Matv11 recombinant adenovirus vector, uses, methods of manufacture, and hpv therapeutic vaccines

Abstract

The present application relates to the technical field of HPV therapeutic vaccine, in particular to MATV11 recombinant adenovirus vector, application, preparation method and HPV therapeutic vaccine, the MATV11 recombinin adenovirus vector includes the nucleotide sequence as described in SEQ ID No.1 and the nucleotide sequence as shown in SEQ ID No.2.The HPV therapeutic vaccine has good immune effect to HPV16 / 18 / 52, and can also produce effective specific immune response to HPV56E7, HPV51E7, HPV68E7 and HPV51E6, and can produce effective cross immune response, the immune effect is good, so that the effect of the vaccine is effectively improved, and the application range is wide.

Description

Technical Field

[0001] This invention relates to the field of HPV therapeutic vaccine technology, and more specifically, to the MATV11 recombinant adenovirus vector, its application, preparation method, and HPV therapeutic vaccine. Background Technology

[0002] Cervical cancer is a leading cause of death among women. In 2020, an estimated 604,000 women worldwide were diagnosed with cervical cancer, and approximately 342,000 died from it. Cervical cancer is now the most common cancer in 23 countries and a leading cause of cancer death in 36 countries. The incidence of cervical cancer in my country is alarming. In 2018, 106,430 new cases and 47,739 deaths were reported, accounting for 18.2% and 17.3% of the global cervical cancer incidence and mortality rates, respectively. According to the National Cancer Center's "2020 Annual Work Report of National Cancer Centers," cervical cancer remains the sixth most common cancer among women in my country, and its mortality rate remains the eighth highest among female malignant tumors.

[0003] HPV is now considered the most prevalent sexually transmitted disease worldwide, with 50%-80% of sexually active individuals contracting HPV in their lifetime. Nearly 95% of cervical cancer cases are related to HPV infection, and the majority of HPV-related morbidity and mortality are caused by cervical cancer. Widespread HPV vaccination has significantly reduced cancer incidence worldwide; currently, at least 118 million women have received one dose of the HPV vaccine. While this number is encouraging, it still represents only 3.5% of the world's population. Approximately 530,000 new cases of cervical cancer are caused by HPV each year, resulting in 265,700 deaths. Furthermore, other genital mucosal tissues, including the anus and rectum, are also susceptible to HPV infection. The incidence of anal squamous cell carcinoma has been rising over the past thirty years. Currently, there are approximately 35,000 cases of anal cancer annually, with similar incidence rates in men and women. Therefore, both in China and internationally, the number of people infected with HPV, as well as those with precancerous cervical lesions, vulvar and anal lesions, and subsequent related cancers caused by HPV, is alarming.

[0004] Mucous membranes and skin are the most common sites of HPV infection. Especially sexually active women of childbearing age can contract HPV through sexual contact with an infected partner. HPV infects basal epithelial cells through damaged epithelial cells, causing cervical dysplasia and CIN. Persistent infection with high-risk HPV types leads to cervical cancer. Most natural HPV infections are confined to the inner lining of the mucous membrane and do not develop into cancer. Approximately 90% of HPV-infected individuals clear the virus within a few months of infection through innate immune responses and specific humoral immunity. 10% of patients experience persistent infection, and about 1% have an increased risk of developing cancer. Persistent HPV infection is a significant cause of cervical cancer. Therefore, timely clearance of HPV infection and reversal of cervical intraepithelial neoplasia are crucial to preventing the development of cervical cancer. However, there is currently no specific treatment for HPV. All existing HPV vaccines are preventative and have no therapeutic effect on existing infections or lesions. Even after HPV vaccination, regular cervical cancer screening is necessary as advised by relevant authorities. Therefore, the development of therapeutic vaccines has broad application prospects in the treatment of HPV.

[0005] Studies have confirmed that the genotype distribution and infection rate of high-risk HPV vary significantly across different countries, regions, geographical environments, ethnicities, and populations. High-risk HPV types 16 and 18 are the two genotypes with the strongest known carcinogenicity. HPV 16 / 18 / 58 / 33 and 52 are the five most prevalent subtypes of cervical cancer in my country. HPV 52 has a particularly high infection rate in North America, Asia, and Africa. Domestic studies report an overall HPV 52 infection rate as high as 2.4% in women, and even higher at 3.1% in Central China, making it the genotype with the highest infection rate among all high-risk HPV types. Research indicates that the rate of mixed infection with high-risk HPV types increases with the severity of cervical lesions. Therefore, treatment and testing for patients with multiple mixed infections should be strengthened. Currently, most therapeutic HPV vaccines in clinical trials target only HPV types 16-18 (E6-E7 or E6-E7) or both HPV 16 and 18. They do not target other genotypes, especially HPV 52 / 58 / 33 / 31. Therefore, developing therapeutic HPV vaccines targeting multiple types is equally important for improving the cure rate of high-grade cervical intraepithelial neoplasia.

[0006] However, existing vaccines targeting HPV16 / 18 only target a single HPV genotype, which limits their effectiveness. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a recombinant adenovirus vector, its application, preparation method and HPV therapeutic vaccine, so as to solve the problems of single genotype coverage, poor immune effect and memory effect of existing vaccines.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0009] The present invention provides a MATV11 recombinant adenovirus vector, the MATV11 recombinant adenovirus vector comprising the nucleotide sequence as described in SEQ ID No. 1 and the nucleotide sequence as shown in SEQ ID No. 2.

[0010] Based on the above technical solution, the present invention can be further improved as follows.

[0011] Furthermore, the adenovirus is a replication-defective human adenovirus type 5 with a combined deletion of the E1 and E3 regions.

[0012] Furthermore, the nucleotide sequence described in SEQ ID No. 1 is located in the E1 region, and the nucleotide sequence described in SEQ ID No. 2 is located in the E2 region.

[0013] The present invention also provides a method for preparing the MATV11 recombinant adenovirus vector as described above. After constructing the recombinant plasmid, the recombinant plasmid is then cloned, screened, identified and transfected to obtain the recombinant adenovirus vector. The recombinant plasmid contains the nucleotide sequence as described in SEQ ID No. 1 and the nucleotide sequence as shown in SEQ ID No. 2.

[0014] Furthermore, the construction includes the step of amplifying the nucleotide sequence as shown in SEQ ID No. 1 and the nucleotide sequence as shown in SEQ ID No. 2 using primer pairs, wherein the forward primer is shown in SEQ ID No. 3 and the reverse primer is shown in SEQ ID No. 4.

[0015] Furthermore, the transfected cells are HEK293A cells.

[0016] The present invention also provides a fusion protein composition comprising a first fusion protein and a second fusion protein, wherein the first fusion protein and the second fusion protein are prepared using the MATV11 recombinant adenovirus vector as described above.

[0017] Furthermore, the amino acid sequence of the first fusion protein is shown in SEQ ID No. 5, and the amino acid sequence of the second fusion protein is shown in SEQ ID No. 6.

[0018] The present invention also provides the use of the MATV11 recombinant adenovirus vector as described above in the preparation of HPV16 / 18 / 52 therapeutic vaccines.

[0019] The present invention also provides an HPV therapeutic vaccine comprising the MATV11 recombinant adenovirus vector as described above or the fusion protein composition as described above.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) The MATV11 recombinant adenovirus vector of the present invention includes two nucleotide sequences encoding HPV16 / 18 / 52E6 and HPV16 / 18 / 52E7 respectively, and each sequence contains a nucleotide sequence encoding CD40L, so that the vaccine prepared by the recombinant adenovirus vector can cover HPV16 / 18 / 52, and also has a good cross-immune response effect against other HPV genotypes;

[0022] (2) The MATV11 recombinant adenovirus vector of the present invention is a replication defective human adenovirus type 5 with joint deletion of E1 and E3 regions. The early genes of E1 and E3 regions of the Ad5 vector are deleted, resulting in replication defects, but it can transfect host cells without killing host cells, expand gene loading space, and has high safety, making it suitable for community populations.

[0023] (3) The HPV therapeutic vaccine of the present invention has a good immune effect on HPV16 / 18 / 52, and can also produce an effective specific immune response to HPV56 E7, HPV51 E7, HPV68 E7 and HPV51 E6.

[0024] (4) The HPV therapeutic vaccine of the present invention can target multiple HPV virus genotypes and has a good immune effect, which effectively improves the vaccine's efficacy and has a wide range of applications. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the nucleotide sequence structure of the MATV11 recombinant adenovirus vector of the present invention;

[0026] Figure 2 A schematic diagram of the MATV11 non-replicating adenovirus vector structure in Example 1, which is the MATV11 recombinant adenovirus vector of the present invention.

[0027] Figure 3 As the MATV11 recombinant adenovirus vector of the present invention, in Example 2, the changes of the target antigen, p53, and pRB after transfection of 293 with MATV11 were detected by in vitro Western blotting. Figure 3Figure A shows the results of Western blotting (WB) analysis of the expression of the E6 fusion protein of HPV16 / 18 / 52 after transfection with MATV0 and MATV11 into 293 cells. Figure 3 Figure B in the middle shows the expression results of the E7 fusion protein of HPV16 / 18 / 52 after transfection of 293 with MATV11. Figure 3 The middle image (C) shows the results of Western blotting (WB) detection of the expression of p53 and pRB after transfection with MATV0, MATV3, and MATV11 at 293 cells.

[0028] Figure 4 The MATV11 recombinant adenovirus vector of the present invention, in Example 3, is a comparison of the results of specific IFN-γ production of mouse lymphocytes against the antigen in the single-needle intramuscular injection experimental group and the control group.

[0029] Figure 5 The MATV11 recombinant adenovirus vector of the present invention, in Example 3, is a comparison of the production of antigen-specific IFN-γ by spleen lymphocytes of C57BL / 6J mice in the experimental group and the control group 1 week and 2 weeks after single injection.

[0030] Figure 6 The MATV11 recombinant adenovirus vector of the present invention, in Example 4, growth curves of mouse TC-1 subcutaneous tumors in the experimental group and the control group;

[0031] Figure 7 The MATV11 recombinant adenovirus vector of the present invention, in Example 4, shows the results of the tumor-suppressive effect of HPV18 / 52 in C57BL / 6J mice. Figure 7 In section A, the growth curve of U14-HPV18 E6 / E7 subcutaneous tumors is shown. Figure 7 The growth curve of subcutaneous tumor B is for U14-HPV52 E6 / E7.

[0032] Figure 8 The MATV11 recombinant adenovirus vector of the present invention, in Example 4, shows the results of the immune response induced in C57BL / 6J mice after a booster injection four weeks later. Figure 8 In the middle A, there is a comparison of the IFN-γ content specific to E6 and E7 cells of HPV16 / 18 / 52 in mouse spleen lymphocytes in the experimental group and the control group. Figure 8 Figure B shows a comparison of the IFN-γ content specific to mouse spleen lymphocytes targeting HPV56 E7, HPV51 E7, HPV68 E7, and HPV51 E6 in the experimental and control groups.

[0033] Figure 9As the MATV11 recombinant adenovirus vector of the present invention, Example 5 shows a comparison of the activation status of specific CTLs, Th1 and Th2 cells in vivo by flow cytometry detection with different numbers of viral particles. Figure 9 A represents the control group. Figure 9 Group B is the low-dose group. Figure 9 Group C represents the medium-dose group. Figure 9 Group D is the high-dose group. Detailed Implementation

[0034] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0035] The MATV11 recombinant adenovirus vector of the present invention includes nucleotide sequences for encoding E6 and E7 antigens of HPV 16, HPV18 and HPV52, and also includes nucleotide sequences for encoding CD40L.

[0036] The MATV11 recombinant adenovirus vector of the present invention has a nucleotide sequence designed for HPV16 / 18 / 52E6-E7 protein antigens and also contains a nucleotide sequence encoding CD40L, so that the fusion protein expressed therein uses CD40L as an adjuvant, thereby improving the immune effect and memory effect of the vaccine.

[0037] The MATV11 recombinant adenovirus vector of this invention exerts its immune effect in vivo after intramuscular injection through the following mechanism: the antigen encoded by the adenovirus vaccine is presented to T cells via antigen-presenting cells. Activated CD4+ cells differentiate into specific Th1 cells, activated CD8+ cells differentiate into specific CTL cells, and under the action of secreted cytokines such as IFN-γ and IL-4, they specifically attack free HPV16 / 18 / 52 viruses and cervical epithelial cells integrating HPV16 / 18 / 52 E6 and E7 genes, ultimately clearing the virus and reversing the progression of CIN. The primary target population is individuals with persistent HPV infection and cervical, anal, and vulvar neoplasia.

[0038] The TNFSFL molecule CD40L, a natural ligand of CD40, participates in the activation of dendritic cells (DCs). CD40L, also known as gp39, TNF-associated activation protein (TRAP), and T-B cell-activating molecule (T-BAM), is a 33 kDa type II transmembrane glycoprotein with 266 amino acids, of which 214 are extracellular amino acids. It exhibits high homology with TNF at the amino acid level. CD40L can induce dendritic cells to produce interleukin-12p70 (IL-12p70) and other cytokines, and promote the differentiation of effector T cells.

[0039] Adenoviruses are non-enveloped, double-stranded DNA viruses, measuring 80-100 nm, and are among the most promising vaccine vector platforms. Adenovirus vector vaccines exhibit good safety and efficacy. Type 5 recombinant adenovirus vectors offer advantages such as a wide host cell range, large insertable fragments, high viral titers, and mature industrial production systems. Adenovirus vectors include replicating and non-replicating types. The main advantages of non-replicating adenovirus vectors are high-titer growth capability, ease of handling, non-integration into the human genome, strong immunogenicity, and ease of large-scale clinical production.

[0040] Specifically, the schematic diagram of the nucleotide sequence structure of the MATV11 non-replicating adenovirus vector of the present invention is shown below. Figure 1 As shown, the adenovirus of the present invention is a replication-defective human adenovirus type 5 (Ad5) with a combined deletion of the E1 and E3 regions. The nucleotide sequence described in SEQ ID No. 1 is located in the E1 region, and the nucleotide sequence described in SEQ ID No. 2 is located in the E2 region.

[0041] The early gene in the E1 region of the Ad5 vector is deleted, resulting in replication defects. However, it can transfect host cells without killing them, expand the gene loading space, and has high safety, making it suitable for community populations.

[0042] The method for preparing the MATV11 recombinant adenovirus vector of the present invention involves constructing a recombinant plasmid, followed by cloning, screening, identification, and transfection to obtain a recombinant adenovirus vector. The recombinant plasmid contains the nucleotide sequence as described in SEQ ID No. 1 and the nucleotide sequence as shown in SEQ ID No. 2.

[0043] Preferably, in the method of the present invention, the construction process includes the step of amplifying the nucleotide sequence as shown in SEQ ID No. 1 and the nucleotide sequence as shown in SEQ ID No. 2 using primer pairs, wherein the forward primer is shown in SEQ ID No. 3 and the reverse primer is shown in SEQ ID No. 4.

[0044] Preferably, the transfected cells are HEK293A cells.

[0045] The fusion protein composition of the present invention includes a first fusion protein and a second fusion protein, which are prepared using the MATV11 recombinant adenovirus vector as described above.

[0046] Preferably, the amino acid sequence of the first fusion protein is shown in SEQ ID No. 5, and the amino acid sequence of the second fusion protein is shown in SEQ ID No. 6. The recombinant vector of the present invention can be used in the preparation of HPV16 / 18 / 52 therapeutic vaccines.

[0047] The HPV16 / 18 / 52 therapeutic vaccine of the present invention includes the MATV11 recombinant vector as described above or the fusion protein composition as described above.

[0048] The present invention will be described below through specific embodiments.

[0049] Example 1: Construction of MATV11 non-replicating adenovirus vector

[0050] This embodiment utilizes gene synthesis and in vitro ligation technologies to construct a non-replicating adenovirus type 5 vector for MATV11. The specific steps are as follows:

[0051] (1) Resuscitate the pad-CMV-BstBI strain and extract plasmids.

[0052] (2) The nucleotide sequence shown in SEQ ID No.1 was synthesized in vitro and loaded into the PUC57 sequence (HPV16 / 18 / 52E7-CD40L_pUC57).

[0053] (3) Constructing the pad-HPV16 / 18 / 52E7-CD40L backbone plasmid:

[0054] a. Digest the pad-CMV-BstBI plasmid with BstBI enzyme;

[0055] b. Using HPV16 / 18 / 52E7-CD40L_pUC57 as a template, PCR was performed to obtain the in vitro ligation fragment. The primers are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0056] SEQ ID No. 3 (MATV11-F):

[0057]

[0058] SEQ ID No.4 (MATV11-R):

[0059]

[0060] The bolded section represents the PCR primer HPV16 / 18 / 52E7-CD40L segment, and the marked section represents the homologous recombination overlap segment.

[0061] c. The products obtained by in vitro linking of a and b are shown in Table 1:

[0062] Table 1

[0063] Material Name volume Pad-CMV-BSTBI plasmid 2μl HPV16 / 18 / 52E7-CD40L PCR purification 5μl NEBuilder HiFi DNA Assembly Master Mix 10μl <![CDATA[Deionized H2O]]> 3μl Total Volume 20μl

[0064] d. Transfer 5 μL of the above product into T1 for clone screening and identification;

[0065] e. Obtain the pad-HPV16 / 18 / 52E7-CD40L plasmid.

[0066] (4) Construct the final backbone plasmid MATV11:

[0067] a. Gene synthesis of the delE3-CMV-HPV16 / 18 / 52E6-CD40L fragment containing the nucleotide sequence shown in SEQ ID No.2, loading it into the PUC57 vector, PCR to obtain the target fragment, and then purification;

[0068] b. Digest the pad-HPV16 / 18 / 52E7-CD40L plasmid with SpeI and purify the product;

[0069] c. Co-transform products a and b into the BJ5183 homologous recombinant plasmid, and select clones for identification;

[0070] d. Transfect the correctly identified MATV11 plasmid into the high-copy infectious state T1 to obtain the final strain for plasmid extraction;

[0071] (5) After PacI digestion of the MATV11 plasmid, the plasmid was purified and transfected into HEK293A cells to finally obtain a non-replicating adenovirus vector of MATV11, the structure of which is shown below. Figure 2 As shown, its nucleotide sequence is shown in SEQ ID No. 7.

[0072] Example 2: Expression of MATV11 antigen

[0073] 293 cells were seeded onto plates and transfected with the virus in complete culture medium 16-24 hours later (moi=1). Cell residue was collected 48 hours later, and the cleavage proteins were detected using Western blotting to identify the MATV11 fusion protein. The results are as follows: Figure 3 As shown in A and B, the red boxes represent the positions of the first and second fusion proteins, respectively. The fusion protein expressing the exogenous gene inserted in the E1 region, detected using HPV16 E7, is approximately 100 kDa. The fusion protein expressing the exogenous gene inserted in the E3 region, detected using HPV18 E6 antibody, is also around 100 kDa, consistent with the size of fusion proteins.

[0074] To further demonstrate the safety of the MATV11 vaccine after antigen point mutation, this embodiment also tested the expression of the pRB and p53 genes after transfection with the virus. Figure 3 The graph in center (C) shows the Western blotting results of p53 and pRB expression after transfection of 293 cells with MATV0, MATV3, and MATV11. MATV3 is a bivalent therapeutic HPV16 / 18 vaccine. The controls are plasmids of wild-type HPV16 E6, HPV16 E7, HPV18 E6, and HPV18 E7, HPV16 E7 invitrogen 28-0006 mouse antibody 1:100, and HPV18 E6 genetex 1:100.

[0075] according to Figure 3 As can be seen from the data in Figure C, compared with the cells transfected with wild-type HPV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7 plasmids, the pRB and p53 of the cells transfected with MATV11 were basically unchanged, indicating that the antigen point mutation carried by MATV11 is not tumorigenic.

[0076] Example 3: Identification of the immune effects of MATV11 in C57BL / 6J mice

[0077] (1) Immunogenicity of MATV11 by single intramuscular injection in C57BL / 6J mice.

[0078] Healthy 8-week-old female C57BL / 6J mice were intramuscularly injected with MATV11 vaccine at a dose of 10^8 pfu / mouse. The control group received empty vector MATV0 virus. One week after injection, the mice were sacrificed, and spleen lymphocytes were isolated. Lymphocytes were stimulated with a peptide pool, and the production of antigen-specific IFN-γ by lymphocytes was detected using an Elispot assay. The results are as follows: Figure 4 As shown.

[0079] pass Figure 4It can be seen that the MATV11 experimental group can produce a specific immune response against the antigen in mice, most notably HPV16 E7 and HPV18 E6, which is similar to the response of HPV therapeutic vaccines in C57BL / 6J mice reported in the literature.

[0080] (2) Changes in immune response in C57BL / 6J mice after 1 and 2 weeks of single-dose MATV11 injection.

[0081] To further investigate the changes in immunogenicity in mice after intramuscular injection of MATV11, mice were sacrificed at 1 and 2 weeks after vaccination, and splenic lymphocytes were isolated. Lymphocytes were stimulated with a peptide pool, and the Elispot assay was used to detect the production of antigen-specific IFN-γ by the lymphocytes. Figure 5 As shown, the specific immune response against HPV18 E6 decreased slightly in the second week after vaccination, while the specific responses to other antigens did not change much, indicating that the immune response induced by the vaccine was still at its peak.

[0082] Example 4: Antitumor effect of MATV11 in C57BL / 6J mice

[0083] (1) Comparison of tumor inhibition rates between MATV11 group and MATV0 group:

[0084] TC-1 cells are transgenic mouse lung epithelial cells containing HPV16 E6, HPV16 E7, and the ras gene. A subcutaneous tumor model was established in the buttocks. Patients were randomly divided into MATV11, MATV0, and PBS groups. On day 5 after subcutaneous tumor inoculation, each mouse received MATV11 vaccine at a dose of 3*10^8 pfu / mouse. Tumor size was measured and recorded every other day. Figure 6 As shown, the results indicated that MATV11 at 3*10^8 pfu / mouse effectively inhibited the growth of TC-1 tumors. Two weeks after vaccination, the tumors in the experimental group shrank significantly, reaching a volume exceeding 2000 mm in the control group. 3 MATV11 has a tumor inhibition rate of over 80%.

[0085] (2) The antitumor effect of MATV11 against HPV18 / 52 in C57BL / 6J mice.

[0086] MATV11 is a trivalent vaccine targeting HPV16 / 18 / 52 E6 and E7 proteins. Currently, most preclinical studies of therapeutic HPV vaccines focus on HPV16, using the TC-1 cell line. To further demonstrate the immune effects of MATV11 against HPV18 and HPV52, this embodiment uses lentiviruses to construct U14 cell lines overexpressing HPV18 E6 / E7 and HPV52 E6 / E7.

[0087] Subcutaneous tumors of U14-HPV18 E6 / E7 and U14-HPV52 E6 / E7 were established. Mice were inoculated with MATV11 on days 7 and 14, and the growth of the subcutaneous tumors was measured. Figure 7 As shown, compared with the control group, the tumor volume growth in the experimental group mice was significantly slower, while the control group mice showed greater individual variation, with tumors exceeding 600 mm². 3 The tumor grows rapidly. For example... Figure 7 As shown, the experimental group mice were generally of similar size with little individual variation, and the tumor inhibition rate was over 70%.

[0088] The experimental results above show that MATV11 can generate a specific immune response against HPV16 / 18 / 52, effectively inhibiting the growth of the corresponding tumors.

[0089] (3) Immune response induced in C57BL / 6J mice after MATV11 booster injection.

[0090] Since most current HPV therapeutic vaccines are booster shots, based on literature and previous comparisons of the changes in specific immune responses induced by MATV11 at 1 and 2 weeks after injection, it can be concluded that the immune response is still at its peak at week 2. A booster shot at the peak may not be as effective. Therefore, in this embodiment, the immune response in C57BL / 6J mice was detected 1 week after the MATV11 booster shot at 4 weeks interval.

[0091] This experiment involved three groups: MATV0, MATV11 I (5 weeks post-MATV11 injection, followed by sacrifice 5 weeks later), and MATV11 II (1 week post-MATV11 booster injection, followed by sacrifice 1 week later). Lymphocytes were stimulated with the peptide pool and then subjected to an Elispot assay. The results are as follows: Figure 8 As shown.

[0092] according to Figure 8 It can be seen that the booster shot after a 4-week interval significantly enhanced the immune response to the vaccine, producing an effective specific immune response. Cross-reactivity results showed that HPV56 E7, HPV51 E7, HPV68 E7, and HPV51 E6 could produce effective cross-immune responses.

[0093] Example 5: Immunogenicity and dose-relatedness of MATV11 vaccine

[0094] The theoretical dose of the vaccine used in the above examples was 1*10^11 vp. To further investigate the differences in immune responses induced by different vaccine doses, this example selected high-dose (1*10^11 vp), medium-dose (1*10^10 vp), and low-dose (1*10^9 vp) vaccines, respectively, and then used flow cytometry to detect the activation of specific CTLs, Th1, and Th2 cells in vivo. In the experiment, mice were vaccinated with the control group, high-dose, medium-dose, and low-dose vaccines, respectively, and were sacrificed one week after a booster injection one month later.

[0095] Test results as follows Figure 9 As shown, the high-dose group effectively activated specific CTLs and Th1 cells. The low-dose group showed virtually no activation of specific Th1 cells, and CTL activation was limited to HPV52 E6 and HPV52 E7 cells; other cells were not effectively activated. The results for the medium-dose group were similar to those for the low-dose group, but the activation of specific CTLs increased slightly.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A MATV11 recombinant adenovirus vector, characterized in that, The MATV11 recombinant adenovirus vector comprises the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence shown in SEQ ID No.

2.

2. The MATV11 recombinant adenovirus vector according to claim 1, characterized in that, The adenovirus in question is a replication-defective human adenovirus type 5 with a combined deletion of the E1 and E3 regions.

3. The MATV11 recombinant adenovirus vector according to claim 2, characterized in that, The nucleotide sequence shown in SEQ ID No. 1 is located in the E1 region, and the nucleotide sequence shown in SEQ ID No. 2 is located in the E3 region.

4. A method for preparing the MATV11 recombinant adenovirus vector as described in claim 3, characterized in that, A recombinant plasmid was constructed, then cloned, screened, identified, and transfected to obtain the MATV11 recombinant adenovirus vector. The recombinant plasmid contains the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence shown in SEQ ID No.

2.

5. The method for preparing the MATV11 recombinant adenovirus vector according to claim 4, characterized in that, The transfected cells were HEK293A cells.

6. The use of the MATV11 recombinant adenovirus vector as described in any one of claims 1 to 3 in the preparation of a therapeutic HPV vaccine.

7. An HPV therapeutic vaccine, characterized in that, Includes the MATV11 recombinant adenovirus vector as described in claim 1 or 2.

Images