Drug for treating HPV infection-related diseases

Abstract

Provided are an antigen combination, a fusion protein, and a coding nucleic acid. The materials are used for preparing a self-replicating RNA vaccine for treating persistent cervical HPV infection and induced intraepithelial neoplasia and cervical cancer, which can extend the indication to include long-term HPV-infected individuals. Therefore, virus-infected cells can be eliminated to treat long-term infection, and precancerous lesions and cervical cancer can also be treated more effectively.

Description

Drugs for treating HPV infection-related diseases

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on March 6, 2024, with application number 202410257224.4 and invention name “Drugs for treating HPV infection-related diseases”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to a drug for treating HPV infection-related diseases. Background Art

[0003] Cervical cancer poses a significant threat to women's health worldwide, ranking fourth in incidence among all malignant tumors. Particularly in developing countries, it is the second most common cancer among women and the leading cause of cancer-related death in women under 35. According to 2018 data from the World Health Organization, there are approximately 570,000 new cases of cervical cancer and 311,000 deaths worldwide each year. In China, in 2020, there were approximately 110,000 new cases of cervical cancer, accounting for 18.3% of new cases worldwide, and approximately 59,000 deaths, accounting for 17.3% of the global total. Notably, the incidence of cervical cancer among young women in China is on the rise, with the age of onset trending younger, posing a significant public health challenge.

[0004] Unlike other cancers, cervical cancer is almost always caused by persistent infection with high-risk human papillomavirus (HR-HPV), of which approximately 60% is attributed to the high-risk HPV16 subtype. Therefore, clearing HR-HPV infection or treating the cervical intraepithelial neoplasia (CIN) caused by HR-HPV is a key strategy for preventing cervical cancer. Currently approved preventive HPV vaccines effectively prevent new HPV infections by targeting the HPV late protein L1 as an antigen to stimulate the production of neutralizing antibodies against this protein. However, this vaccine is ineffective in treating individuals already infected with HPV16 and the lesions it causes. Currently, there is a lack of effective viral clearance methods for patients with persistent HPV16 infection. Therefore, this unmet medical need urgently requires the development of new therapeutic agents to clear persistent HPV16 infection in the cervix and treat the cervical intraepithelial neoplasia (CIN) caused by HR-HPV.

[0005] HPV, a circular, double-stranded DNA virus with a diameter of 50-55 nm, is one of the main causes of cervical cancer. High-risk HPV16, in particular, accounts for 50%-60% of cervical cancer cases. While most HPV infections clear naturally, a small number of persistent infections progress to cervical intraepithelial neoplasia (CIN) or even invasive cervical carcinoma (ICC). CIN is categorized into three grades, CIN1 to CIN3, depending on the severity of the lesion. CIN1 is considered low-grade intraepithelial neoplasia, while CIN2 and CIN3 are collectively considered high-grade intraepithelial neoplasia. Studies have shown that approximately 30% of untreated patients will progress to cervical cancer. This emphasizes the crucial importance of preventing and treating persistent HPV16 infection in the development of cervical cancer. The HPV genome encodes the early proteins E1, E2, E4, E5, E6, and E7, and the late proteins L1 and L2. L1 and L2 form the viral outer coat and are the primary antigens used in preventive vaccines to prevent HPV infection, but they are ineffective against those already infected. HPV expresses different early proteins at different stages of host infection. During the latent infection phase, before lesions develop, the E1, E2, E4, and E5 proteins are primarily expressed. When infection progresses to CIN and cervical cancer, the expression of E1, E2, E4, and E5 is suppressed, leaving E6 and E7 proteins primarily expressed. Therefore, E6 and E7 antigens are primarily used in the development of therapeutics for CIN and cervical cancer.

[0006] Currently, HPV therapeutic drugs mainly target high-grade cervical intraepithelial neoplasia (CIN3) and have entered clinical research internationally, including: 1. The VGX-3100 product developed by Inovio Corporation of the United States: It consists of a therapeutic double plasmid expressing HPV16 E6 and E7 proteins and HPV18 E6 and E7 proteins, respectively, and has completed two Phase III clinical studies; 2. The GX-188E product developed by Genexine Corporation of South Korea: It consists of a therapeutic single plasmid expressing Flt-3L and HPV 16 and 18 subtypes E6 and E7 proteins in series, forming a fusion protein, and has completed Phase II clinical studies; 3. The VB10.16 product developed by Nykode Therapeutics of Norway: It consists of a therapeutic single plasmid expressing CCL3L1 and HPV 16 and 18 subtypes E6 and E7 proteins in series, forming a fusion protein, and has completed Phase I / IIa clinical studies. It can be seen that the current cervical HPV therapeutic vaccines abroad are all aimed at replacing surgical treatment for CIN3. There is no therapeutic vaccine for people with long-term HPV infection. Moreover, because these drugs were studied at an early stage, the technology used has limitations and the therapeutic effect is limited, and still needs continuous improvement. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a drug for treating diseases related to HPV infection.

[0008] The present invention aims to provide an HPV antigen combination, a fusion protein, and related biomaterials. The antigen combination can eliminate the virus at an earlier stage of viral infection and prevent the occurrence of neoplasia.

[0009] Another object of the present invention is to provide a vaccine and method that can not only eliminate viral host cells and treat long-term infections, but also more effectively treat precancerous lesions and cervical cancer.

[0010] Therefore, the present invention provides:

[0011] The antigen combination includes the HPV virus E1, E2, E6 and E7 proteins. E1 and E2 are expressed during the viral infection period and can be used to eliminate the viral infection.

[0012] The present invention screened the antigenic proteins of the HPV virus and combined antigens with higher affinity. It was ultimately found that the antigen fragments obtained by combining E1, E2, E6 and E7 proteins and screening fragments of E1 and E2 proteins had higher immunogenicity than other antigen combinations and showed better antigen affinity.

[0013] The present invention also provides an immunogenic fragment of the E1 protein, which has the amino acid sequence shown in SEQ ID NO:3.

[0014] The present invention also provides an immunogenic fragment of the E2 protein, which has an amino acid sequence as shown in SEQ ID NO:4.

[0015] In the present invention, the fragments are connected by a linker. The length of the linker is 1 to 10 amino acid residues, for example, the linker is AGA or G5SG5.

[0016] In the antigen combination described herein, the number of any one of the four antigenic proteins (E1, E2, E6, or E7 proteins) is 1, or an integer greater than 1, which is not limited in the present invention. The same protein may be repeated two or more times in a consecutive sequence in the antigen combination, or two or more proteins may be combined into a unit and repeated two or more times.

[0017] For example, the antigen combination from N-terminus to C-terminus is (E1) a (E2) b (E6) c (E7) d 、(E1) a (E2) b (E7) c (E6)d 、(E1) a (E6) b (E2) c (E7) d 、(E1) a (E6) b (E7) c (E2) d 、(E1) a (E7) b (E6) c (E2) d 、(E1) a (E7) b (E2) c (E6) d 、(E2) a (E1) b (E6) c (E7) d 、(E2) a (E1) b (E7) c (E6) d 、(E2) a (E6) b (E1) c (E7) d 、(E2) a (E6) b (E7) c (E1) d 、(E2) a (E7) b (E1) c (E6) d 、(E2) a (E7) b (E6) c (E1) d 、(E6) a (E1) b (E2) c (E7) d 、(E6) a (E1) b (E7) c (E2) d 、(E6) a (E2) b (E1) c (E7) d 、(E6) a (E2) b (E7) c (E1) d 、(E6) a (E7)b (E1) c (E2) d 、(E6) a (E7) b (E2) c (E1) d 、(E7) a (E1) b (E2) c (E6) d 、(E7) a (E1) b (E6) c (E2) d 、(E7) a (E2) b (E1) c (E6) d 、(E7) a (E2) b (E6) c (E1) d 、(E7) a (E6) b (E1) c (E2) d or (E7) a (E6) b (E2) c (E1) d Wherein, a, b, c, d are the number of times the antigen protein is repeated, and a, b, c, d are independently selected from non-zero integers. In the embodiment of the present invention, the effect is verified by using an antigen combination in which a, b, c, d are all 1.

[0018] In addition, taking the antigen combination consisting of E6 protein, E7 protein, E1 protein and E2 protein as an example, the antigen combination from N-terminus to C-terminus is: [(E6) a (E7) b ] x (E1) c (E2) d 、[(E6) a (E7) b (E1) c ] x (E2) d 、(E6) a [(E7) b (E1) c ] x (E2) d 、(E6) a [(E7) b (E1) c (E2) d ]x 、(E6) a (E7) b [(E1) c (E2) d ] x 、[(E6) a (E7) b ] x [(E1) c (E2) d ] y or [(E6) a (E7) b ] x [(E1) c (E2) d ] y Wherein, a, b, c, d are the number of times the antigen protein is repeated, and a, b, c, d are independently selected from non-zero integers. x, y are the number of times the antigen protein component unit is repeated, and x, y are independently selected from non-zero integers.

[0019] The present invention has experimented with antigen combinations with different arrangement orders. In some examples, the antigen combinations included, from N-terminus to C-terminus, the HPV E6, E7, E1, and E2 proteins, respectively. Compared with antigen combinations with other connection orders, these antigen combinations exhibited better immunogenicity and greater antigen affinity.

[0020] In some specific embodiments, the HPV virus is HPV16 subtype. The amino acid sequence of its E6 protein is shown in SEQ ID NO: 1; the amino acid sequence of its E7 protein is shown in SEQ ID NO: 2; the amino acid sequence of its E1 protein is shown in SEQ ID NO: 3; and the amino acid sequence of its E2 protein is shown in SEQ ID NO: 4. More specifically, the amino acid sequence of the antigen combination is shown in SEQ ID NO: 5.

[0021] In order to further enhance the immune response of the antigen fragment in vivo, the present invention connects the aforementioned antigen combination to a chemokine, wherein the chemokine is selected from: XCL-1, CCL11, CCL13, CCL14 and / or CCL26. In the present invention, the chemokine is derived from humans or other animals, and is a full-length sequence or a partial fragment with chemotactic activity. Experiments have shown that compared with other chemokines, CCL11 can more effectively deliver the antigen combination to DC cells, thereby improving the presentation effect and enhancing the immune response. In addition, previous studies have shown that CCL11 has the effect of enhancing immunity in the human body.

[0022] The present invention provides a fusion protein comprising the chemokine CCL11 and the antigen combination. The chemokine is located at the N-terminus or C-terminus of the antigen combination, which is not limited by the present invention. In some specific embodiments, CCL11 is located at the N-terminus of the antigen combination, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:6.

[0023] The present invention combines E1E2, E6E7, and E6E7 antigens. Compared to vaccines using only E6E7 protein or combinations of E6E7 linked to other antigens, the antigen combination provided by the present invention can eliminate the virus at an earlier stage of infection and prevent neoplasia. Furthermore, the addition of E1E2 and the selection of the order of arrangement enhance the immunogenicity of the original E6E7, thereby improving its ability to treat cervical intraepithelial neoplasia and cervical cancer.

[0024] Furthermore, the present invention also provides a nucleic acid encoding the aforementioned antigen combination or the aforementioned fusion protein.

[0025] In the present invention, the nucleic acid includes deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), wherein DNA includes cDNA or genomic DNA; and the RNA includes mRNA.

[0026] The present invention does not limit the nucleic acid sequence, and any nucleic acid that can encode the antigen combination or fusion protein as described above can be used. In the embodiment of the present invention, in order to further improve the immune effect of the antigen, the nucleic acid sequence is codon optimized. Among multiple optimization schemes, the optimized sequence described below has more significant advantages compared to other schemes. In a specific embodiment, the nucleic acid encoding the E6 protein has a sequence as shown in SEQ ID NO:7; the nucleic acid encoding the E7 protein has a sequence as shown in SEQ ID NO:8; the nucleic acid encoding the E1 protein has a sequence as shown in SEQ ID NO:9; the nucleic acid encoding the E2 protein has a sequence as shown in SEQ ID NO:10.

[0027] In a further embodiment, the nucleic acid encoding the antigen combination having an amino acid sequence as shown in SEQ ID NO:5 has a sequence as shown in SEQ ID NO:11.

[0028] In a further embodiment, the nucleic acid encoding the antigen combination having an amino acid sequence as shown in SEQ ID NO:6 has a sequence as shown in SEQ ID NO:12.

[0029] In some embodiments, the nucleic acid further comprises a 5' end Cap structure and / or a 3' end poly A tail.

[0030] The 5'-end Cap structure is CAP type 0, CAP type 1, CAP type 2, CapAG, CapAU or Cap GG, which is not limited in the present invention.

[0031] The length of the polyA is 50 to 200 bp, for example, 50 bp, 75 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 150 bp or 200 bp, which is not limited in the present invention.

[0032] Furthermore, the present invention also provides a plasmid vector, comprising a backbone vector and the nucleic acid as described above.

[0033] The plasmid vector described in the present invention is used for storing the nucleic acid as described above, amplifying the nucleic acid as described above, or expressing the fusion protein and / or antigen combination as described above.

[0034] In the embodiment of the present invention, the vector used for nucleic acid amplification is an mRNA vector, comprising a T7 promoter, a 5' untranslated region (UTR), a 3' UTR, a 31+10 nt spacer, and a poly A tail. As a feasible example, the sequence of the mRNA vector is shown in SEQ ID NO: 13.

[0035] The present invention also provides a host cell, which comprises the plasmid vector as described above or has the nucleic acid as described above integrated into its genome.

[0036] The host cell of the present invention is used for storage of the aforementioned nucleic acid or plasmid, amplification of the aforementioned nucleic acid or plasmid, or expression of the aforementioned fusion protein and / or antigen combination.

[0037] In an embodiment of the present invention, the host cell is a bacterial cell or a mammalian cell. In an embodiment of the present invention, the host cell is a TC-1 cell or a B16 cell.

[0038] Furthermore, the present invention also provides a method for preparing the antigen combination or the fusion protein, comprising: inserting a nucleic acid encoding the antigen combination or the fusion protein into a plasmid vector, and expressing the nucleic acid in a host cell. After the expression, the obtained expression product is further enriched and purified.

[0039] Furthermore, the present invention also provides a method for preparing the nucleic acid, comprising inserting a nucleic acid encoding the antigen combination or fusion protein into a self-replicating mRNA vector to prepare a nucleic acid with a polyA structure. After linearization and in vitro transcription, the nucleic acid described above is obtained. In an embodiment of the present invention, a 5'-terminal cap structure is also added during the in vitro transcription process.

[0040] Furthermore, the present invention also provides the use of the aforementioned antigen combination, the fusion protein, and / or the nucleic acid in the preparation of a vaccine. In the present invention, the vaccine is a product for preventing HPV infection and / or a vaccine for preventing or treating diseases caused by HPV infection. The vaccine of the present invention is used to prevent or treat HPV infection, wherein the HPV is HPV16.

[0041] Furthermore, the present invention also provides a vaccine comprising the antigen combination, the fusion protein and / or the nucleic acid.

[0042] In the present invention, the vaccine is a protein vaccine, a DNA vaccine, or an mRNA vaccine. Depending on the antigen, the vaccine also includes a pharmaceutically acceptable carrier, excipient, and / or adjuvant. The drug also includes a pharmaceutically acceptable excipient.

[0043] In the embodiment of the present invention,

[0044] The vaccine comprises the aforementioned antigen combination and aluminum adjuvant;

[0045] The vaccine comprises the fusion protein and aluminum adjuvant as described above;

[0046] The vaccine includes the nucleic acid as described above, wherein the nucleic acid is DNA, and further includes a TLR or STING agonist such as Poly I:C, CpG;

[0047] The vaccine comprises the aforementioned nucleic acid, which is mRNA, and further comprises a lipid adjuvant, which comprises SM102, DSPC, Cholesterol and DMG-PEG2000.

[0048] Furthermore, the present invention also provides the use of the aforementioned vaccine in the preparation of products for preventing HPV infection and / or products for preventing and treating diseases caused by HPV infection. The HPV is HPV16.

[0049] Furthermore, the present invention also provides a method for preventing HPV infection and / or preventing and treating diseases caused by HPV infection, which comprises administering the vaccine as described above. The modes of administration include: injection, oral administration, rectal administration or vaginal administration. The subject of the prevention and / or treatment of the present invention is a mammal receiving treatment for a disease or condition, such as a human, a primate (e.g., a monkey) or a non-primate mammal. The non-primate mammal includes bovines, equines, ovines, porcines, canines, felines or rodents. Among them, the preferred mammal is a mouse, a cat, a dog or a pig.

[0050] The present invention provides an antigen combination, a fusion protein, and encoding nucleic acid. These materials are used to prepare a self-replicating RNA vaccine for treating persistent HPV16 infection of the cervix, induced intraepithelial neoplasia, and cervical cancer. This approach combines E1, E2, E6, and E7, expanding its indications to include those with chronic HPV16 infection. Furthermore, by predicting and extracting epitope-rich regions in the E1E2 protein, its combination with E6E7 significantly enhances immunogenicity. This approach not only eliminates viral host cells and treats chronic infection, but also more effectively treats precancerous lesions and cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 shows the predicted epitopes of E1 and E2;

[0052] Figure 2 shows the expression detection diagram of each sequence;

[0053] FIG3 shows a comparison of the proportion of E7-specific CD8+ T cells induced by CCL11-E6E7E1E2 and CCL11-E6E7 in mice immunized with CCL11-E6E7 by flow cytometry;

[0054] FIG4 shows a comparison of the immunogenicity of CCL11-E6E7E1E2 and CCL11-E6E7;

[0055] Figure 5 shows the anti-tumor effects of each group;

[0056] FIG6 T cells induced by CCL11-E6E7E1E2 lyse B16 cells infected with E1E2-expressing lentivirus in vitro. DETAILED DESCRIPTION

[0057] The present invention provides a drug for treating diseases related to HPV infection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0059] The antigen combination described in the present invention is a combination of HPV E1, E2, E6, and E7 proteins. The antigens in this combination can be linked to each other, mixed as fragments, or partially linked and partially mixed, which is not limited by the present invention. The fragments can be directly linked or connected via a linker, which is also not limited by the present invention.

[0060] The fusion protein described in the present invention is a new protein molecule formed by fusing two or more protein molecules together. Multiple protein fragments can be directly connected or connected through a linker, which is not limited in the present invention.

[0061] The protein-encoding nucleic acid of the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The RNA is in the form of mRNA. The nucleic acid can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be linear or circular in topology. The nucleic acid can be, for example, a part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0062] In the present invention, the nucleic acid sequence for expressing the fusion protein is optimized, and these optimizations include but are not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0063] The present invention also provides a transcription unit for a fusion protein, which refers to a DNA sequence starting from a promoter and ending with a terminator. Regulatory segments may also be included on either side of or between the promoter and terminator. These regulatory segments may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence, such as a promoter enhancer and a poly(A) signal. The transcription unit provided by the present invention includes a CMV or CMV / R promoter, a CMV enhancer, and a nucleic acid segment encoding the fusion protein.

[0064] The recombinant vectors of the present invention refer to recombinant nucleic acid vectors, which are recombinant DNA molecules that contain a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences necessary for expression in prokaryotes include a promoter, and optionally an operator sequence, a ribosome binding site, and possibly other sequences. Prokaryotes are known to utilize promoters, enhancers, and termination and polyadenylation signals. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself.

[0065] In this manual, "plasmid" and "vector" can sometimes be interchanged and general, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, and which are known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or are only potential genome inserts. In a specific embodiment, the nucleic acid encoding the fusion protein provided by the invention can be constructed in various eukaryotic expression vectors or mRNA expression vectors.

[0066] The host cells described herein are prokaryotic or eukaryotic hosts containing nucleic acid vectors and / or target genes. The host cells are transformed or transfected with vectors constructed using recombinant DNA technology. Such transformed host cells are capable of replicating protein-encoding vectors or expressing the desired protein.

[0067] In the embodiment of the present invention, the method for preparing the fusion protein adopts the method of inducing recombinant host expression, and the culture can be cultured bacteria, cells, culture fluid, or substances extracted and / or purified from the above culture.

[0068] Prevention, as used herein, refers to administering the vaccine before a disease develops to reduce the risk of developing the disease. Prevention, as used herein, refers to eliminating the virus during the viral infection stage. Treatment, as used herein, refers to administering the vaccine after the disease develops to inhibit tumor growth, i.e., eliminate the virus, reduce tumor volume, or slow tumor growth. The tumor is cervical intraepithelial neoplasia and / or cervical cancer.

[0069] The sequence involved in the present invention is as follows:

[0070] E6 protein (SEQ ID NO: 1):

[0071] E7 protein (SEQ ID NO: 2):

[0072] E1 protein (SEQ ID NO: 3):

[0073] E2 protein (SEQ ID NO: 4):

[0074] E6E7E1E2 (SEQ ID NO: 5):

[0075] CCL11-E6E7E1E2(SEQ ID NO:6):

[0076] Nucleic acid encoding E6 protein (SEQ ID NO: 7):

[0077] Nucleic acid encoding E7 protein (SEQ ID NO: 8):

[0078] Nucleic acid encoding E1 protein (SEQ ID NO: 9):

[0079] Nucleic acid encoding E2 protein (SEQ ID NO: 10):

[0080] Encoding E6E7E1E2 (SEQ ID NO: 11):

[0081] Encoding CCL11-E6E7E1E2 (SEQ ID NO: 12):

[0082] mRNA vector (SEQ ID NO: 13):

[0083] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0084] Example 1 Selection of epitope-enriched regions of HPV16 virus E1 and E2 proteins.

[0085] Using NetMHCpan-4.1 software, based on artificial neural networks (ANNs), and TepiTool software, based on the Stabilized Matrix Method (SMM), we predicted epitopes within the full-length amino acid sequences of the HPV16 E1 and E2 proteins. During the prediction process, we set the lengths of the E1 and E2 epitopes to 8, 9, 10, 11, and 12 amino acids, respectively. For HLA genotypes with a frequency of more than 5% in the human population (HLA-A01:01, HLA-A02:01, HLA-A03:01, HLA-A11:01HLA-A24:02, HLA-B07:02, HLA-B35:01, HLA-B40:01, HLA-B51:01, HLA-C01:02, HLA-C03:03, HLA-C03:04, HLA-C04:01, HLA-C06:02, HLA-C07:01and HLA-C07:02), the affinity values ​​(IC50) of all antigenic epitopes of E1 and E2 proteins were predicted, and antigenic epitopes with affinity values ​​less than 50nM were considered effective antigenic epitopes. Next, we selected segments from the E1 and E2 proteins that contained more than 10 effective epitopes per 100 amino acids using both prediction methods and defined them as epitope-rich regions. The prediction results are shown in Figure 1, A and B. Based on these predictions, we selected segments 270aa-500aa from E1 and 150aa-360aa from E2.

[0086] Example 2: Assembling E1E2 sequences with E6E7 to form a new vaccine

[0087] Our company has previously applied for a patent based on the fusion of the E6E7 antigen and the chemokine CCL11 (patent application number PCT / CN2022 / 097092). The fusion with CCL11 significantly enhanced the immunogenicity of E6E7. Therefore, this application intends to further add the E1E2 sequence based on the previous patent to expand the indication to patients with HPV infection. Therefore, we constructed six sequence patterns: CCL11-E1E2E6E7, CCL11-E1E6E7E2, CCL11-E2E1E6E7, CCL11-E6E7E1E2, CCL11-E2E6E7E1, and CCL11-E6E7E2E1, numbered 1, 2, 3, 4, 5, and 6, respectively. The nucleotides encoding the corresponding amino acid sequences were codon-optimized and constructed into a vector that can transcribe self-replicating mRNA. The sequence is as described above.

[0088] Self-replicating mRNA is prepared and packaged into LNP formulations. The specific process is to clone a synthetic DNA fragment encoding the protein of interest into a plasmid vector containing sequences corresponding to the T7 promoter, 5' untranslated region (UTR), 3' UTR, and a 31+10nt spacer + 71 nucleotide poly A tail. The plasmid is linearized with the class II restriction endonuclease BspQI to produce a template without additional nucleotides other than poly A. The linearized plasmid DNA purified by ethanol precipitation is subjected to in vitro transcription (IVT) using T7 RNA polymerase (Genscript). In the presence of 10mM N1-methyl pseudodihydropyridine-5'-triphosphate, 5'-adenosine triphosphate, 5'-cytidine triphosphate, and 5'-guanosine triphosphate. The IVT process also includes a co-transcriptional capping agent capable of forming a cap1 structure. The RNA is further purified using magnetic beads. The concentration and quality of the RNA are evaluated by spectrophotometry and capillary gel electrophoresis system. SM102, DSPC, Cholesterol and DMG-PEG2000 were prepared into lipid-ethanol solution according to the concentration and ratio; the mRNA concentration was calculated according to the N / P ratio and the flow rate ratio FRR and dissolved in the mRNA-citrate buffer.

[0089] The lipid-ethanol solution and mRNA-citrate buffer were filtered separately through 0.22-μm membrane filters. The lipid-ethanol solution and mRNA-citrate buffer were drawn into syringes, and the air in the syringes was expelled. The syringes were connected to the lipid and aqueous sample inlets of the microfluidic LNP synthesizer and secured to syringe pumps. The flow rate and mixing volume of the syringe pumps were adjusted, and mRNA-LNP synthesis was completed using a microfluidic chip. Dialysis or ultrafiltration was subsequently performed as needed. 1 μg of each coated replicate mRNA sample was transfected into 293T cells, and protein expression was assayed. The results showed that CCL11-E6E7E1E2 had the highest expression level, as shown in Figure 2. Therefore, the combined sequence was determined to be CCL11-E6E7E1E2.

[0090] Example 3 Comparison of the immunogenicity of CCL11-E6E7E1E2 and original CCL11-E6E7

[0091] Explore the effect of the addition of E1E2 on the cell-specific T cell response induced by the original CCL11-E6E7 fusion gene vaccine (using the vaccine with HPV16 E6 and E7 proteins as the antigen as an example; the steps for inducing T cell responses for vaccines containing other antigens are the same):

[0092] Given that the fusion gene can be expressed normally in mammalian cells, we prepared an mRNA vaccine combining CCL11-E6E7E1E2 and the original CCL11-E6E7 and immunized mice at a dose of 1 μg, with five mice per group. After a single injection, blood was collected from each group on days 7, 14, 17, 21, 24, and 28, and added to heparinized PBS. The entire sample was centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded, and the remaining pellet was dispersed by vortexing and lysed in 1 mL of lysis buffer at room temperature for 1 minute. The entire sample was then centrifuged at 1200 rpm for 6 minutes. The supernatant was discarded, and the pellet was washed once with 700 μL of PBS and centrifuged a second time at 1200 rpm for 6 minutes. After the supernatant was discarded, 300 μL of inactivated 10% FBS-containing 1640 medium was added, the pellet was resuspended, and stained with 1 μL of E7 protein tetramer (E7-tetramer) for 1 hour. Flow cytometry staining: CD8-FITC; E7-tetramer-PE. The flow cytometry results are shown in Figure 3.

[0093] The results showed that the number of E7-specific T cells in all CCL11-E6E7E1E2 groups was significantly higher than that in the CCL11-E6E7 group at D17 and later time points. This suggests that the addition of E1E2 to the C-terminus effectively enhances the immunogenicity of the original E7 antigen, allowing CCL11-E6E7 to ultimately induce a stronger specific immune response to the antigen molecule. Following the evaluation of E7 immunogenicity, we analyzed the immunogenicity of E1E2 and E6 antigens using ELISPOT analysis in mouse spleens at the end of the experiment (D28). The results, shown in Figure 4, demonstrate that E1E2 itself indeed generates a corresponding immune response, providing the basis for the expansion of the present invention's indications to individuals with chronic HPV infection. Furthermore, the number of E6 antigen spots was significantly higher than that of E6 in CCL11-E6E7. This demonstrates that the addition of E1E2 not only generates an immune response itself but also unexpectedly enhances the immunogenicity of E6E7.

[0094] Example 4 Comparison of the anti-tumor effects of CCL11-E6E7E1E2 and original CCL11-E6E7

[0095] The therapeutic effect of the fusion gene vaccine on TC-1 homologous transplanted tumor cells in mice (using the fusion gene vaccine containing HPV16 E6 and E7 proteins as an example; the verification steps for the tumor intervention effect of vaccines containing other antigens are the same):

[0096] Given the excellent efficacy of the CCL11-E6E7E1E2 vaccine in cellular immunization experiments, we further investigated the therapeutic efficacy of the fusion gene in TC-1 homograft tumors. Each immunization dose was 1 μg. Around day 10 after implantation, when the average tumor volume reached 150 mm³, mice were immunized with a single dose. Tumors were subsequently measured and volume calculated using the same method. Tumor growth curves are plotted and shown in Figure 5. The results showed that the CCL11-E6E7E1E2 mRNA group had a stronger tumor-suppressing effect than the CCL11-E6E7 mRNA group. Because the TC-1 cell line expresses the E6E7 antigen, the addition of E1E2 unexpectedly enhanced the anti-tumor effect of CCL11-E6E7.

[0097] Example 5: T cells induced by CCL11-E6E7E1E2 lyse B16 cells infected with lentivirus expressing E1E2 in vitro

[0098] Because TC-1 cells do not express the E1E2 antigen, although an E1E2 immune response was detected in Example 3, whether this response effectively cleared the viral infection remained to be verified. We encapsulated E1E2 into a lentivirus and infected B16 cells. Splenocytes from mice immunized with CCL11-E6E7E1E2 or CCL11-E6E7 were then isolated and co-incubated with B16 cells infected with the E1E2 lentivirus. The results showed that a 5:1 ratio of splenocytes from mice immunized with CCL11-E6E7E1E2 to B16 cells infected with the E1E2 lentivirus effectively killed the virus, whereas splenocytes from mice immunized with CCL11-E6E7 did not. These results suggest that the addition of E1E2 lyses the virus-infected cells (see Figure 6).

[0099] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antigen combination comprising an immunogenic fragment of the E1 protein, an immunogenic fragment of the E2 protein, and E6 and E7 proteins of the HPV virus.

2. The antigen combination according to claim 1, characterized in that From N-terminus to C-terminus, it includes HPV virus E6 protein, E7 protein, E1 protein immunogenic fragment and E2 protein immunogenic fragment.

3. The antigen combination according to claim 1 or 2, characterized in that The HPV virus is HPV16 virus; The amino acid sequence of the E6 protein is shown in SEQ ID NO: 1; The amino acid sequence of the E7 protein is shown in SEQ ID NO: 2; The amino acid sequence of the immunogenic fragment of the E1 protein is shown in SEQ ID NO: 3; The amino acid sequence of the immunogenic fragment of the E2 protein is shown in SEQ ID NO:

4.

4. The antigen combination according to claim 3, characterized in that Its amino acid sequence is shown in SEQ ID NO:

5.

5. An immunogenic fragment of E1 protein having the amino acid sequence shown in SEQ ID NO:

3.

6. An immunogenic fragment of the E2 protein having the amino acid sequence shown in SEQ ID NO:

4.

7. A fusion protein comprising the chemokine CCL11 and the antigen combination according to any one of claims 1 to 4.

8. The fusion protein according to claim 7, characterized in that Its amino acid sequence is shown in SEQ ID NO:

6.

9. A nucleic acid encoding the antigen combination according to any one of claims 1 to 4 or the fusion protein according to claim 7 or 8.

10. The nucleic acid according to claim 9, characterized in that The nucleic acid encoding the E6 protein has the sequence shown in SEQ ID NO: 7; The nucleic acid encoding the E7 protein has the sequence shown in SEQ ID NO: 8; The nucleic acid encoding the E1 protein has the sequence shown in SEQ ID NO:9; The nucleic acid encoding the E2 protein has the sequence shown in SEQ ID NO:

10.

11. The nucleic acid according to claim 9, characterized in that A nucleic acid encoding an antigen combination having an amino acid sequence as shown in SEQ ID NO: 5, having a sequence as shown in SEQ ID NO: 11; The nucleic acid encoding the antigen combination with the amino acid sequence shown in SEQ ID NO:6 has the sequence shown in SEQ ID NO:

12.

12. The nucleic acid according to any one of claims 9 to 11, characterized in that It also includes a 5' end cap structure and / or a 3' end poly A tail.

13. A plasmid vector comprising a backbone vector and the nucleic acid according to any one of claims 9 to 12.

14. A host cell comprising the plasmid vector according to claim 13 or a host cell in which the nucleic acid according to any one of claims 9 to 11 is integrated into its genome.

15. A method for preparing the antigen combination according to any one of claims 1 to 4 or the fusion protein according to claim 7 or 8, comprising: The nucleic acid encoding the antigen combination according to any one of claims 1 to 4 or the fusion protein according to claim 7 or 8 is inserted into a plasmid vector and expressed in a host cell.

16. A method for preparing the nucleic acid according to claim 12, comprising inserting a nucleic acid encoding the antigen combination according to any one of claims 1 to 4 or the fusion protein according to claim 7 or 8 into a self-replicating mRNA vector, preparing a nucleic acid with a polyA structure, and obtaining the nucleic acid according to claim 10 after linearization and in vitro transcription.

17. Use of the antigen combination according to any one of claims 1 to 4, the fusion protein according to claim 7 or 8, and / or the nucleic acid according to any one of claims 9 to 12 in the preparation of a vaccine.

18. A vaccine comprising the antigen combination according to any one of claims 1 to 4, the fusion protein according to claim 7 or 8, and / or the nucleic acid according to any one of claims 9 to 12.

19. The vaccine according to claim 18, characterized in that The vaccine comprises the antigen combination according to any one of claims 1 to 4, and further comprises an aluminum adjuvant; The vaccine comprises the fusion protein according to claim 7 or 8, and further comprises an aluminum adjuvant; The nucleic acid according to any one of claims 9 to 12 is DNA, and the vaccine further comprises Poly I:C, CpG, or a STING agonist; The vaccine comprises the nucleic acid according to any one of claims 9 to 12, which is mRNA, and the vaccine further comprises SM102, DSPC, Cholesterol and DMG-PEG2000.

20. Use of the vaccine according to claim 19 in the preparation of products for preventing HPV infection and / or products for preventing and treating diseases caused by HPV infection.

21. A method for preventing HPV infection and / or preventing and treating diseases caused by HPV infection, comprising administering the vaccine according to any one of claims 18 to 20.