MATV11 recombinant adenovirus vector, application, preparation method and HPV therapeutic vaccine

Abstract

The invention relates to the technical field of HPV therapeutic vaccines, in particular to an MATV11 recombinant adenovirus vector, application, a preparation method and an HPV therapeutic vaccine. The MATV11 recombinant adenovirus vector comprises a nucleotide sequence as shown in SEQ ID No. 1 and a nucleotide sequence as shown in SEQ ID No. 2. The invention further discloses a preparation method of the MATV11 recombinant adenovirus vector. The HPV therapeutic vaccine has a good immune effect on HPV16 / 18 / 52, can generate effective specific immune response, can generate effective cross immune response on HPV56E7, HPV51E7, HPV68E7 and HPV51E6, and is good in immune effect, so that the effect of the vaccine is effectively improved, and the application range is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of HPV therapeutic vaccines, and in particular to a MATV11 recombinant adenovirus vector, application, preparation method and HPV therapeutic vaccine. Background Art

[0002] Cervical cancer is the leading cause of death among women. In 2020, an estimated 604,000 women were diagnosed with cervical cancer and about 342,000 women died from cervical cancer worldwide. Cervical cancer has become a common cancer in 23 countries and the leading cause of cancer death in 36 countries. The incidence of cervical cancer in my country is not optimistic. In 2018, 106,430 new cases of cervical cancer and 47,739 deaths were reported, accounting for 18.2% and 17.3% of the global incidence and mortality of cervical cancer. According to the National Cancer Center's "2020 National Cancer Center Annual Work Report", cervical cancer is still the sixth most common cancer among women in my country, and the mortality rate is still ranked eighth among female malignant tumors.

[0003] HPV is now considered the most prevalent sexually transmitted disease in the world, with 50%-80% of sexually active individuals infected with HPV in their lifetime. Almost 95% of cervical cancer cases are related to HPV infection, and most of the HPV-related morbidity and mortality are caused by cervical cancer. The incidence of cancer worldwide has been greatly reduced through widespread HPV vaccination, and at least 118 million women have received a dose of HPV vaccine. Although this number is encouraging, it only accounts for 3.5% of the world's population. About 530,000 new cases of cervical cancer are caused by HPV each year, resulting in 265,700 deaths. In addition, other genital mucosal tissues, including the anus and rectum, are also susceptible to HPV infection. Over the past three decades, the incidence of anal squamous cell carcinoma has been increasing. Currently, there are about 35,000 cancer cases of anal cancer each year, with similar incidence rates in men and women. Therefore, the number of HPV infections, HPV-induced cervical precancerous lesions, vulvar and anal precancerous lesions, and subsequent related cancers is not optimistic, both in China and internationally.

[0004] Mucous membranes and skin are the most common areas of HPV infection. Especially women of childbearing age who are sexually active may be infected with HPV through sexual contact with infected partners. HPV infects basal epithelial cells through damaged epithelial cells, causing cervical dysplasia and CIN. Persistent infection with high-risk HPV leads to the occurrence of cervical cancer. Most natural HPV infections are confined to the epithelial lining of the mucosa and will not develop into cancer. About 90% of HPV-infected patients clear the virus within a few months after viral infection under the mediation of innate immune response and specific humoral immunity. 10% of patients are persistently infected, and about 1% of patients have an increased risk of cancer. Persistent HPV infection is an important cause of the eventual occurrence of cervical cancer. Therefore, timely elimination of HPV virus infection and reversal of cervical intraepithelial neoplasia are crucial to ultimately prevent the development of cervical cancer. However, there is no specific drug for HPV so far. All current HPV vaccines are preventive vaccines and have no therapeutic effect on existing infections or lesions. Even if you have received the HPV vaccine, you should still follow the recommendations of relevant departments and undergo regular cervical cancer screening. 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 in different countries, regions, geographical environments, races and populations. High-risk HPV16 and HPV18 are the two most carcinogenic genotypes identified in studies. HPV16 / 18 / 58 / 33 and 52 are the five main subtypes of cervical cancer in my country. The infection rate of HPV52 is particularly high in North America, Asia and Africa. Domestic studies have reported that the overall infection rate of HPV52 in women is as high as 2.4%, and in central China it is as high as 3.1%, making it the genotype with the highest infection rate of all high-risk HPVs. Studies have shown that as cervical lesions worsen, the mixed infection rate of high-risk HPV increases. Therefore, the treatment and testing of patients with multiple mixed infections should be strengthened. Currently, the antigens of HPV therapeutic vaccines in clinical trials are basically all concentrated on single HPV16 E6-E7 or HPV18 E6-E7 and two types of HPV16 and HPV18, and no other genotypes except HPV16 and HPV18 are targeted, especially HPV52 / 58 / 33 / 31, etc. Therefore, the development of therapeutic HPV vaccines targeting multiple types is equally important to improve the cure rate of high-grade cervical intraepithelial neoplasia.

[0006] However, the existing vaccines against HPV16 / 18 target a single HPV genotype, which leads to major limitations of the vaccine. Summary of the invention

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

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

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

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

[0011] Furthermore, the adenovirus is a replication-deficient human adenovirus type 5 in which the E1 region and the E3 region are jointly deleted.

[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, cloning, screening, identification and transfection are performed to obtain the recombinant adenovirus vector, and the recombinant plasmid contains the nucleotide sequence shown in SEQ ID No.1 and the nucleotide sequence 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 a primer pair, 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, which includes 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 as SEQ ID No.5, and the amino acid sequence of the second fusion protein is shown as SEQ ID No.6.

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

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

[0020] The beneficial effects of the present invention are:

[0021] (1) The MATV11 recombinant adenovirus vector of the present invention comprises 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 reaction effect against other HPV genotypes;

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

[0023] (3) The HPV therapeutic vaccine of the present invention has a good immune effect against HPV16 / 18 / 52, and can also produce an effective specific immune response and an effective cross-immune response against 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, thereby effectively improving the effectiveness of the vaccine and having a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 The MATV11 recombinant adenovirus vector of the present invention is a schematic diagram of the structure of the MATV11 non-replicating adenovirus vector of Example 1;

[0027] Figure 3 The MATV11 recombinant adenovirus vector of the present invention, in Example 2, the changes of target antigen, P53 and pRB after 293 transfection with MATV11 were detected by in vitro WB. Figure 3A in the middle is the result of WB detection of the expression of HPV16 / 18 / 52 E6 fusion protein after MATV0 and MATV11 were transfected into 293 cells. Figure 3 Figure B in the middle shows the expression results of HPV16 / 18 / 52 E7 fusion protein after 293 was transfected with MATV11. Figure 3 Middle C is the result of WB detection of P53 and pRB expression after MATV0, MATV3 and MATV11 were transfected into 293 cells;

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

[0029] Figure 5 The MATV11 recombinant adenovirus vector of the present invention, in Example 3, is a comparison of the production results 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 is a growth curve of TC-1 subcutaneous tumor of mice in the experimental group and the control group in Example 4;

[0031] Figure 7 The MATV11 recombinant adenovirus vector of the present invention, in Example 4, is a result diagram of the tumor inhibition effect of HPV18 / 52 in C57BL / 6J mice. Figure 7 A in the middle is the growth curve of U14-HPV18 E6 / E7 subcutaneous tumor. Figure 7 Middle B is the growth curve of U14-HPV52 E6 / E7 subcutaneous tumor;

[0032] Figure 8 The MATV11 recombinant adenovirus vector of the present invention, in Example 4, the immune response results induced in C57BL / 6J mice after a booster injection four weeks apart, Figure 8 A is a comparison of the IFN-γ content of mouse spleen lymphocytes specific to E6 and E7 of HPV16 / 18 / 52 in the experimental group and the control group; Figure 8 Middle B is a comparison of IFN-γ content specific to HPV56 E7, HPV51E7, HPV68 E7 and HPV51 E6 in mouse spleen lymphocytes in the experimental group and the control group;

[0033] Fig. 9The MATV11 recombinant adenovirus vector of the present invention, in Example 5, is a comparison chart of the activation of specific CTL, Th1 and Th2 cells in vivo detected by cell flow cytometry with different numbers of virus particles. Fig. 9 A is the control group, Fig. 9 Middle B is the low-dose group, Fig. 9 Medium C is the medium dose group. Fig. 9 D is the high-dose group. DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0035] The MATV11 recombinant adenovirus vector of the present invention comprises nucleotide sequences for encoding E6 and E7 antigens of HPV 16, HPV18 and HPV52, and also comprises a nucleotide sequence 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 for encoding CD40L, so that the fusion protein expressed by it uses CD40L as an adjuvant, thereby improving the immune effect and memory effect of the vaccine.

[0037] The MATV11 recombinant adenovirus vector of the present invention exerts an immune effect in the body after intramuscular injection by presenting the antigen encoded by the adenovirus vaccine 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 IFN-γ, IL-4 and other cytokines, specifically attack the free viruses of HPV16 / 18 / 52 and the cervical epithelial cells integrated with HPV16 / 18 / 52E6 and E7 genes, and finally eliminate the virus and reverse the progression of CIN. The main application population is HPV persistent infection, cervical, anal and vulvar neoplasia.

[0038] TNFSFL molecule CD40L, which is the natural ligand of CD40, participates in the activation of dendritic cells (DC). CD40L, also known as gp39, tumor necrosis factor-associated activation protein (TNF associated activation protein, TRAP), T cell-B cell-activating molecule (T-BAM), is a 33kDa type II transmembrane glycoprotein with a total of 266aa, including 214 amino acids in the extracellular membrane region, which has a 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] Adenovirus is a non-enveloped, double-stranded DNA virus with a size of 80-100nm. It is one of the most promising vaccine vector platforms. Adenovirus vector vaccines have good safety and efficacy. Type 5 recombinant adenovirus vectors have the advantages of a wide range of host cells, large insertable fragments, high viral titers, and a mature industrial production system. Adenovirus vectors include replicative and non-replicative. The main advantages of non-replicative adenovirus vectors are high titer growth ability, easy operation, no integration into the human genome, strong immunogenicity, and easy clinical large-scale production.

[0040] Specifically, the nucleotide sequence structure diagram of the MATV11 non-replicating adenovirus vector of the present invention is as follows: Figure 1 It can be seen that the adenovirus of the present invention is a replication-deficient human adenovirus type 5 (Ad5) with a combined deletion of the E1 region and the E3 region, the nucleotide sequence as described in SEQ ID No.1 is located in the E1 region, and the nucleotide sequence as described in SEQ ID No.2 is located in the E2 region.

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

[0042] The preparation method of the MATV11 recombinant adenovirus vector of the present invention comprises constructing a recombinant plasmid, cloning, screening, identifying and transfecting to obtain a recombinant adenovirus vector, wherein 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 using a primer pair to amplify the nucleotide sequence as shown in SEQ ID No.1 and the nucleotide sequence as shown in SEQ ID No.2, in which 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 comprises a first fusion protein and a second fusion protein, and the first fusion protein and the second fusion protein are prepared by using the above-mentioned MATV11 recombinant adenovirus vector.

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

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

[0048] The present invention is described below by means of specific examples.

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

[0050] This example constructs a non-replicative adenovirus type 5 vector of MATV11 based on gene synthesis technology and in vitro ligation technology. The specific steps are as follows:

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

[0052] (2) In vitro gene synthesis of the nucleotide sequence shown in SEQ ID No. 1 was carried out and loaded into the pUC57 sequence (HPV16 / 18 / 52E7-CD40L_pUC57).

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

[0054] a.BstBI digested pad-CMV-BstBI plasmid;

[0055] b. Using HPV16 / 18 / 52E7-CD40L_pUC57 as a template, PCR was performed to obtain the in vitro ligation fragment, and the primers were shown in the sequences 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 bold part is the PCR primer HPV16 / 18 / 52E7-CD40L segment, and the marked line is the homologous recombination overlap segment.

[0061] c. The products of a and b above are connected in vitro, and the system is 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. Take 5ul of the above product and transfer it into T1 for clone screening and identification;

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

[0066] (4) Construction of 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, loaded into the PUC57 vector, and purified after obtaining the target fragment by PCR;

[0068] b. SpeI digested pad-HPV16 / 18 / 52E7-CD40L plasmid and purified the product;

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

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

[0071] (5) The MATV11 plasmid was digested with PacI and purified, and then transfected into HEK293A cells to obtain the MATV11 non-replicating adenovirus vector, the structure of which is shown in the following figure. Figure 2 As shown, its nucleotide sequence is shown in SEQ ID No.7.

[0072] Example 2 Expression of MATV11 antigen

[0073] 16-24 hours after 293 cells were plated, complete medium was used to transfect the virus with MOI=1. After 48 hours, cell residues were collected and the lysate was used to detect the expression of MATV11 fusion protein by WB. The test results were as follows: Figure 3 As shown in A and B, the red boxes represent the positions of the first fusion protein and the second fusion protein, respectively. The fusion protein expressed by the exogenous gene inserted in the E1 region detected by HPV16 E7 is around 100KD, and the fusion protein expressed by the exogenous gene inserted in the E3 region detected by HPV18 E6 antibody is also around 100KD, which is consistent with the size of the fusion protein.

[0074] In order to further demonstrate the safety of the vaccine MATV11 after antigenic point mutation, this example also detected the expression of pRB and p53 genes after virus transfection. Figure 3 Middle C is the result of WB detection of P53 and pRB expression after MATV0, MATV3 and MATV11 were transfected into 293 cells. MATV3 is a bivalent HPV16 / 18 therapeutic vaccine. The controls are wild-type HPV16 E6, HPV16 E7, HPV18E6 and HPV18 E7 plasmids, HPV16 E7 invitrogen 28-0006 mouse antibody 1:100, HPV18 E6 genetex 1:100.

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

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

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

[0078] Healthy 8-week-old C57BL / 6J female mice were intramuscularly injected with MATV11 vaccine at a dose of 10^8 pfu / mouse. The control group was given an empty virus MATV0. The mice were killed 1 week after the injection, spleen lymphocytes were isolated, and the lymphocytes were stimulated with peptide pools. The Elispot experiment was used to detect the production of specific IFN-γ by lymphocytes against antigens. The results are as follows: Figure 4 shown.

[0079] pass Figure 4It can be seen that the experimental group MATV11 can produce specific immune responses against antigens in mice, the most significant of which are 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 1 week and 2 weeks after a single injection of MATV11.

[0081] In order to further investigate the changes in immunogenicity in mice after intramuscular inoculation of MATV11, mice were killed at 1 and 2 weeks after vaccination, spleen lymphocytes were isolated, lymphocytes were stimulated with peptide pools, and Elispot assay was used to detect the production of specific IFN-γ by lymphocytes against antigens. Figure 5 As shown, the specific immune response to HPV18 E6 decreased slightly in the second week after vaccination, while the specific responses of other antigens did not change much, indicating that the in vivo immune response stimulated by the vaccine is still at its peak.

[0082] Example 4 Anti-tumor effect of MATV11 in C57BL / 6J mice

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

[0084] TC-1 cells are transgenic mouse lung epithelial cells with HPV16 E6 and HPV16 E7 and ras genes. A subcutaneous tumor model of the buttocks was established and randomly divided into MATV11, MATV0 and PBS groups. The MATV11 vaccine was inoculated at 3*10^8pfu / mouse on the 5th day after the inoculation of the subcutaneous tumor. The size of the subcutaneous tumor was measured and recorded every other day. Figure 6 The results showed that MATV11 could effectively inhibit the growth of TC-1 tumors at 3*10^8pfu / mouse. Two weeks after vaccination, the tumors in the experimental group were significantly reduced, and the volume of the mice in the control group exceeded 2000mm. 3 The tumor inhibition rate of MATV11 is over 80%.

[0085] (2) The tumor-suppressing 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 on HPV therapeutic vaccines focus on HPV16, and the TC-1 cell line is also used. In order to further demonstrate the immune effect of MATV11 against HPV18 and HPV52, this example uses lentivirus to construct U14 overexpressing HPV18 E6 / E7 cell lines and HPV52 overexpressing E6 / E7 cell lines.

[0087] Subcutaneous tumors of U14-HPV18 E6 / E7 and U14-HPV52 E6 / E7 were established, and MATV11 was inoculated on days 7 and 14. The growth of subcutaneous tumors in mice was measured as follows: Figure 7 As shown in the figure, compared with the control group, the tumor volume of the experimental group mice grew significantly slower, and the individual differences of the control group mice were large. 3 , the tumor grows rapidly. Figure 7 As shown, the volume of mice in the experimental groups was basically consistent, with small individual differences, and the tumor inhibition rate was above 70%.

[0088] According to the above experimental results, it can be seen that MATV11 can produce a specific immune response against HPV16 / 18 / 52 and effectively inhibit the growth of corresponding tumors.

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

[0090] Since current HPV therapeutic vaccines are basically booster shots, according to the literature and previous comparisons of changes in specific immune responses caused by MATV11 at 1 week and 2 weeks after injection, it can be concluded that the immune response is still at its peak at the 2nd week, and boosting at the peak may not be effective. Therefore, this example selected MATV11 to boost the injection at an interval of 4 weeks to detect the immune response in C57BL / 6J mice 1 week after.

[0091] The experimental groups were MATV0, MATV11 I 5W (MATV11 was given I injection and then killed 5 weeks later) and MATV11 II injection 1W (MATV11 was given II injection and then killed 1 week later after 4 weeks of booster injection). Elispot experiment was performed after stimulating lymphocytes with peptide pool. The results are shown in the figure. Figure 8 shown.

[0092] according to Figure 8 It can be seen that the booster injection after 4 weeks significantly enhanced the immune response of the vaccine in vivo and produced an effective specific immune response. The cross-reaction results showed that HPV56 E7, HPV51 E7, HPV68 E7 and HPV51 E6 can produce effective cross-immune reactions.

[0093] Example 5 Immunogenicity of MATV11 vaccine and correlation with dose

[0094] The theoretical value of the vaccine dose in the above examples is 1*10^11vp. In order to further investigate the difference in the immune response caused by the vaccine virus dose, this example selects high-dose (1*10^11vp), medium-dose (1*10^10vp) and low-dose (1*10^9vp) vaccines to inoculate mice respectively, and then detects the activation of specific CTL, Th1 and Th2 cells in vivo by cell flow cytometry. In the experiment, mice were inoculated with a control group, a high dose, a medium dose and a low dose, respectively, and were killed one week after the booster injection one month apart.

[0095] Test results such as Fig. 9 As shown, it can be seen that the high-dose group can effectively activate specific CTL and Th1 cells. There is basically no specific Th1 cell activation in the low-dose group, and CTL activation is only in the HPV52 E6 and HPV52 E7 groups, and the others are basically not effectively activated. The results of the medium-dose group and the low-dose group are similar, but the activation of specific CTL is slightly increased.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

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

2.

2. A MATV11 recombinant adenovirus vector according to claim 1, characterized in that: The adenovirus is a replication-deficient human adenovirus type 5 with E1 region and E3 region jointly deleted.

3. A MATV11 recombinant adenovirus vector according to claim 2, characterized in that: 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.

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

2.

5. The method for preparing the MATV11 recombinant adenovirus vector according to claim 4, characterized in that: The construction includes the steps of amplifying the nucleotide sequence as shown in SEQ ID No.1 and the nucleotide sequence as shown in SEQ ID No.2 using a primer pair, wherein the forward primer is shown in SEQ ID No.3 and the reverse primer is shown in SEQ ID No.

4.

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

7. A fusion protein composition, characterized in that: The fusion protein composition comprises a first fusion protein and a second fusion protein, and the first fusion protein and the second fusion protein are prepared by using the MATV11 recombinant adenovirus vector according to any one of claims 1 to 3.

8. A fusion protein according to claim 7, characterized in that: 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.

9. Use of the MATV11 recombinant adenovirus vector according to any one of claims 1 to 3 in the preparation of HPV therapeutic vaccines.

10. A HPV therapeutic vaccine, characterized in that: It includes the MATV11 recombinant adenovirus vector as described in claim 1 or 2 or the fusion protein composition as described in claim 7 or 8.

Images