Recombinant HPV16 E6E7 adenovirus as well as vaccine, preparation method and application thereof

Abstract

The invention belongs to the technical field of cancer immunotherapy, and particularly relates to a recombinant HPV16E6E7 adenovirus as well as a vaccine, a preparation method and application thereof. In order to develop more safe and effective HPV vaccines with long-term immune efficacy, the invention provides a recombinant HPV16E6E7 adenovirus which contains an HPV16E6E7 antigen sequence and SARS-CoV-2 virus HR1 and HR2 segment sequences of the HPV16E6E7 antigen sequence. The recombinant HPV16E6E7 adenovirus vaccine prepared from the adenovirus and the combination of the recombinant HPV16E6E7 adenovirus vaccine and other anti-tumor drugs can significantly inhibit the growth of tumors in cervical cancer and head and neck squamous cell carcinoma, and can trigger an obvious cellular immune response in a mouse body, thereby providing a candidate scheme for the transformation research of cancer immunotherapy in the future.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer immunotherapy, and specifically relates to a recombinant HPV16 E6E7 adenovirus and a vaccine thereof, as well as a preparation method and application thereof. Background Art

[0002] Human papillomavirus (HPV) is considered one of the most common sexually transmitted viruses, infecting the skin and mucous membranes. Currently, 15 high-risk HPV types have been linked to the development and progression of cervical cancer, with HPV16 and HPV18 accounting for nearly 70% of cervical cancer cases. While the incidence of cervical cancer has declined dramatically due to widespread screening and prevention efforts, the incidence of head and neck squamous cell carcinoma (HNSCC) in the United States continues to rise and is expected to surpass that of cervical cancer. Notably, oropharyngeal squamous cell carcinoma (OSC), a specific type of HNSCC, has been linked to persistent infection with high-risk HPV. Nearly 70% of ONSCC cases are caused by HPV, particularly HPV16. Furthermore, approximately 90% of anal cancers are caused by persistent infection with high-risk HPV types. Consequently, persistent infection with high-risk HPV can lead to a variety of cancers, including HNSCC, cervical, and anal cancers, as well as less common cancers of the vulva, penis, and vagina.

[0003] Cancer immunotherapy aims to trigger the host immune response to fight tumor cells. A variety of immunotherapy methods have been developed to stimulate innate and adaptive immunity in the tumor microenvironment. + T lymphocytes (CTLs) are considered the most effective killer cells. CTL activation primarily relies on antigen-presenting cells, such as macrophages and dendritic cells, which present tumor-associated antigens to immature T cells in a process known as cross-presentation. Once activated, CTLs eliminate tumor cells by secreting cytokines such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα), as well as releasing cytotoxic granules, thereby preventing and treating tumors. Therefore, various types of tumor vaccines have been extensively developed and are undergoing clinical trials based on cancer immunotherapy.

[0004] Persistent HPV16 infection can cause high-grade intraepithelial neoplasia, which can subsequently develop into tumors. HPV16 oncoproteins E6 and E7 play a crucial role in tumor development, progression, and metastasis. E6 and E7 trigger cancer development by promoting cell proliferation, inhibiting apoptosis, enhancing cell migration, suppressing cellular immune responses, and disrupting cellular metabolic balance.

[0005] It can be seen that the preparation of tumor vaccines based on cancer immunotherapy targeting HPV16 E6E7 has great potential in the prevention and treatment of tumors. Currently, Gardasil, Cervarix, and Gardasil-9 are all marketed HPV vaccines that have shown excellent results in the prevention of HPV-induced tumors, especially cervical cancer. Therapeutic vaccines for HPV-induced tumors have been widely developed, such as ADXS11-001, TA-HPV, and PRGN-2009. Although they have been put into various phases of clinical trials, no vaccine has yet been successfully marketed and widely promoted for use. Therefore, the preparation of a safe, effective, and long-term HPV vaccine is of great significance for the prevention, treatment, and prognosis of HPV-induced tumors. Summary of the Invention

[0006] In order to develop more safe, effective and long-term immune efficacy HPV vaccines, the present invention provides a recombinant HPV16E6E7 adenovirus vaccine and its preparation method and application.

[0007] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0008] In a first aspect, the present invention provides a recombinant HPV16 E6E7 adenovirus, the antigen expressed by which contains the HPV16 E6E7 antigen sequence and the SARS-CoV-2 virus HR1 and HR2 segment sequences.

[0009] Furthermore, the amino acid sequence of the HPV16 E6E7 antigen is as shown in SEQ ID No. 1, or has more than 80% homology with SEQ ID No. 1 and has the same or similar biological activity.

[0010] Furthermore, the nucleotide sequence of the HPV16 E6E7 antigen is shown as SEQ ID No. 2.

[0011] SEQ ID No. 1: Amino acid sequence of HPV16 E6E7 antigen

[0012] HQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGIMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP。

[0013] SEQ ID No.2: Nucleotide sequence of HPV16 E6E7 antigen

[0014] .

[0015] Furthermore, the amino acid sequences of the HR1 and HR2 segments of the SARS-CoV-2 virus are as shown in SEQ ID No. 3, or have more than 80% homology with SEQ ID No. 3 and have the same or similar biological activity.

[0016] Furthermore, the nucleotide sequences of the HR1 and HR2 segments of the SARS-CoV-2 virus are shown in SEQ ID No. 4.

[0017] SEQ ID No.3: Amino acid sequence of HR1 and HR2 segments of SARS-CoV-2 virus

[0018] LYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPD VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL.

[0019] SEQ ID No.4: Nucleotide sequence of HR1 and HR2 segments of SARS-CoV-2 virus

[0020] CTGTACGAGAATCAGAAGCTGATCGCCAATCAGTTCAACAGCGCCATCGGCAAGATCCAAGACAGCCTGAGCAGCACCGCTAGCGCCCTGGGCAAGCTGCAAGACGTGGTGAATCAGAACGCCCAAGCCCTGAACACCCTGGTGAAG CAGCTGAAGAACCACACAAGCCCCGACGTGGACCTGGGCGACATCAGCGGCATCAACGCTAGCGTGGTGAACATTCAGAAGGAGATCGACAGACTGAACGAGGTGGCCAAGAACCTGAACGAGAGCCTGATCGACCTGCAAGAGCTG.

[0021] Preferably, the amino acid sequence of the antigen expressed by the recombinant HPV16 E6E7 adenovirus is shown as SEQ ID No.5.

[0022] Preferably, the nucleotide sequence of the antigen expressed by the recombinant HPV16 E6E7 adenovirus is shown as SEQ ID No.6.

[0023] SEQ ID No.5: Amino acid sequence of the recombinant HPV16 E6E7 adenovirus expressing the antigen of the present invention

[0024] HQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGIMHGDTPTLHEYMLDLQPET TDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL.

[0025] SEQ ID No.6: Nucleotide sequence of the recombinant HPV16 E6E7 adenovirus expressing antigen of the present invention

[0026]

[0027] Furthermore, the HPV16 E6E7 antigen forms a trimer structure with the HR1 and HR2 segments of the SARS-CoV-2 virus.

[0028] In a second aspect, the present invention provides an adenovirus vector comprising the nucleic acid sequence for expressing the antigen of the recombinant HPV16 E6E7 adenovirus.

[0029] Furthermore, the adenovirus vector is selected from human Ad5 vector, Ad35 vector or Ad26 vector and / or chimpanzee AdC68 vector, AdC7 vector or ChAdOx1 vector.

[0030] Preferably, it is selected from the human type 5 replication-deficient adenovirus in which E1 and E3 are jointly deleted.

[0031] In a third aspect, the present invention provides a method for preparing the above-mentioned recombinant HPV16 E6E7 adenovirus, which comprises the following steps: synthesizing the HPV16 E6E7-HR gene, and constructing an adenovirus shuttle plasmid containing the HPV16 E6E7-HR gene using molecular cloning technology, co-transfecting the shuttle plasmid with the backbone plasmid of the AdMax adenovirus system into host cells for packaging the recombinant adenovirus to obtain a replication-defective recombinant adenovirus, and then expanding the culture and purifying it.

[0032] In the above preparation method:

[0033] Furthermore, the nucleotide sequence of the HPV16 E6E7-HR gene is shown in SEQ ID No.6.

[0034] Furthermore, the vector used by the adenovirus is selected from human Ad5 vector, Ad35 vector or Ad26 vector and / or chimpanzee AdC68 vector, AdC7 vector or ChAdOx1 vector.

[0035] Preferably, it is selected from the human type 5 replication-deficient adenovirus in which E1 and E3 are jointly deleted.

[0036] Furthermore, the shuttle plasmid is selected from at least one of pDC516, pDC316, pDC311, pDC312, pDC315, pDC511, pDC512, pDC515, pShuttle, pShuttle-CMV, pCTAP-Shuttle series plasmids, pNTAP-Shuttle series plasmids, pAdTrack, pAdTrack-CMV, pacAd5 series plasmids, pHBAd series plasmids or pXC1 plasmids.

[0037] Furthermore, the backbone plasmid is selected from at least one of pBHGfrtdelE13FLP, pBHGloxdelE13cre, pAdEasy-1, pAdEasy-2, pBHGE3i or pBHGE10i.

[0038] Furthermore, the host cell is selected from at least one of HEK293, PER.C6, HeLa, A549 or HT-1080.

[0039] In a fourth aspect, the present invention provides a recombinant HPV16 E6E7 adenovirus vaccine comprising the above-mentioned recombinant HPV16 E6E7 adenovirus or adenovirus vector.

[0040] Furthermore, the recombinant HPV16 E6E7 adenovirus vaccine is in the form of an injection, nasal drops, spray or inhaler.

[0041] Preferably, the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection or intraperitoneal injection.

[0042] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned recombinant HPV16 E6E7 adenovirus, adenovirus vector or vaccine, and other anti-tumor drugs.

[0043] In a sixth aspect, the present invention provides a combination drug, which comprises administering the above-mentioned recombinant HPV16 E6E7 adenovirus, adenovirus vector or vaccine separately or simultaneously with other anti-tumor drugs.

[0044] Furthermore, the other anti-tumor drugs are selected from at least one of cisplatin, paclitaxel, carboplatin, topotecan, bevacizumab, cetuximab, pembrolizumab, nivolumab, medroxyprogesterone acetate or 5-fluorouracil (5-FU).

[0045] Preferably, the other anti-tumor drugs are cisplatin and paclitaxel.

[0046] Furthermore, the pharmaceutical composition or the combined drug is in the form of an injection, nasal drops, spray or inhalant.

[0047] Preferably, the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection or intraperitoneal injection.

[0048] In a sixth aspect, the present invention provides the use of the above-mentioned recombinant HPV16 E6E7 adenovirus, adenovirus vector, recombinant HPV16 E6E7 adenovirus vaccine, pharmaceutical composition or combination drug in preventing and / or treating tumors caused by HPV infection.

[0049] Furthermore, the tumor includes at least one of cervical cancer, head and neck squamous cell carcinoma, anal cancer, vulvar cancer, vaginal cancer or penile cancer.

[0050] Preferably, the head and neck squamous cell carcinoma includes oropharyngeal squamous cell carcinoma, hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma.

[0051] Beneficial Effects: Based on the HPV16 E6E7 target, the present invention has developed a recombinant HPV16 E6E7 adenovirus vaccine for the prevention and treatment of various tumors caused by HPV infection, including head and neck squamous cell carcinoma, cervical cancer, and anal cancer. The vaccine contains the HPV16 E6E7 antigen sequence and the HR1 and HR2 segments of the SARS-CoV-2 virus. Animal experiments have shown that the recombinant HPV16 E6E7 adenovirus vaccine can effectively activate the cellular immune response of mice, and has a good preventive and therapeutic effect on both the mEERL orthotopic tumor model and the TC-1 subcutaneous tumor model, significantly improving the survival rate of tumor-bearing mice. Moreover, its combination with cisplatin / paclitaxel has achieved significant therapeutic effects in the TC-1 subcutaneous tumor treatment model. Therefore, the recombinant HPV16 E6E7 adenovirus vaccine of the present invention provides a good candidate vaccine for translational research on the treatment and prevention of tumors caused by HPV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a graph showing the tumor growth curves of the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) and the HPV16 E6E7 adenovirus vaccine (without HR) in Example 2.

[0053] Figure 2 These are the evaluation results of the cellular immune response induced by the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) in Example 3; mice immunized with the recombinant HPV16 E6E7 adenovirus group (containing HR) and the control and empty adenovirus groups showed: A) the proportion of E6 tetramer-positive CD8+ cells in peripheral blood mononuclear cells; B) the proportion of E7 tetramer-positive CD8+ cells in peripheral blood mononuclear cells; C) the proportion of E6 tetramer-positive CD8+ cells in spleen cells; D) the proportion of E7 tetramer-positive CD8+ cells in spleen cells; E) the proportion of CD69+CD4+ cells in spleen cells; F) the proportion of CD69+CD8+ cells in spleen cells.

[0054] Figure 3These are the evaluation results of the cellular immune response induced by the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) in Example 3; mice immunized with the recombinant HPV16 E6E7 adenovirus group (containing HR) and the control and empty adenovirus groups: A) the proportion of central memory CD8+ cells in splenocytes; B) the proportion of effector memory CD8+ cells in splenocytes; C) the proportion of CD8+ cells secreting interferon-γ after stimulation with the E6 peptide library; D) the proportion of CD8+ cells secreting interferon-γ after stimulation with the E7 peptide library; E) the proportion of CD8+ cells secreting tumor necrosis factor-α after stimulation with the E7 peptide library; F) statistical analysis of interferon-γ spot counts.

[0055] Figure 4 This is an image of an enzyme-linked immunosorbent spot (ELISPOT) assay after stimulation with the E7 peptide library in Example 3.

[0056] Figure 5 This is the survival curve of the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) in the mEERL orthotopic tumor treatment model of Example 4.

[0057] Figure 6 These are the anti-tumor effect results of the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) in the TC-1 subcutaneous tumor treatment model of Example 5: A) tumor growth curve; B) tumor weight; C) survival curve of mice in the control group, adenovirus empty vector group, and recombinant HPV16 E6E7 adenovirus group (containing HR).

[0058] Figure 7 These are the anti-tumor effect results of the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) in the TC-1 subcutaneous tumor treatment model of Example 5; A) the proportion of E6 tetramer-positive CD8+ cells in peripheral blood mononuclear cells, B) the proportion of E7 tetramer-positive CD8+ cells in peripheral blood mononuclear cells, C) the proportion of E6 tetramer-positive CD8+ cells in spleen cells, D) the proportion of E7 tetramer-positive CD8+ cells in spleen cells, E) the proportion of myeloid-derived immunosuppressive cells in tumor cells, and F) the proportion of M2 macrophages in tumor cells in the control group, the empty adenovirus group, and the recombinant HPV16 E6E7 adenovirus group (containing HR).

[0059] Figure 8 This is the anti-tumor effect of the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) in the TC-1 subcutaneous tumor prevention model of Example 6: A) Tumor growth curve; B) Tumor weight; C) Survival curve of mice in the control group, adenovirus empty vector group, and recombinant HPV16 E6E7 adenovirus group (containing HR); D) Tumor growth curve; E) Survival curve of mice in the control group and recombinant HPV16 E6E7 adenovirus group in the TC-1 re-challenge tumor model.

[0060] Figure 9 This is the tumor growth curve of Example 7, which shows the recombinant HPV16 E6E7 adenovirus vaccine (containing HR) combined with cisplatin / paclitaxel in the TC-1 subcutaneous tumor treatment model.

[0061] Figure 10 Specific sequence information for the HPV16 E6 and E7 peptide libraries.

[0062] Data are expressed as mean ± SEM, with statistical significance as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. DETAILED DESCRIPTION

[0063] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, this application is further described in detail below in conjunction with the embodiments. Unless otherwise defined herein, the meanings of all technical terms and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art.

[0064] The terms "include," "comprising," "having," "containing," or "involving," and their variations herein, are inclusive or open-ended and do not exclude other unrecited elements or method steps. Those skilled in the art will understand that the above terms, such as "comprising," encompass the meaning of "consisting of."

[0065] In the present invention, "a", "an", "at least one" and "one or more" are used interchangeably. When the lower limit and upper limit of a numerical range are disclosed, any value and any included range falling within the range are specifically disclosed. In particular, each range of values ​​disclosed herein (in the form of "about a to b", or equivalently, "approximately a to b", or equivalently, "about a b") should be understood to represent each value and range encompassed in the broader range.

[0066] The terms "HPV16 E6 and E7" refer to two key viral oncoproteins that play a central role in the malignant transformation of cells following HPV-16 infection. HPV-16 is a high-risk HPV type that is closely associated with the development of various cancers, including cervical cancer, head and neck squamous cell carcinoma, and anal cancer. E6 and E7 interfere with the normal regulatory mechanisms of host cells, promoting cell proliferation and inhibiting apoptosis, thereby contributing to the development of cancer.

[0067] HPV16 E6 and E7 interfere with key tumor suppressor pathways, such as p53 and pRb, promoting cell proliferation, inhibiting apoptosis, and inducing genomic instability, ultimately leading to cancer. They are important targets for HPV-related cancer research and represent a breakthrough in the development of new treatments.

[0068] The term "TC-1 cells" refers to cells derived from lung epithelial cells of C57BL / 6 mice transformed with HPV-16 E6 and E7 oncogenes. These cells express HPV-16 E6 and E7 proteins, which play an important role in the development of cervical cancer.

[0069] The term "mEERL cells" refers to oral epithelial cells derived from C57BL / 6 mice and transformed with the HPV-16 E6 and E7 oncogenes. It is a cell line commonly used in head and neck cancer research, particularly HPV-associated head and neck squamous cell carcinoma.

[0070] Cisplatin and Paclitaxel are two commonly used chemotherapy drugs. Cisplatin and Paclitaxel work synergistically through different mechanisms to enhance their ability to kill cancer cells. They are widely used to treat a variety of cancers, including testicular cancer, ovarian cancer, bladder cancer, lung cancer, head and neck cancer, breast cancer, pancreatic cancer, and non-small cell lung cancer.

[0071] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0072] The main materials used in the following examples are as follows:

[0073] The E6 peptide library and the E7 peptide library were commissioned to Wuhan Dangang Biotechnology Co., Ltd. for synthesis; the specific information of the peptide library is shown in the attached Figure 10 .

[0074] mEERL cells were purchased from ABM, and TC-1 cells were purchased from Xiamen Yimo Biotechnology Co., Ltd.

[0075] C57BL / 6 mice (6–8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All animal experiments were performed in accordance with the guidelines evaluated and approved by the Ethics Committee of Sichuan University.

[0076] Example 1 Preparation of recombinant HPV16 E6E7 adenovirus vaccine based on human type 5 replication-deficient adenovirus with combined deletion of E1 and E3

[0077] 1. Acquisition of HPV16 E6E7-HR gene

[0078] The HPV16 E6 stop codon was mutated to express E6 and E7 as a fusion protein, and amino acids 24 and 26 of the HPV16 E7 protein were mutated from C and E to G and G, respectively. The HR1 (916-966aa) and HR2 (1157-1203aa) regions of the SARS-CoV-2 S protein were added to the C-terminus of the E6E7 fusion protein to form a trimer structure, thereby increasing the immunogenicity of the antigen. A signal peptide was added or not to the E6E7-HR fusion protein (amino acid sequence shown in SEQ ID No. 5) to express the E6E7-HR fusion protein as a secreted protein or an intramembrane protein. The HPV16 E6E7-HR sequence (shown in SEQ ID No. 5) was converted into a nucleotide sequence, and Suzhou Jinweizhi Technology Co., Ltd. was commissioned to perform humanization optimization of the HPV16 E6E7-HR codon and synthesize the HPV16 E6E7-HR gene (nucleotide sequence shown in SEQ ID No. 6). HPV16 E6E7-HR was cloned into the adenovirus shuttle plasmid pDC516 using molecular cloning technology to obtain the pDC516-E6E7-HR shuttle plasmid.

[0079] The acquisition of the HPV16 E6E7 gene was the same as the above operation, except that the E6E7 fusion protein (amino acid sequence as shown in SEQ ID No. 1) was directly codon-optimized, and the operation of adding HR1 and HR2 of the SARS-CoV-2 virus S protein to its C-terminus was not performed. The nucleotide sequence of the HPV16 E6E7 gene is shown in SEQ ID No. 2. Molecular cloning technology was also used to clone HPV16 E6E7 into the adenovirus shuttle plasmid pDC516 plasmid to obtain the pDC516-E6E7 shuttle plasmid.

[0080] 2. Packaging of recombinant HPV16 E6E7 adenovirus vaccine

[0081] The constructed pDC516-E6E7-HR or pDC516-E6E7 shuttle plasmid was co-transfected with the backbone plasmid pBHGfrtdelE13FLP of the AdMax adenovirus system into HEK293 cells for recombinant adenovirus packaging. The process is as follows:

[0082] 1) 8×10 5 HEK293A cells were seeded in six-well plates with high-glucose DMEM + 10% FBS medium and cultured overnight in a cell culture incubator at 37°C containing 5% CO2.

[0083] 2) The next day, the medium was replaced with high-glucose DMEM + 2% FBS, and the adenovirus backbone plasmid (pBHGfrtdelE13FLP) with the adenovirus E1 and E3 regions deleted and the shuttle plasmid were co-transfected into HEK293A cells using lipofectamine 3000. The specific steps were as follows: 4 μg of backbone plasmid and 2 μg of shuttle plasmid were taken for each transfection well, diluted with 125 μL Opti-MEM medium, and then 12 μL P3000 reagent was added; in another 1.5 ml EP tube, 7.5 μL lipofectamine 3000 was diluted with 125 μL Opti-MEM medium; the diluted plasmid and diluted lipofectamine 3000 were mixed in a 1:1 ratio, incubated at room temperature for 10-15 minutes, and then added to the cells. The cells were cultured continuously and passaged on 25 cm 2 In the cell culture flask, observe the signs of cell toxicity every day, and when the cells have grown to the bottom of the flask, transfer them to 75cm 2 In the cell culture flask, the virus is collected until obvious plaques appear on the cells and most of the cells become diseased and fall off from the bottom.

[0084] 3) Collect the virus-free cell culture and centrifuge at 1200 rpm for 3 minutes. The virus-containing supernatant is aspirated and the cell pellet is resuspended in 1 / 10 the culture volume of the virus-containing supernatant. The cell pellet is placed in a -80°C refrigerator and then frozen and thawed three times in a 37°C water bath. Centrifuge at 3000 rpm for 20 minutes, collect the virus-containing supernatant, and combine it with the virus-containing supernatant above. This is the adenovirus seed for the vaccine.

[0085] 4) Take 50 μL of the vaccine candidate strain seed liquid, add 2 μL of proteinase K, and digest at 50°C for 30 min to release the viral genome. Use this as a template for PCR amplification of the HPV16 E6E7-HR gene or HPV16 E6E7 gene sequence. The PCR product is recovered from the electrophoresis gel and sequenced for identification.

[0086] The conditions for PCR amplification are as follows:

[0087] Denaturation: 98°C, 2 min; Denaturation: 98°C, 10 s; Annealing: 60°C, 15 s; Extension: 72°C, 55 s; Extension: 72°C, 5 min; Cycle number: 35 times; PCR amplification primers are as follows:

[0088] IM05-F:GAGCTGCGTTCTACGTGGGTATAAG(SEQ ID No.7)

[0089] IM05-R:TCCATCAAACGAGTTGGTGCTCATG(SEQ ID No.8)

[0090] 3. Amplification of recombinant HPV16 E6E7 adenovirus vaccine

[0091] The identified recombinant adenovirus vaccine strain was amplified in 293-H cells step by step. The specific process was as follows: at an MOI of 3, the cell density was 4×10 6 Adenovirus seed is added to a 293-H cell suspension of 10 cells / ml. After 48-72 hours, the virus culture is collected and the master virus seed bank and the working virus seed bank are prepared according to the repeated freeze-thaw method mentioned above. The recombinant adenovirus vaccine is amplified on a large scale using a cell shake flask or bioreactor, and the virus culture is collected after most of the cells are diseased. The process of amplifying cells and viruses in a bioreactor is as follows: First, after the bioreactor is sterilized, cell culture medium is added to the bioreactor. When the operating conditions are stable at 37°C, pH 7.0, DO 50%, 50-150rpm, the 293-H cells amplified in the shake flask are collected and inoculated into the bioreactor at an inoculation cell density of 1.0×10 6 Cells / ml were added to 10L of cell culture medium. The conditions for cell culture in the reactor were temperature 37°C, rotation speed 50-150rpm, pH 7.15-7.25, DO 30-50%. Glucose concentration, cell density and cell morphology were measured daily. When the cell density in the bioreactor reached 1.0-5.0×10 6 When the cell viability reaches 60-70%, the culture is terminated and virus lysis buffer is added to the bioreactor with a final concentration of 0.05%-1% Tween 80. The virus is lysed at 37°C for 2-4 hours and the virus liquid is collected.

[0092] 4. Purification of recombinant HPV16 E6E7 adenovirus vaccine

[0093] The collected virus is purified by cesium chloride ultracentrifugation or ion exchange chromatography. The specific process is as follows:

[0094] (1) Purification of recombinant HPV16 E6E7 adenovirus vaccine by cesium chloride ultracentrifugation

[0095] The collected virus culture was centrifuged at 1200 g for 10 minutes, the virus-containing culture supernatant was aspirated, the cell pellet was resuspended with 1 / 10 culture volume of the virus-containing supernatant, and repeatedly frozen and thawed three times in a -80°C refrigerator and a 37°C water bath, centrifuged at 3000 rpm for 10-20 minutes, and the supernatant was aspirated. The virus-containing culture supernatant was concentrated 10-fold using a 100K-300K ultrafiltration membrane pack; a 1.4 g / ml cesium chloride solution (53 g cesium chloride + 87 ml 10 mM Tris-HCl, pH 7.9) and a 1.2 g / ml cesium chloride solution (26.8 g cesium chloride + 92 ml 10 mM Tris-HCl, pH 7.9) were prepared; 8 ml of the 1.4 g / ml cesium chloride solution was slowly added to an ultracentrifuge tube, followed by the gentle addition of 6 ml of the 1.2 g / ml cesium chloride solution; and finally, 20 ml of the virus-containing supernatant was added to the top of the discontinuous gradient. After balancing, the mixture was centrifuged at 100,000 × g at 4° C. for 90 minutes; after centrifugation, the blue virus band was aspirated with a syringe, dialyzed to remove cesium chloride, and stored at −80° C.

[0096] (2) Ion exchange chromatography purification of recombinant HPV16 E6E7 adenovirus vaccine

[0097] The viral culture was collected and lysed with 0.05%-1% Tween 20 at 37°C for 2-4 hours. The lysed culture was clarified by filtration through 1.2μm and 0.45μm capsule filters. The sample was concentrated 10-fold using a 100-300kD molecular weight tangential flow membrane bag and then washed with 10 volumes of filtration buffer (50mM Tris-HCl, 2mM MgCl2, 0-500mM NaCl, pH 8.0). The washed sample was collected; nuclease was added to the washed sample at a final concentration of 10-50U / ml and digested at 37°C for 1-3 hours. The sample was then purified using Q Sepharose XL, Source 30Q, or Source Anion exchange chromatography with 15Q fillers follows the following procedure: equilibration with equilibration buffer for 5 column volumes at a flow rate of 20 ml / min. Sample is then loaded at a rate of 10 ml / min. After loading, the equilibration buffer is equilibrated to a conductivity level. The sample is then eluted using a linear gradient from 100% low-salt buffer to 100% high-salt buffer at a flow rate of 10 ml / min, with each elution peak collected. After elution, the column is regenerated with 2M NaCl buffer for 5-10 column volumes at a flow rate of 20 ml / min. The virus peak is then collected, and the eluted virus sample is then subjected to buffer exchange by dialysis or tangential flow filtration.

[0098] The recombinant HPV16 E6E7 adenovirus vaccine obtained by HPV16 E6E7-HR gene recombination is defined as "recombinant HPV16 E6E7 adenovirus vaccine (containing HR)", and the recombinant HPV16 E6E7 adenovirus vaccine obtained by HPV16 E6E7 gene recombination is defined as "HPV16 E6E7 adenovirus vaccine (excluding HR)", and animal experiments are used to verify the efficacy.

[0099] Example 2 Tumor Therapy Assay of Recombinant HPV16 E6E7 Adenovirus Vaccine

[0100] To compare the therapeutic efficacy of recombinant HPV16 E6E7 adenovirus vaccine (with HR) and HPV16 E6E7 adenovirus vaccine (without HR) in tumor cell therapy, 6-8 week-old female C57BL / 6 mice were subcutaneously inoculated with 2×10 5 TC-1 cells. On the third day after tumor cell inoculation, the mice were randomly divided into four groups. The mice in each group were treated with PBS, empty adenovirus, recombinant HPV16 E6E7 adenovirus vaccine (with HR), and HPV16 E6E7 adenovirus vaccine (without HR), respectively (5×10 5 VP / mouse, intramuscular injection). Tumor growth was monitored starting on day 5, and tumor volume was measured every 3 days. Figure 1 Compared with the HPV16 E6E7 adenovirus group (without HR), the recombinant HPV16 E6E7 adenovirus group (with HR) could significantly slow down tumor growth.

[0101] Example 3 Evaluation of cellular immune responses induced by recombinant HPV16 E6E7 adenovirus vaccine

[0102] To evaluate the cellular immune effect induced by the recombinant HPV16 E6E7 adenovirus vaccine (containing HR), 6-8 week-old female C57BL / 6 mice were divided into three groups on day 0 and given PBS, empty adenovirus vector, or recombinant HPV16 E6E7 adenovirus vaccine (containing HR) (5×10 5 VP / mouse, intramuscular injection). The mice were sacrificed on the 7th day and the cellular immune response of the mice was detected. Compared with the PBS control group and the adenovirus empty vector group, the recombinant HPV16 E6E7 adenovirus group (containing HR) significantly increased the E6 tetramer-positive and E7 tetramer-positive CD8 + Cell ratio, such as Figure 2 As shown in A, B, C, and D. Figure 2 As shown in E and F, the recombinant HPV16 E6E7 adenovirus vaccine can also significantly increase the activation of CD4 + 、CD8 + The proportion of T cells (CD69 + CD4+ 、CD69 + CD8 + In addition, central memory T cells (CD44 + CD62L + CD8 + ) and effector memory T cells (CD44 + CD62L - CD8 + ) ratio has also increased significantly ( Figure 3 A, B). The spleen cells of each group of mice were extracted at 1.5×10 6 Cells were plated in a 12-well plate overnight and stimulated with E6 peptide library (4 μg / ml) and E7 peptide library (2 μg / ml). Brefeldin A was added to each well at a concentration of 1 μg / ml 6 hours before cell collection. After cell collection, extracellular and intracellular staining was performed. Flow cytometry analysis showed that after stimulation with E6 peptide library, mice in the recombinant HPV16 E6E7 adenovirus group secreted IFNγCD8 + The cell ratio increased significantly ( Figure 3 C); After stimulation with the E7 peptide library, mice in the recombinant HPV16 E6E7 adenovirus group secreted IFNγ, TNFα, and CD8 + The cell ratio increased significantly ( Figure 3 D, E). The spleen cells of each group of mice were extracted at 1.5×10 5 Density plated in ELISPOT plates, stimulated with E7 peptide library (2 μg / ml), incubated for 12-48 hours, and then Figure 4 It can be seen that the mice in the recombinant HPV16 E6E7 adenovirus group secreted IFNγCD8 + The cell ratio and IFNγ secretion ability were significantly improved. Figure 4 The statistical analysis of the results confirmed this ( Figure 3 F).

[0103] Example 4 Antitumor Effect of Recombinant HPV16 E6E7 Adenovirus Vaccine in the mEERL Carcinoma in Situ Model

[0104] To evaluate the efficacy of a recombinant HPV16 E6E7 adenovirus vaccine (containing HR) for oropharyngeal HPV16 infection after intranasal immunization. + Tumor (HPV16 E6E7 adenovirus vaccine immunization can produce HPV16 E6E7 specific cytotoxic T lymphocytes, targeting HPV16 + Tumor, here " + ” refers to positive) treatment effect, 50 μL containing 4×10 4mEERL cells were suspended in PBS and placed at the base of the tongue of 6-8 week old C57BL / 6 mice. On the 4th day, the mice were divided into three groups and given PBS, adenovirus empty vector, or recombinant HPV16 E6E7 adenovirus vaccine (containing HR) (1.0×10 10 VP / mouse, intranasal immunization), and closely monitor the growth of mice. Figure 5 It can be seen that the recombinant HPV16 E6E7 adenovirus nasal spray vaccine has a significant anti-tumor effect and improves the survival rate of mice.

[0105] Example 5 Antitumor Effect of Recombinant HPV16 E6E7 Adenovirus Vaccine in TC-1 Subcutaneous Tumor Therapy Model

[0106] To evaluate the therapeutic effect of recombinant HPV16 E6E7 adenovirus vaccine (containing HR) for cervical cancer after intramuscular injection, 6-8 week-old female C57BL / 6 mice were subcutaneously inoculated with 2×10 5 TC-1 cells. On the third day after tumor cell inoculation, the mice were randomly divided into three groups. The mice in each group were treated with PBS, empty adenovirus vector, or recombinant HPV16 E6E7 adenovirus vaccine (5×10 5 VP / mouse, intramuscular injection), and the tumor growth of mice was monitored. Recombinant HPV16 E6E7 adenovirus vaccine (containing HR) can significantly delay tumor growth ( Figure 6 A), and at the same time, it can improve the survival rate of tumor-bearing mice ( Figure 5 C). Mice were killed on day 21, and tumor tissues were removed and weighed. The recombinant HPV16 E6E7 adenovirus vaccine significantly reduced tumor weight ( Figure 6 B) Flow cytometry showed that compared with the control group and the adenovirus empty vector group, the recombinant HPV16 E6E7 adenovirus vaccine significantly increased the number of E6 tetramer-positive and E7 tetramer-positive CD8 + Cell ratio ( Figure 7 A, B, C, D). After preparing tumor cells into single-cell suspensions for tumor microenvironment analysis, the recombinant HPV16 E6E7 adenovirus vaccine significantly reduced the proportion of myeloid-derived immunosuppressive cells and M2 macrophages ( Figure 7 E, F).

[0107] Example 6 Anti-tumor Effect of Recombinant HPV16 E6E7 Adenovirus Vaccine in TC-1 Subcutaneous Tumor Prevention Model

[0108] To evaluate the preventive effect of recombinant HPV16 E6E7 adenovirus vaccine (containing HR) for cervical cancer after intramuscular injection, 6-8 week-old female C57BL / 6 mice were divided into three groups. On day 0, mice in each group were treated with PBS, empty adenovirus vector, or recombinant HPV16 E6E7 adenovirus vaccine (containing HR) (5×10 5 VP / mouse, intramuscular injection), 2×10 5 TC-1 cells. Monitoring tumor growth in mice. Figure 8 A shows that the recombinant HPV16 E6E7 adenovirus vaccine has a good preventive effect on tumors and significantly improved the survival rate of mice ( Figure 8 C). On day 21, the mouse tumor tissue was removed and weighed. The recombinant HPV16 E6E7 adenovirus vaccine significantly reduced the tumor weight ( Figure 8 B). When mice were re-challenged with TC-1 on day 100, the recombinant HPV16 E6E7 adenovirus vaccine still had a strong and effective immune effect and delayed tumor growth ( Figure 8 D) and continued to improve the survival rate of mice ( Figure 8 E).

[0109] Example 7 Antitumor Effect of Recombinant HPV16 E6E7 Adenovirus Vaccine Combined with Cisplatin / Paclitaxel in the TC-1 Subcutaneous Tumor Therapy Model

[0110] To evaluate the therapeutic effect of recombinant HPV16 E6E7 adenovirus vaccine (containing HR) combined with cisplatin / paclitaxel for cervical cancer, 6-8 week-old female C57BL / 6 mice were subcutaneously inoculated with 2×10 5 TC-1 cells. Four days after tumor cell inoculation, the patients were randomly divided into 6 groups: control group, adenovirus empty vector group, recombinant HPV16 E6E7 adenovirus group (containing HR), cisplatin / paclitaxel group, adenovirus empty vector combined with cisplatin / paclitaxel group, and recombinant HPV16 E6E7 adenovirus group (containing HR) combined with cisplatin / paclitaxel. The patients were treated with PBS, adenovirus empty vector, and recombinant HPV16 E6E7 adenovirus vaccine (5×10 5 VP / mouse, intramuscular injection), and cisplatin / paclitaxel (cisplatin: 2 mg / kg, paclitaxel: 20 mg / kg, intraperitoneal injection) were administered on days 4 and 11, respectively, and the tumor growth of the mice was monitored. Figure 9 It can be seen that the recombinant HPV16 E6E7 adenovirus combined with cisplatin / paclitaxel can delay tumor growth in mice and have a certain protective effect on mice.

Claims

1. A recombinant HPV16 E6E7 adenovirus, characterized in that: The expressed antigen contains the HPV16 E6E7 antigen sequence and its SARS-CoV-2 virus HR1 and HR2 segment sequences.

2. The adenovirus according to claim 1, characterized in that: The amino acid sequence of the HPV16 E6E7 antigen is as shown in SEQ ID No.1, or has more than 80% homology with SEQ ID No.1 and has the same or similar biological activity; preferably, the nucleotide sequence of the HPV16 E6E7 antigen is as shown in SEQ ID No.

2.

3. The adenovirus according to claim 1, characterized in that: The amino acid sequence of the HR1 and HR2 segments of the SARS-CoV-2 virus is as shown in SEQ ID No.3, or has more than 80% homology with SEQ ID No.3 and has the same or similar biological activity; preferably, the nucleotide sequence of the HR1 and HR2 segments of the SARS-CoV-2 virus is as shown in SEQ ID No.

4.

4. The adenovirus according to claim 1, characterized in that: The amino acid sequence of the antigen expressed by the recombinant HPV16 E6E7 adenovirus is shown as SEQ ID No.5; preferably, the nucleotide sequence of the antigen expressed by the recombinant HPV16 E6E7 adenovirus is shown as SEQ ID No.

6.

5. The adenovirus according to claim 1, characterized in that: The HPV16 E6E7 antigen forms a trimer structure with the HR1 and HR2 segments of the SARS-CoV-2 virus.

6. An adenovirus vector, characterized in that: A nucleic acid sequence for expressing an antigen comprising the recombinant HPV16 E6E7 adenovirus according to any one of claims 1 to 5.

7. The adenoviral vector according to claim 6, characterized in that: The adenovirus vector is selected from human Ad5 vector, Ad35 vector or Ad26 vector and / or chimpanzee AdC68 vector, AdC7 vector or ChAdOx1 vector; preferably, it is selected from human type 5 replication-deficient adenovirus with combined deletion of E1 and E3.

8. The method for preparing the recombinant HPV16 E6E7 adenovirus according to any one of claims 1 to 5, characterized in that: The following steps are involved: The HPV16 E6E7-HR gene is synthesized, and an adenovirus shuttle plasmid containing the HPV16 E6E7-HR gene is constructed using molecular cloning technology. The shuttle plasmid and the backbone plasmid of the AdMax adenovirus system are co-transfected into host cells to package the recombinant adenovirus to obtain a replication-defective recombinant adenovirus, which is then expanded and cultured and purified.

9. A recombinant HPV16 E6E7 adenovirus vaccine, characterized in that: Contains the recombinant HPV16 E6E7 adenovirus according to any one of claims 1 to 5 or the adenovirus vector according to any one of claims 6 to 7.

10. The recombinant HPV16 E6E7 adenovirus vaccine according to claim 9, characterized in that: The recombinant HPV16E6E7 adenovirus vaccine is in the form of an injection, nasal drops, spray or inhaler; preferably, the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection or intraperitoneal injection.

11. A pharmaceutical composition, characterized in that: Contains the recombinant HPV16 E6E7 adenovirus according to any one of claims 1 to 5, the adenovirus vector according to any one of claims 6 to 7, or the vaccine according to any one of claims 9 to 10, and other anti-tumor drugs.

12. A combined drug, characterized in that: The recombinant HPV16 E6E7 adenovirus according to any one of claims 1 to 5, the adenovirus vector according to any one of claims 6 to 7, or the vaccine according to any one of claims 9 to 10, and other anti-tumor drugs are administered separately or simultaneously.

13. The pharmaceutical composition according to claim 11 or the combined drug according to claim 12, characterized in that: The other anti-tumor drugs are selected from at least one of cisplatin, paclitaxel, carboplatin, topotecan, bevacizumab, cetuximab, pembrolizumab, nivolumab, medroxyprogesterone or 5-fluorouracil; preferably, the other anti-tumor drugs are cisplatin and paclitaxel.

14. Use of the recombinant HPV16 E6E7 adenovirus according to any one of claims 1 to 5, the adenovirus vector according to any one of claims 6 to 7, the vaccine according to any one of claims 9 to 10, the pharmaceutical composition according to claim 11 or 13, or the combined drug according to claim 12 or 13 in preventing and / or treating tumors caused by HPV infection.

15. The use according to claim 14, characterized in that: The tumor comprises at least one of cervical cancer, head and neck squamous cell carcinoma, anal cancer, vulvar cancer, vaginal cancer or penile cancer.

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