[0174] 5.1 Comparison of immunogenicity of LNP / repHPV-002 and HPV-004 in C57BL / 6 mice

[0175] 6-8 weeks old 15-20g C57BL / 6 mice were selected and adaptively raised for one week, and 5.0μg mRNA doses of complex LNP / repHPV-002 and HPV-004 were administered to the right hind leg for immunization on days 0 and 28 (D0, D28, the same below), and the control group was injected with an equal volume of PBS solution (referred to as the PBS group), with 5 mice in each group. On day 42 (D42, the same below), the spleen of the mice was collected and stimulated with a mixture of HPV16 E6 / HPV18 E6 / HPV18 E6 / HPV18 E7 peptide library (synthesized by Nanjing GenScript, each peptide consists of 15 amino acids and has 7 amino acid repeats) and then IFN-γ ELISPOT (IFN-γ ELISPOT kit, Abcam, ab64029) and tetramer analysis (MBL, #TS-5008-1) were performed.

[0176] 5.1.1 IFN-γ ELISpot analysis of mouse spleen cells

[0177] 5.1.1.1 Spleen cell processing

[0178] Fresh spleens from mice immunized twice with LNP / repHPV-002 and HPV-004 or PBS were collected on D42 and placed in a culture dish containing 5 mL of HBSS (Hanks balanced salt solution) buffer. Using a surgical blade, carefully cut the spleen into small pieces (~0.2 cm2). Place a sterile cell screen on top of a 50 mL centrifuge tube and transfer the spleen to the cell screen with forceps. Mash or crush the spleen with the end of a syringe plunger to pass it through the screen. Add 5–10 mL of PBS for washing if necessary. Centrifuge the cells at 1000 g for 10 min at 4°C and discard the supernatant. Resuspend the cells with 2–5 mL of pre-chilled 1× RBC lysis buffer. Place the resuspension on ice for 5 min, centrifuge the cells at 1000 g for 10 min, and discard the supernatant. Wash the cell suspension with 10–20 mL of ice-cold PBS. Centrifuge at 1000 g for 10 min at 4°C and discard the supernatant. Resuspend the cells in 1640 medium to a cell concentration of 3 × 10 6 cells / mL.

[0179] 5.1.1.2 Peptide library stimulation

[0180] Add 100 μL PBS to each well and incubate at room temperature for 10 min. Remove PBS and pat the plate dry. Add 100 μL of treated spleen cell sample to each well, add S1 and S2 peptide libraries to the sample wells, 0.2 μg per well, and repeat 3 times for each sample. Add Concavlin A to the positive wells, 0.2 μg per well, and add PBS to the negative wells. Incubate in a 37°C cell culture incubator for 15-18 h.

[0181] 5.1.1.3 ELISpot

[0182] After 15-18 hours of incubation of spleen cells with peptide library stimulation, remove the medium, add 100μL PBST to each well, incubate at 4℃ for 10min, and remove PBST. Wash three times with PBST. Add 100μL detection antibody to each well and incubate at room temperature for 1h 30min. Remove the supernatant and wash 3 times with PBST. Add 100μL diluted Streptavidin-AP conjugate to each well and incubate at room temperature for 1h. Remove the supernatant and wash 3 times with PBST. Remove the plastic base of the 96-well plate, wash the front and back of the plate 3 times with running water, and pat dry. Add 100μL BCIP / NBT buffer to each well and incubate at room temperature in the dark for 15-30min until the positive wells can show sufficient spots. Remove the BCIP / NBT buffer, wash the front and back of the plate 3 times with running water, pat dry, and blow dry all wells. Read the number of spots using an ELISPOT plate reader.

[0183] 5.1.1.4 Experimental results

[0184] The spleens of mice immunized with LNP / repHPV-002 and HPV-004 were collected on D42 and stimulated with a mixture of HPV16 and HPV18 E6 / E7 peptide libraries to detect the release of IFN-γ. Figure 3 ) showed that compared with the control group, both the replicating vaccine repHPV-002 and the non-replicating HPV-004 could produce stronger cellular immunity in C57BL / 6 mice, with the number of immune spots exceeding 3500, and there was no significant difference between the two (P>0.05).

[0185] 5.1.2 HPV16E7 tetramer analysis

[0186] The spleen cells collected on D42 were induced overnight with the same peptide library as above, and detected using the kit of MBL (MBL, #TB-5008-1). For specific details, please refer to the kit manual.

[0187] The experimental results show that ( Figure 4 ), repHPV-002 and HPV-004 can induce antigen-specific CD8 + Compared with the self-replicating repHPV-002, the non-self-replicating HPV-004 can produce higher levels of antigen-specific CD8 + T cells, there was no significant difference between the two.

[0188] 5.2 Comparison of immunogenicity of LNP / repHPV-002 and HPV-004 in Balb / c mice

[0189] 6-8 weeks old 15-20g Balb / c mice were selected and adaptively raised for one week, and 1.0μg mRNA dose of complex LNP / repHPV-002 and HPV-004 were given in the right hind leg for immunization on D0 and D28, respectively. The control group was injected with an equal volume of PBS solution (referred to as PBS group), with 5 mice in each group. On D42, the spleen of the mice was collected and stimulated with HPV16 E6 / HPV18 E6 / HPV18E6 / HPV18 E7 peptide library mixture (synthesized by Nanjing GenScript, each peptide consists of 15 amino acids and has 7 amino acid repeats) and then IFN-γ ELISPOT analysis was performed.

[0190] The experimental results show that ( Figure 5 ), LNP / repHPV-002 and HPV-004 both had good immunogenicity in Balb / c mice.

[0191] The results in Example 5 show that self-replicating and non-self-replicating (LNP / repHPV-002 and HPV-004) mRNA vaccines can produce good immune effects in both strains of mice.

[0192] Example 6 Preventive efficacy of LNP-delivered repHPV-002 or HPV-004 against TC-1 tumors in C57BL / 6 mice

[0193] First tumor bearing: 6-8 weeks old, 15-20 g C57BL / 6 mice were selected and adaptively raised for one week. 1.0 μg mRNA of LNP / repHPV-002 and HPV-004 were administered to the right hind leg for immunization on D0 and D28, respectively. The control group was injected with an equal volume of PBS solution (denoted as PBS group), with 5 mice in each group. On the 35th day of the first immunization (D35, the same below), 1×10 5 The mice were challenged with TC-1 cells (ATCC No.: CRL-2493). The tumor size was observed every 3-4 days, and the mice were weighed. On day 59 (D59, the same below), the mice in the PBS group were euthanized, while the mice in the LNP / repHPV-002 and HPV-004 groups were kept as no tumors were found.

[0194] Second tumor bearing: On day 69 (D69, the same below), mice in the LNP / repHPV-002 and HPV-004 groups were subcutaneously inoculated with 1×10 6 TC-1 cells and inoculated with 1×10 6TC-1 cells (denoted as Control group). The tumor size was detected every 3-4 days, and the mice were weighed. All mice were euthanized on the 90th day (D90, the same below).

[0195] result( Figure 6 and Figure 7 ) showed that LNP-delivered repHPV-002 and HPV-004 showed 100% preventive tumor inhibition effect in the first TC-1 tumor challenge experiment; and in the second TC-1 cell challenge experiment with increased tumor loading, they still showed good tumor inhibition effect. This shows that LNP / repHPV-002 and HPV-004 can produce more memory T cells and have a strong and sustained killing effect on TC-1 cells.

[0196] Example 7 Preventive efficacy of LNP-delivered repHPV-002 and HPV-004 against CT26-HPV18 tumors in BALB / c mice

[0197] Balb / c mice aged 6-8 weeks were selected and adaptively raised for one week. They were immunized with 5.0 μg mRNA of LNP / repHPV-002 and HPV-004 in the right hind leg at D0 and D28, respectively. The control group was injected with an equal volume of PBS solution (denoted as PBS group). There were 5 mice in each group. On the 49th day of the first immunization, 1×10 5 CT26 cells were used to attack mice. Among them, CT26-HPV18 cells are CT26 from ATCC (CRL-2638TM) and a stable cell line constructed by stably transfecting HPV18E6 and E7 genes with lentivirus. The tumor size was detected every 3-4 days, and the mice were weighed. All mice were euthanized on day 77 (D77, the same below).

[0198] result( Figure 8 ) showed that in Balb / c mice inoculated with HPV18 E6 and E7 stably transfected tumor cell line CT26 (HPV18), the tumor inhibition rate of the repHPV-002 group was >80%, while HPV-004 also exceeded 50% of the tumor inhibition rate. The above results show that LNP / repHPV-002 and HPV-004 also have good therapeutic effects against CT26-HPV18 tumors in BALB / c mice.

[0199] Example 8 Comparison of therapeutic efficacy of repRNA and mRNA delivered by different doses of LNP on TC-1 tumors

[0200] First tumor bearing: 6-8 weeks old, 15-20 g C57BL / 6 mice were selected and fed for one week. 1×105 TC-1 cells were grouped according to tumor size on day 6. After grouping, mice were given 1.0ug or 5.0μg mRNA doses of complex LNP / repHPV-001 and 002 or HPV-004 or HPV-006 in the right hind leg for immunotherapy on days 7, 14 and 21 (D7, D14, D21, the same below), and the control group was injected with an equal volume of PBS solution (recorded as PBS group), with 9 mice in each group, and immunized 3 times in total. The tumor size was observed every 3-5 days, and the mice were weighed.

[0201] On day 27 (D27, the same below), the average tumor size of mice in the PBS group exceeded 2000 mm 3 , euthanasia is required.

[0202] The results are shown in the figure ( Fig. 9 ), in the first therapeutic trial against TC-1 tumors, the tumor inhibition rates of the 1μg and 5μg dose groups of repHPV-001 and repHPV-002 were close to 100% at D27. Among them, the CR in the repHPV-0011.0μg group and the repHPV-001 5.0μg group were both 9 / 9; the CR in the repHPV-002 1.0μg group was 7 / 9, and the CR in the repHPV-0025.0μg group was 8 / 9; and the CR in the HPV-004 5.0μg group and the HPV-006 5.0μg group was 2 / 9. The results showed that the self-replicating group (repHPV-001 / 002) showed better efficacy than the non-replicating group (HPV-004 and HPV-006).

[0203] Continue to observe until the 53rd day. Fig.10 As shown, the survival rate of mice in the repHPV-001 1.0μg group was 100%, while the survival rates of the repHPV-001 5.0μg, repHPV-002 1.0μg and repHPV-002 5.0μg groups were all 89%, the survival rates of the HPV-004 5.0μg group were 67%, and the survival rates of the HPV-006 5.0μg group were 11%. These results can all indicate that the self-replicating mRNA vaccine group (repHPV-001 / 002) has a better tumor inhibition effect than the non-replicating mRNA vaccine (HPV-004 / 006).

[0204] Second tumor bearing: The mice with complete tumor suppression (CR) in the repHPV-001 5.0 μg group, repHPV-002 5.0 μg group, repHPV-001 1.0 μg group and repHPV-002 1.0 μg group on day 53 were selected and 1×10 5The control group was a newly purchased C57BL / 6 mouse inoculated with 1×10 5 TC-1 tumors (referred to as the Control group). Fig.11 )(17 days after the second tumor challenge), there was still no tumor growth in the mice in the four groups (Note: Since the tumor growth size in the four groups was 0, Fig.11 The four tumor growth curves on the curve overlap completely). This shows that the LNP-coated self-replicating HPV vaccine (repHPV-001 / 002) can not only produce strong effector T cells (T eff ) can also produce strong memory T cells (T CM ), able to maintain continuous killing capability against TC-1.

Claims

1. An mRNA molecular construct, characterized in that: The mRNA molecules are self-replicating and / or non-self-replicating; Optionally, the mRNA is circular RNA and / or TaRNA.

2. The mRNA molecule construct according to claim 1, characterized in that The mRNA molecular construct comprises a linked coding segment, and the polypeptide encoded by the coding segment has HPV virus antigen epitope immunogenicity; Preferably, the HPV virus antigen epitopes include proteins E6 and E7; Preferably, the HPV virus antigen epitopes include E6 protein and E7 protein from HPV16 and HPV18 subtypes; More preferably, in the mRNA molecule construct, the E6 protein and E7 protein sequences from HPV16 and HPV18 subtypes are connected in series.

3. The mRNA molecule construct according to any one of claims 1 to 2, characterized in that: The mRNA molecule construct further includes a 5'UTR; Preferably, the mRNA molecule construct further includes a 3'UTR; Preferably, the mRNA molecule construct further comprises a poly(A) sequence; Preferably, the mRNA molecule construct also includes a signal peptide sequence; Preferably, the signal peptide sequence is an IgE signal peptide; More preferably, the self-replicating mRNA molecule construct further comprises non-structural proteins nsP1-4.

4. The mRNA molecule construct according to any one of claims 1 to 3, characterized in that: The nsP1-4 comprises the sequence shown in SEQ ID NO: 9; Preferably, the 5'UTR comprises a sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 11; Preferably, the 3'UTR comprises a sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 13; Preferably, the poly(A) sequence comprises a sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 15; More preferably, the IgE signal peptide comprises the sequence shown in SEQ ID NO:

16.

5. The mRNA molecule construct according to any one of claims 1 to 4, characterized in that: wherein the protein encoded by the mRNA comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; Preferably, the protein encoded by the mRNA comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; More preferably, the protein encoded by the mRNA comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:

8.

6. The mRNA molecule construct according to any one of claims 1 to 5, characterized in that: wherein the protein encoded by the mRNA independently comprises 1 to 12 amino acid substitutions relative to at least one of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; Preferably, the 1-12 amino acid substitutions are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions; Preferably, the amino acid substitution is a conservative amino acid substitution; Preferably, the substitution is a highly conservative amino acid substitution; More preferably, the protein encoded by the mRNA comprises an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:

8.

7. The mRNA molecule construct according to any one of claims 1 to 6, characterized in that: It contains the coding nucleotide sequence corresponding to the antigen protein; Preferably, the coding nucleotide sequence corresponding to the antigen protein is a codon-optimized sequence; More preferably, the coding nucleotide sequence corresponding to the antigen protein comprises SEQ ID NO: 17, SEQ ID NO: 18, The nucleotide sequence shown in SEQ ID NO:19 or SEQ ID NO:

20.

8. The mRNA molecule construct according to any one of claims 1 to 7, characterized in that: The mRNA comprises a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

9. A vaccine, characterized in that The vaccine comprises the mRNA molecule construct according to any one of claims 1 to 8.

10. The vaccine according to claim 9, characterized in that The vaccine also includes a delivery formulation; Preferably, the delivery formulation is a nanoparticle; Preferably, the delivery preparation includes lipid nanoparticles (LNP), lipopolyplex (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc. More preferably, the nanoparticles are lipid nanoparticles (LNP).

11. The vaccine according to any one of claims 9 to 10, characterized in that The vaccine uses the mRNA molecule construct described in any one of claims 1 to 9 or a combination thereof as an immunogenic component.

12. The vaccine according to any one of claims 9 to 11, characterized in that The vaccine is an HPV vaccine.

13. A carrier, characterized in that Comprising the mRNA construct according to any one of claims 1 to 8, or a combination thereof.

14. A cell, characterized in that Comprising the mRNA construct according to any one of claims 1 to 8, or a combination thereof, or the vector according to claim 13.

15. A nanoparticle, characterized in that: Comprising the mRNA construct according to any one of claims 1 to 8, or a combination thereof; Preferably, the nanoparticles include lipid nanoparticles (LNP), lipid multipolymers (LPP), polymer nanoparticles (PNP), inorganic nanoparticles (INP), cationic nanoemulsion (CNE), exosomes, biological microvesicles, protamine, etc. More preferably, the nanoparticles are lipid nanoparticles (LNP).

16. Use of the mRNA construct according to any one of claims 1-8, the vaccine according to any one of claims 9-12, the vector according to claim 13, the cell according to claim 14, or the nanoparticle according to claim 15 in the preparation of a medicament for inducing an immune response in a subject in need thereof.

17. Use of the mRNA construct according to any one of claims 1-8, the vaccine according to any one of claims 9-12, the vector according to claim 13, the cell according to claim 14, or the nanoparticle according to claim 15 in the preparation of a medicament for treating or preventing HPV-related diseases in a subject in need thereof. Preferably, the HPV-related disease includes cancer or tumor; More preferably, the cancer or tumor includes, for example, cervical cancer, anal cancer, penile cancer, vaginal cancer, vulvar cancer, and oropharyngeal cancer at the back of the throat.

18. A method for inducing an immune response in a subject in need thereof, characterized in that: Administering the vaccine of any one of claims 9 to 12 to a subject; Preferably, the method induces an immune response against HPV by using a replicating or non-replicating virus.

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