CN118344491B

CN118344491B - HPV16/18 therapeutic vaccine, preparation method and application

Info

Publication number: CN118344491B
Application number: CN202410543160.4A
Authority: CN
Inventors: 李飞; 魏睿; 黄晓园; 纪腾; 韩迎燕; 代依林
Original assignee: Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2024-05-03
Filing date: 2024-05-03
Publication date: 2025-02-14
Anticipated expiration: 2044-05-03
Also published as: CN118344491A

Abstract

The present invention belongs to the field of bioengineering technology, in particular to a HPV16/18 therapeutic vaccine, preparation method and application. The present invention discloses a fusion protein, which is a protein as follows a) or b): a) the amino acid sequence is a fusion protein of SEQ ID No.2; b) the amino acid sequence shown in SEQ ID No.2 is replaced and/or deleted and/or added with one or more amino acid residues to obtain a fusion protein having the activity of binding HPV16/18E6-E7. The recombinant adenovirus that can express the fusion protein HPV16/18E7 protein provided by the present invention is used as an HPV16/18 therapeutic vaccine (MATV3), which has good immunogenicity in a mouse model and can induce a strong cellular immune response in a short time. The study on the tumor inhibition effect of the TC-1 mouse subcutaneous tumor model showed that the growth of TC-1 tumors can be significantly inhibited after two inoculations of MATV3, and HPV 16E7 and HPV18E7-specific cellular immune responses can be generated in mice. The vaccine preparation method is fast and simple, and can be mass-produced for community populations.

Description

HPV16/18 therapeutic vaccine, preparation method and application

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an HPV16/18 therapeutic vaccine, a preparation method and application.

Background

Human papillomavirus (Human papillomavirus, HPV) is a small, non-enveloped, double-stranded DNA virus with mesophilic and mucosal properties belonging to the papillomaviridae family. Human Papillomaviruses (HPV) are highly prevalent human pathogens in the worldwide population, one of the most common sexually transmitted infectious agents. To date, over 200 HPV types have been identified, of which there are over 40 associated with genital tract infections in women, and some strains can infect mucosal interiors and thus trigger epithelial malignancy, an important causative factor for cervical cancer and Cervical Intraepithelial Neoplasia (CIN). More than 95% of cervical cancer cases are from HPV infection. Cervical cancer is the fourth most common cancer in women worldwide, and 60.4 ten thousand new cases are estimated in year 2020 by WHO. In 34.2 ten thousand deaths from cervical cancer in 2020, about 90% occur in low and medium income countries. Over 2/3 of patients are from less developed areas such as south africa and latin america and become the leading cause of cancer death in that area.

Screening allows pre-cancerous lesions to be found at a stage that is easy to treat. However, in low and medium income countries, there is limited opportunity for people to obtain relevant precautions, which are only found after the disease has progressed to late symptomatology. Furthermore, there is limited availability of mechanisms for the treatment of cancer lesions such as surgery, radiation and chemotherapy, leading to higher mortality of cervical cancer in these countries. If effective intervention is adopted at different stages, the global cervical cancer high mortality rate can be reduced.

Different types cause different clinical manifestations, and can be classified into a skin low-risk type, a skin high-risk type, a mucous membrane low-risk type, a mucous membrane high-risk type and the like according to the affected tissue parts. HPV infecting the cervix can be classified into high-risk type and low-risk type according to its oncogenic potential and its correlation with cervical cancer. While HPV infections are common in all women, most genital HPV infections remain asymptomatic and self-clearing. However, persistent cervical infection with one or more high-risk HPV types has been recognized as an essential and inadequate factor for cervical cancer and its precursors. Among them, HPV16 and HPV18 have been identified as major oncogenic types, resulting in about 70% of cervical cancer cases worldwide.

The high-risk HPV16/18 has the highest detection rate in cervical lesion patients, accounts for about 71% of all subtypes of HPV, and has important significance in early screening and treatment of cervical cancer by researching pathogenic mechanism, detection method, prevention method and the like of the high-risk HPV. HPV infection rates and types in different areas are different, and high-risk HPV16/18/52/58 is the most common infection type in China.

Over the last 3 years, some important guideline updates have been continued, mainly the guideline update in the United states cancer Society 2020, published by the United states cancer Society (AMERICAN CANCER Society, ASC), the screening advice for cervical cancer general risk group, 2019ASCCP, published by the United states colposcope and Pathology Society (American Society for Colposcopy AND CERVICAL Pathology, ASCCP), the guidelines for risk-based cervical cancer screening abnormality and premalignant lesion management, and the guidelines for WHO cervical cancer premalignant lesions (version 2), published by the world health organization, 2021, 7 (New edition guideline). The new edition of guidelines provides different screening and treatment management strategies for different crowds from different angles, determines key points for increasing screening and treatment, and has the core of improving the screening rate of cervical cancer and treating precancerous lesions, enhancing the prevention and treatment of cervical cancer, and realizing the aim of eliminating cervical cancer in year WHO 2030.

However, HPV has heretofore been devoid of specific drugs. The existing treatment method in clinic only achieves the aim of killing viruses by diminishing inflammation, improving immunity and destroying the living environment of the viruses. Some traditional therapies for treating HPV clinically, such as laser, freezing, surgery and the like, have the defects of incomplete treatment, long treatment course, easy scar formation and the like. Among them, the most difficult problem to overcome is the repeated recurrence of HPV after treatment. All HPV vaccines are prophylactic vaccines at present, and have no therapeutic effect on existing infection or lesions. And the existing vaccine can not prevent all high-risk HPV persistent infection and cervical lesions caused by the high-risk HPV persistent infection, and even if the HPV vaccine is inoculated, cervical cancer needs to be screened regularly according to the advice of related departments. Therefore, the development of therapeutic vaccines has a broad application prospect in HPV treatment.

The relevant therapeutic HPV vaccines of the present stage are essentially all in preclinical research stage. Therapeutic vaccines mainly include attenuated vectors, nucleic acids, polypeptides, proteins and cellular vaccines, delivering target genes and target antigens for high-risk HPV subtype-associated tumor proteins via various vectors, and clearing infectious viruses by inducing cd8+ toxic T cell-mediated cellular immune responses while blocking precancerous lesions. The successful development of therapeutic vaccines will be a minimally invasive and efficient means of immunotherapy compared to traditional surgical treatment of HPV infection. Most of the research and development of therapeutic HPV vaccines are focused on developed areas such as Europe and America, and only VGX3100 developed by American vaccine-cancer immunotherapy company Inovio Pharma, an HPV16E6-E7 and HPV 18E 6-E7 HPV therapeutic vaccine of DNA plasmid vector is now subjected to three-phase clinical experiments. The DNA vaccine has the defects that the DNA vaccine is easily taken up by myocardial cells in the intramuscular injection process, and the continuous immunogenicity is blocked. And the cost of the DNA vaccine is high, and the pain of the vaccinated person is obvious during the electric transfer. The polypeptide vaccine is safe, high in stability and easy to produce, but has weak immunogenicity, and the therapeutic efficacy of the polypeptide vaccine is often enhanced by means of an immunoadjuvant or liposome. Protein vaccines are also weaker in immunogenicity and higher in production cost. Live vector attenuated vaccines mainly include recombinant bacterial and viral vectors that replicate in large amounts in host cells by the infectious organism, can induce strong cellular and humoral immunity against vaccine antigens, and then there is still some risk of using live vector vaccines in immunocompromised patients.

Recently non-replicating adenoviral vectors have attracted considerable attention again as vectors that are primarily resistant to new coronavirus vaccines. The main advantages of non-replicating adenovirus vectors are high titer growth ability, ease of handling, non-integration with human genome, strong immunogenicity, ease of clinical mass production, etc. And Ad vector vaccines against COVID-19 are now approved and used worldwide. Large scale clinical data shows the great potential and safety of Ad vectors as a platform for new and recurrent infectious disease vaccines. Based on the infection and treatment status of HPV at the present stage and the research background of our laboratory, an HPV therapeutic vaccine aiming at HPV16/18E6-E7 protein is designed, and the main application population is HPV persistent infection, cervical, anal and vulva internal neoplasia.

Based on the above objects, the present invention provides a gene-optimized polynucleotide for encoding HPV 16E 7 and HPV 18E 7 fusion proteins, the sequence of which is shown in SEQ ID No. 1. The polynucleotide takes replication defective human adenovirus 5 with combined deletion of E1 and E3 as a vector, HEK293 cells integrating adenovirus E1 genes as a packaging cell line, and the novel HPV16/18 therapeutic vaccine of the recombinant adenovirus vector is obtained through packaging.

Disclosure of Invention

The invention firstly provides a fusion protein which is the protein of the following a) or b):

a) A fusion protein with the amino acid sequence of SEQ ID No. 2;

b) Fusion protein with HPV16/18E6-E7 binding activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2.

The present invention also provides a biological material related to the above fusion protein, which is any one of the following B1) to B8):

b1 Nucleic acid molecules encoding the above fusion proteins;

B2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1),

B4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

B8 A recombinant microorganism comprising the recombinant vector of B4);

optionally, the vector comprises an adenovirus vector;

Optionally, the recombinant microorganism comprises HEK293A cells.

In certain embodiments, the nucleic acid molecule is a nucleic acid molecule as set forth in 1) or 2) or 3) or 4) below:

1) The coding sequence is a DNA molecule or a cDNA molecule of SEQ ID No.2 in a sequence table;

2) The nucleic acid sequence is a DNA molecule of SEQ ID No.1 in a sequence table;

3) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the above protein;

4) Hybridizing under stringent conditions with the nucleotide sequence defined in 1) or 2) and encoding a cDNA molecule or genomic DNA molecule of the fusion protein described above.

The invention also provides application of the fusion protein in preparing vaccines or medicaments for treating HPV 16/18.

The invention also provides application of the biological material in preparing vaccines or medicaments for treating HPV 16/18.

The invention also provides a primer pair for amplifying the nucleic acid molecule fragment encoding the fusion protein.

In certain embodiments, the amplification primer pair is a forward primer set forth in SEQ ID NO.3 and a reverse primer set forth in SEQ ID NO. 4.

The invention also provides a preparation method of the biological material, which comprises the following steps of (1) cloning HPV16/18 sequences and (2) carrying out homologous recombination on the sequences in the step 1 to construct new plasmids.

The invention also provides a preparation method of the vaccine or the medicine for treating HPV16/18, which is obtained by cloning, breeding and purifying the biological material.

The invention also provides a vector containing the polynucleotide.

In a preferred embodiment, the shuttle vector is a Padeasy system.

The invention also provides a human replication defective recombinant adenovirus capable of expressing the polynucleotide.

In a preferred embodiment, the recombinant adenovirus is derived from Padeasy adenovirus system.

The invention also provides application of the recombinant adenovirus in preparation of HPV16/18 therapeutic vaccine.

In a preferred embodiment, in the above application, the recombinant adenovirus is prepared as an injection.

In a more preferred embodiment, the recombinant adenovirus is prepared as an intramuscular injection.

The invention also provides a method for preparing the fusion protein recombinant adenovirus capable of expressing the fusion proteins HPV 16E 7, HPV18E7 and reticulin, which comprises the following steps of (1) constructing a shuttle plasmid vector containing polynucleotides for encoding HPV 16E 7 and HPV18E7, (2) transfecting the shuttle plasmid vector and a skeleton plasmid together in the step (1) into host cells, (3) culturing the host cells in the step (2), (4) harvesting human replication defective recombinant adenovirus released from the cells in the step (3), (5) amplifying and culturing the recombinant adenovirus in the step (4), and (6) purifying the culture product in the step (5). Preferably, the shuttle plasmid vector of step (1) is pdc3.1.

Preferably, the backbone plasmid in the step (2) is pad-pshuttle-BstbI, and both plasmids belong to padeasy adenovirus system, and are used for packaging recombinant adenovirus containing encoded fusion protein HPV16/18E7 protein in host cells.

Preferably, the cells of step (3) are HEK293 cells.

Preferably, the expansion culture method in the step (5) is suspension culture.

Preferably, the purification method of step (6) is Source 30Q chromatography.

The invention finally provides a vaccine or medicament for the treatment of HPV16/18, said product comprising a protein as defined above or a biological material as defined above.

Compared with the prior art, the invention has at least the following beneficial effects:

the recombinant adenovirus capable of expressing the fusion protein HPV16/18E7 protein provided by the invention is used as an HPV16/18 therapeutic vaccine (MATV 3), has good immunogenicity on a mouse model, and can induce an organism to generate a strong cellular immune response in a short time. Tumor inhibition effect study in a TC-1 mouse subcutaneous tumor model shows that the growth of TC-1 tumors can be obviously inhibited after two MATV3 inoculations, and HPV 16E7 and HPV 18E7 specific cellular immune responses can be generated in the mice. The vaccine preparation method is quick and simple, and can realize mass production for community groups.

Drawings

FIG. 1 shuttle plasmid and MATV3 genomic map;

FIG. 2 shows a Westernblot identification pattern of intracellular expression of the vaccine MATV3 fusion protein HPV 16E 7-HPV 18E 7-Calreticlein;

FIG. 3 Westernblot identification of the effect of vaccine MATV3 expression fusion proteins on P53 and Rb proteins.

FIG. 4 shows statistics of activation of peripheral blood lymphocytes and spleen lymphocytes of mice with HPV 16E 7 specificity by vaccinating healthy C57 mice with MATV3, two week-spaced booster needles, grouped as MATV0, MATV 3I 14D (sacrificed 14 days after 1 week of vaccination), MATV3 II 7D (sacrificed 7 days after two week-spaced booster needles) and MATV3 II 21D (sacrificed 21 days after two week-spaced booster needles).

FIG. 5 shows a flow chart of specific CD4+ and CD8+ cell responses induced by HPV 16E 7 (grouped with FIG. 4) in healthy C57 mice vaccinated with MATV3 with two week intervals of booster needles.

FIG. 6 statistical activation of peripheral blood lymphocytes and spleen lymphocytes of mice with HPV 18E 7 specificity by multi-color cell flow assay, vaccinated healthy C57 mice with MATV3, two week-old booster needles (group wise as in FIG. 4).

FIG. 7 shows a flow chart of specific CD4+ and CD8+ cell responses induced by HPV 18E 7 (grouped with FIG. 4) in healthy C57 mice vaccinated with MATV3 with two week intervals of booster needles.

FIG. 8 shows comparison of the statistical graphs of the Elispot response of HPV 16E 7 and HPV 18E 7 specificities of healthy C57 mice vaccinated with MATV3 with two week intervals of booster needles (group with FIG. 4).

FIG. 9 shows the activation flow charts of HPV 16E 7-specific CTL, th1, th2, CD8+CD107a+, CD8+Ki67+ cells from healthy C57 mice vaccinated with MATV3, booster needles 1 month apart, MATV 3I 37D grouped as MATV0, MATV3 II 7D (mice sacrificed 7 days post 1 month booster needle apart), isolated mouse spleen lymphocytes.

FIG. 10 shows a flow chart of activation of HPV 18E 7-specific CTL, th1, th2, CD8+CD107a+, CD8+Ki67+ cells by multi-color cell flow assay of healthy C57 mice vaccinated with MATV3, booster needles 1 month apart, grouped as in FIG. 9.

FIG. 11 healthy C57 mice were vaccinated with MATV3, with 1 month-old booster needles, and the groupings were compared to the Elispot response statistics for the HPV16 E7 and HPV18 E7 specificities of FIG. 9.

FIG. 12 shows the trend of changes in HPV 16E 7-specific IFN-gamma production by Elispot after vaccination with MATV3 in healthy C57 mice after boosting the needles at the second and fourth week, respectively.

FIG. 13 shows the trend of changes in HPV 18E 7-specific IFN-gamma production by Elispot after vaccination with MATV3 in healthy C57 mice after boosting the needles for the second and fourth week, respectively.

FIG. 14 shows a graph of the growth trend of TC-1 tumors in healthy C57 mice vaccinated with TC-1 subcutaneous tumors after MATV3 vaccine on days 5 and 12.

FIG. 15. In the animal model of FIG. 14, mice vaccinated with MATV3 group were essentially free of neoplasia, again vaccinated with TC-1 cells at day 40, and growth of TC-1 subcutaneous tumor was observed and recorded.

FIG. 16 shows a graph of the growth trend of TC-1 tumors in healthy C57 mice vaccinated with TC-1 subcutaneous tumors after MATV3 vaccine on day 12 and day 19.

FIG. 17 is a statistical plot of the production of HPV 16E 7 and HPV 18E 7-specific IFN-gamma from Elispot-detected mice taken at day 30 in the animal model of FIG. 16 after spleen lymphocytes have been isolated.

FIG. 18 shows a statistical plot of lymphocyte activation in the animal model of FIG. 16, taken at day 30 from MATV0 and MATV3 mice, after spleen lymphocytes have been isolated, for multicolor flow detection of HPV 16E 7 and HPV 18E 7-specific cellular responses.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.

Nucleotide sequence of recombinant protein SEQ ID NO.1：atgcacggcgatacacctacactgcacgagtacatgctggatctgcagccagagacaaccgatctctacggctacggccagctgaacgacagcagcgaggaggaggacgagatcgatggcccagctggccaggccgagccggaccgcgcccactacaacatcgtgaccttttgttgcaagtgtgacagcacgctgcggctgtgcgtgcagagcacacacgtggacatccgcaccctggaggacctgctgatgggcacactgggcatcgtgggccccatctgtagccagaagccaggatcccacggccctaaggccacactgcaggacatcgtgctgcacctggagccccagaacgagatcccggtggacctgctgggccacggccagctgagcgacagcgaggaggagaacgatgagatcgatggcgtgaaccaccagcacctgccagcccgccgcgccgagccacagcgccacacaatgctgtgtatgtgttgtaagtgtgaggcccgcatcgagctggtggtggagagcagcgccgacgacctgcgcgccttccagcagctgtttctgaacaccctgtcctttgtgggcccgtggtgtgcctcccagcag

ggatcccaccagaagcgcaccgccatgtttcaggacccacaggagcgcccccgcaagctgccacagctgtgcacag

agctgcagacaaccatccacgatatcatcctggagtgtgtgtactgcaagcagcagctgctgcgcagggaggtgtacg

actttgctttccgggacctgtgcatcgtgtaccgcgatgggaacccatacgctgtgggcgataagtgtctgaagttttaca

gcaagatcagcgagtaccgccactactgttacagcctgtacggcacaacactggagcagcagtacaacaagccgctg

tgtgatctgctgatcaggtgtatcaacggccagaagccactgtgtcctgaggagaagcagcgccacctggacaagaa

gcagcgcttccacaacatcaggggccggtggaccggccgctgtatgagctgttgccgcagcagccgcacacgcgga

tccgcgcgctttgaggacccaacacggcgcccctacaagctgcctgatctgtgcacggagctgaacaccagcctgca

ggacatcgagatcacctgtgtgtactgcaagacagtgctggagctgacagaggtgtttgagtttgcctttaaggatctgtt

tgtggtgtaccgcgacagcatcccccacgctgccggccacaagtgtatcgatttttacagccgcatccgcgagctgcgc

cactacagcgacagcgtgtacggcgacacactggagaagctgaccaacaccgggctgtacaacctgctgatcaggtg

cctgcggggccagaagccgctgaacccagccgagaagctgcgccacctgaacgagaagcgccgctttcacaacatc

gctgggcactaccgcggccagtgccactcgtgctgcaaccgcgcccgccaggagcgcctccagcgccgcggatcc

ctgctgtccgtgccgctgctgctcggcctcctcggcctggccgtcgccgagcctgccgtctacttcaaggagcagtttct

ggacggcgacgggtggacctcccgctggatcgagtccaagcacaagagcgattttggcaagttcgtgctcagctccg

gcaagttctacggcgacgaggagaaggataagggcctgcagacaagccaggacgcgcgcttctacgcactgtcggc

cagcttcgagcctttcagcaacaagggccagacgctggtggtgcagttcacggtgaagcacgagcagaacatcgact

gtgggggcgggtacgttaagctgttcccaaacagcctggaccagacagacatgcacggcgacagcgagtacaacat

catgtttggccccgacatctgtggccctggcaccaagaaggtgcacgtcatcttcaactacaagggcaagaacgtgctg

atcaacaaggacatccgctgcaaggatgatgagtttacacacctgtacacactgatcgtgcggccagacaacacctac

gaggtgaagatcgacaacagccaggtggagtccggctccctggaggacgattgggacttcctgccacccaagaagat

caaggaccctgatgctagcaagccggaggactgggatgagcgggccaagatcgatgatcccacagactccaagcct

gaggactgggacaagcccgagcacatccctgaccctgatgctaagaagcccgaggactgggatgaggagatggac

ggcgagtgggagcccccagtgatccagaaccctgagtacaagggcgagtggaagccccggcagatcgacaaccca

gattacaagggcacctggatacacccagagatcgacaaccccgagtacagccccgatcccagcatctacgcctacga

taactttggcgtgctgggcctggacctctggcaggtcaagagcggcaccatctttgacaacttcctcatcaccaacgatg

aggcctacgctgaggagtttggcaacgagacgtggggcgtgacaaaggccgccgagaagcagatgaaggacaagc

aggacgaggagcagaggctgaaggaggaggaggaggacaagaagcgcaaggaggaggaggaggccgaggac

aaggaggatgatgaggacaaggatgaggatgaggaggatgaggaggacaaggaggaggatgaggaggaggatgt

ccccggccaggccaaggacgagctgTGA;

Amino acid sequence of recombinant protein SEQ ID NO.2：MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKPGSHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVGPWCASQQGSHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINGQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRGSARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAGHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRGQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRGSLLSVPLLLGLLGLAVAEPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDEL;

EXAMPLE 1 preparation of recombinant HPV therapeutic vaccine MATV3 Using human replication-defective adenovirus as vector

HPV16 E7 and HPV18 E7 and reticulin gene optimization and synthesis. The target antigen of the recombinant HPV therapeutic vaccine MATV3 vaccine is the fusion protein of HPV 16E 7 and HPV 18E 7. The vaccine adjuvant is reticulin. By optimizing HPV 16E 7 and HPV 18E 7 proteins and the reticulin gene, the expression level of the fusion protein is improved, so that the immunogenicity of the vaccine is improved.

Firstly, the use of Upgene software (Gao et al ,A.UpGene:Application ofaweb-based DNA codon optimization algorithm.Biotechnol Prog,2004.20(2):p.443-8.) for codon optimisation, the most rare codons of HPV 16E 7, HPV 18E 7 and reticulin genes are changed to high frequency codons, secondly, considering that software optimisation may mechanically change codons to the highest frequency of use codons, which may not significantly improve protein translation efficiency due to tRNA efficiency, mRNA secondary structure etc., we use manual analysis to replace part of the high and low frequency codons and at the same time evenly distribute the high and low frequency codons to the fusion protein gene while considering increasing GC content in mRNA, which helps to enhance mRNA stability, we suitably increase GC content of the fusion protein gene and distribute G, C nucleotides as evenly as possible throughout the GP gene.

After the fusion protein gene is optimized, a Kozak sequence is added in front of a translation initiation codon, an EcoRI (restriction enzyme digestion site) is inserted into the whole sequence at the upstream side, a HindIII (restriction enzyme digestion site) is inserted into the whole sequence at the downstream side, and the gene sequence is synthesized.

2. The vector constructs the synthesized gene sequence, the target gene fragment is obtained by a PCR method, and the primers used for PCR are as follows:

SEQ ID NO.3, forwardprimer:

GCTCGAGCCTAAGCTTCGAGCCACCATGGATGCACGGCGATACACCTAC SEQ ID No.4, REVERSE PRIMER:

The GATCGGATATCTTATTTCGTCAGAGCTTGAGCAGGCCAAAGG, PCR products were sequenced and verified, after which the PCR products were purified using a PCR product purification kit (OMEGA, REF, D6492-02), the pad-pshuttle-BstbI recombinant backbone plasmids were digested with BSTBI restriction endonucleases (NEB, REF, R0519S) and verified by DNA agarose gel electrophoresis, the digested plasmids were purified by conventional methods (phenol, chloroform, isopropanol), the genes of interest were ligated to the pad-pshuttle-BstbI recombinant backbone plasmids of the padeasy adenovirus system using DNA ligases (NEB, REF, E2621L), T1 competence was transformed, kanar LB plates were coated, colonies were picked for PCR identification, and DNA agarose gel electrophoresis was performed on the PCR products to verify sequencing of positive clones.

3. Recombinant adenovirus packaging, preparation and identification

3.1 Packaging of recombinant adenovirus. The identified single clone is shaken and the plasmid is greatly extracted (QIAGEN, REF, 12362), pacI restriction endonuclease (NEB, REF, R0547L) is used for enzyme digestion of the plasmid, the enzyme digestion plasmid after electrophoresis verification is purified by a traditional method, and HEK293 cells are transfected by the enzyme digestion purification plasmid for recombinant adenovirus packaging.

The procedure was as follows, one day before transfection, HEK293 cells were seeded in 25cm ² cell culture flasks of 1 x 10 ⁶ cells per flask, medium dmem+10% fbs, and incubated overnight in a 37 ℃ 5% co ₂ cell incubator.

On the day of transfection, the digested and purified plasmid was taken and transfected with Lipofectamine 3000 transfection reagent (Invitrogen, ref, L3000-001) according to the instructions attached thereto. The method comprises the following specific steps:

Each transfection flask was bottled with 5ug of the digested purified plasmid, mixed with 10ul P3000 and diluted with 500 ulOpti-MEM. 15ul Lipofectamine 3000 transfection reagents were diluted with 500 ulOpti-MEM. Lipofectamine 3000 system was added to the digested and purified plasmid, mixed well, allowed to stand at room temperature for 15 minutes, and then added to the cells.

The cell toxin-out phenomenon, namely the swelling and the transparent of the cell rounding, is observed every day, is in a grape cluster shape, and has a large amount of plaques, and the cell toxin-out is carried out after most cytopathy in the transfection bottle is removed.

The toxic cells and the supernatant are repeatedly frozen and thawed three times in a refrigerator at-80 ℃ and a water bath at 37 ℃. Centrifugation at 4000rpm for 20 minutes, the virus-containing supernatant was collected and the virus seed was frozen in a-80 ℃ freezer.

3.2 Identification of recombinant adenoviruses

3.2.1PCR amplification fusion protein and fusion protein complete sequence and sequencing identification

500Ul of viral supernatant was taken, and viral DNA was extracted using DNA extraction kit (QIAGEN, REF, 51104) for PCR identification and sequencing verification using the primers as follows:

SEQ ID NO.5 shows Pshuttle-CMV-F GGTCTATATAAGCAGAGCTG

SEQ ID NO.6 shows that Pshuttle-CMV-R GTGGTATGGCTGATTATGATCAG PCR amplification conditions are:

The reaction procedure is:

the result of DNA agarose gel electrophoresis shows that the virus seed can be amplified to a single target band, and the fragment size is correct. And (3) carrying out glue recovery and sequencing on the target strip, wherein the comparison result shows that the sequencing sequence is completely correct.

3.2.2 Identification of target antigen expression HEK293 cells were infected with different constructs of recombinant adenovirus, and after 48 hours, the cells were collected for Westernblot detection of target antigen, and MATV3 could detect the apparent expression of target protein. The results are shown in FIG. 2, wherein 1 is a pre-transfected Marker, 2 is a blank 293 cell, 3 is an empty control adenovirus vector transfected 293 cell, and 4 is a MATV3 transfected 293 cell;

3.2.3 recombinant adenovirus culture

HEK293 cells were cultured with Corning 10-layer cell factory wall-attached at 37 ℃ under 5% co ₂. The cells were inoculated with a seed when cultured to 90% confluency. The culture medium was changed and the P5-generation recombinant adenovirus was infected with HEK293 cells at moi=1, 37 ℃,5% co ₂, and cultured adherent. 72 hours after inoculation of the seed, 100% of the cells were gently shaken when cytopathic effect (CPE) was present, centrifuged at 100g for 5 minutes, the supernatant was discarded, and the final cell pellet was resuspended in 40mL fresh medium. After repeated freeze thawing 3 times in a-80 ℃ refrigerator and 37 ℃ water bath, the supernatant was taken after centrifugation at 3000RPM for 30 minutes and stored in a 4 ℃ refrigerator for purification.

3.2.4 Recombinant adenovirus purification

Purifying recombinant adenovirus by cesium chloride gradient centrifugation, preparing cesium chloride solution with different densities, namely 1.5g/mLA solution, 1.35g/mL B solution and 1.25g/mL C solution, paving the recombinant adenovirus with different densities in an overspeed centrifuge tube according to the sequence of A solution, B solution and C solution, centrifuging for 1 hour by using a Beckmen overspeed centrifuge and a SW41 Ti horizontal rotor at 35000RPM, sucking a lower viral ring, mixing the sucked recombinant adenovirus with cesium chloride B solution 1:1, continuing ultracentrifugation, centrifuging for 18 hours at 35000RPM, sucking the viral ring, and dialyzing. Dialyzate is 2mM magnesium chloride+10 mM Tris-base+5% sucrose solution, dialyzate is changed for 3 times every 6 hours by using a dialysis bag with the cut-off molecular weight of 14kDa and the rotating speed of a magnetic stirrer of 600RPM, the recombinant adenovirus preservation medium is dialyzed into the dialyzate by cesium chloride, and the dialyzed recombinant adenovirus is subpackaged and preserved to a refrigerator with the temperature of minus 80 ℃ for standby.

3.3 Identification and titre determination of Ad5-nCoV

3.3.1PCR amplification target protein gene complete sequence and sequencing identification

The experimental method and procedure were the same as 3.2.1. Agarose gel electrophoresis results show that the virus seeds can be amplified to single target bands, and the fragment size is correct. And (3) carrying out glue recovery and sequencing on the target strip, wherein the comparison result shows that the sequencing result is completely correct.

3.3.2 Infection titre determination

Recombinant adenovirus titers were determined using TakaraAdeno-X TM RAPID TITER KIT. The operation is carried out according to the instruction book attached to the kit, and the specific method is as follows:

a) HEK293 cells were seeded in 24-well plates. The cell density was 2.5X10 ⁵ cells/mL, 1mL was inoculated per well, and the medium was DMEM+10% FBS.

B) A series of dilutions of virus samples were prepared by 10-fold dilution of the virus to be detected from 10 ^-2 to 10 ^-7 using medium, and 50. Mu.L per well was added to the cells.

C) Cells were cultured in a 5% CO ₂ incubator at 37℃for 48 hours.

D) The medium of the cells was aspirated and the cells were allowed to air dry slightly (without excessive drying). The cells were fixed by gently adding 0.5mL of ice methanol per well and leaving at-20℃for 10 minutes.

E) Methanol was pipetted off and the cells were gently washed 3 times with pbs+1% bsa. 0.25mL of anti-Hexon antibody diluent (1:1000 dilution) was added to each well and incubated at 37℃for 1 hour.

F) anti-Hexon antibodies were blotted and cells were gently washed 3 times with PBS+1% BSA, 0.25mL HRP-labeled RatAnti-MouseAntibody (1:500 dilution) was added to each well, and incubated at 37℃for 1 hour.

G) Before pipetting 0.25mL of HRP-labeled RatAnti-MouseAntibody, 10 XDAB substrate was diluted with 1X Stable Peroxidase Buffer to 1 XDAB working solution and allowed to reach room temperature.

H) The RatAnti-MouseAntibody dilutions were blotted and the cells gently washed 3 times with PBS+1% BSA. 0.25mL DAB working solution was added to each well and left at room temperature for 10 minutes.

I) DAB working solution was pipetted off and the cells were gently washed 2 times with PBS.

J) Brown/black positive cells were counted under a microscope. At least 3 fields per well were counted randomly and the average positive cell number was calculated.

K) The infection titer (ifu/mL) was calculated.

The formula is infection titer (ifu/mL) =number of positive cells per field x number of fields per well/(viral volume (mL) ×dilution).

The titer measurement result shows that the infection titer of the purified recombinant adenovirus after concentration is more than 1.0X10 ¹⁰ ifu/mL.

3.3.3 Viral particle count assay

A virus lysate was prepared by mixing 20mmol/LTris-Cl, 2mmol/L EDTA (pH 7.5) solution and 2.0% SDS solution in equal volumes. And taking a proper volume of virus sample to be tested, adding 1/19 volume of virus lysate, repeatedly blowing and beating for 10 times by using a pipette, uniformly mixing, and swirling for 1 minute. The mixture was digested by shaking in a thermostatic water bath at 56℃for 10 minutes, centrifuged at 12000rpm for 5 minutes, and the supernatant was collected to determine OD values at 260nm and 280 nm. The number of adenovirus particles was counted.

The number of virus particles is determined, and the purified recombinant adenovirus reaches more than 1.0X10 ¹¹ VP/mL after concentration.

Example 2 immunological evaluation of HPV therapeutic vaccine of recombinant adenovirus vector MATV3 on mouse model.

Specific cellular immune response induced after two weeks apart booster needle vaccination was detected.

1.1 Healthy C57BL/6 female mice 40, 10 per group. Intramuscular injection of MATV3 virus 1 x10 ¹¹ vp, two weeks apart, followed by the same dose as the booster needle. The control group was empty virus MATV0. The following MATV0 groups, MATV 3I 14D (sacrificed 14 days after 1 needle inoculation), MATV3 II 7D (mice sacrificed 7 days after needle boosting) and MATV3 II 21D (mice sacrificed 21 days after needle boosting). Mice were sacrificed, spleen and peripheral blood lymphocytes were isolated, and the HPV 16E 7 protein overlapping peptide pool and HPV 18E 7 protein overlapping peptide pool were used to stimulate culture for 6 hours, respectively, while protein secretion blockers were added to block cytokine secretion. After 6 hours, fc receptors were blocked, dead cells and cell surface molecular markers were stained, and intracellular cytokines were stained after cells were fixed and perforated. Cell surface markers include CD3, CD4, CD8 and CD45 molecules, and intracellular cytokines include ifnγ and IL4. The levels of ifnγ and IL4 expressed after specific peptide stimulation of cd4+ T cells and cd8+ T cells were analyzed using a flow cytometer (BD FACS CantoTM). As shown in fig. 4, 5, 6 and 7, spleen lymphocytes and peripheral blood lymphocytes were activated to HPV 16E 7-specific CTLs and Th1 cells, but Th2 cell activation was not different after vaccination compared to MATV0 (fig. 4 and 5). The strongest group was MATV 3I 14D, then MATV3 II 7D, and finally MATV3 II 21D the pool of HPV 18E 7 polypeptides activated effector T cells slightly less than the antigenicity of HPV 16E 7, but the spleen lymphocytes and peripheral blood lymphocytes of MATV 3I 14D group were significantly activated CTL compared to the control group (FIGS. 6 and 7). It was thus seen that specific cellular immune responses against HPV 16E 7 and HPV 18E 7 could be generated in vivo 1 week after vaccination, reaching a peak at 2 weeks of vaccination, with reduced efficacy of the immune response over time, and no substantial enhancement at the second week.

1.2 In the animal model of 1.1 above, isolated spleen lymphocytes were subjected to Elispot experiments to further explore the production of IFN-gamma specific for HPV 16E 7 and HPV 18E 7 after vaccination, and after vaccination, IFN-gamma specific for HPV 16E 7 was produced 7-fold compared to control MATV0 after vaccination with MATV 3. However, the generation of specific IFN-gamma for HPV 18E 7 was not significantly different from the control and experimental groups, which may be that the epitope for HPV 18E 7 was not as large in mice as that of HPV 16E 7, as reported in other literature, as shown in FIG. 8.

Specific cellular immune response induced after 1 month-old booster vaccination was detected.

Healthy C57BL/6 female mice were 30, 10 per group. MATV3 virus was injected intramuscularly 1X10 ¹¹ vp with 1 month-old booster needles. The experimental groups were MATV0, MATV3 II 7D (7 days after 1 month of booster needle sacrifice) and MATV 3I 37D (37 days after I needle vaccination). In the same manner as the spleen and peripheral blood of the mice, lymphocytes were isolated, and activation of lymphocytes was examined by multicolor flow after stimulation with HPV 16E 7 and HPV 18E 7 polypeptide pools, it was seen that the enhancement of specific CTL activation by the needle was significantly enhanced after 1 month interval and specific activated cd8+ cells were also significantly increased, as shown in fig. 9 and 10. The Elispot results of spleen lymphocytes are shown in FIG. 11, in which MATV3 II 7D and MATV 3I 37D produced IFN-gamma production against HPV 16E 7 and HPV 18E 7, and 1 month-spaced reinforcing needles significantly enhanced specific IFN-gamma production, as compared to control MATV 0.

3. The change in cellular immune response in mice at various time points after vaccination was examined.

Healthy mice were sacrificed one week, two weeks, three weeks and five weeks after C57 intramuscular injection of MATV 3I needle, and isolated spleen lymphocytes Elispot experiments detected changes in antigen-specific IFN- γ production. And changes in cellular immune responses in mice were detected at Elipot following the week 2 and week 4 booster needles, respectively. As in fig. 12 and 13. IFN-gamma production specific for HPV 16E 7 was shown to be positive for one week after vaccination, and peak 2 weeks after vaccination, and gradually declined, as compared to the control group. The production of large amounts of specific IFN-gamma was again activated 1 week after the booster needle at week 2, and gradually decreased thereafter. The production of a large amount of IFN-gamma can be activated again after the needle is reinforced at the 4 th week, and the effect is obviously higher than that of a single needle. The timing of specific IFN-gamma production against HPV 18E 7 is substantially similar to that of HPV 16E 7, but to a significantly lesser extent, possibly associated with a lesser number of epitopes in mice against HPV 18E 7.

Example 3 anti-tumor effect of HPV therapeutic vaccine of recombinant adenovirus vector MATV3 on mouse subcutaneous tumor model.

3.1MATV3 inhibitory effect on TC-1 early stage subcutaneous tumor.

Selecting 20 healthy female C57 mice, subcutaneously inoculating TC-1 cells on the right buttocks, 1 x10 ⁵ cells per mouse, randomly dividing the 5 th day of subcutaneous tumor inoculation into two groups, namely MATV0 and MATV3, intramuscular injecting virus 1 x10 ¹¹ vp, inoculating the same dose of virus on the 12 th day of subcutaneous tumor inoculation booster needle, observing and measuring the size of the TC-1 subcutaneous tumor of the mice on every other day, and drawing a tumor growth curve. As shown in fig. 14. 86% (13/15) of the mice in MATV3 group had no tumor formation at all during the whole observation period, and the control group showed rice grain-like-sized rumen from day 12, with a tumor formation rate of 100%. The tumor inhibition rate of the MATV3 vaccine is up to more than 95%.

3.2MATV3 memory protection effect.

In the animal model 3.1 described above, the MATV3 group of non-tumorigenic mice was again transplanted with TC-1 cells at day 40, 1X 10 ⁵ cells per mouse, healthy C57 female mice of the same week age were selected for transplantation with TC-1 cells, and the size of the mouse TC-1 subcutaneous tumor was observed and measured at intervals and tumor growth curves were drawn. As shown in fig. 15, the memory effect of the MATV3 vaccine was 100% protective for the development of mouse tumors.

3.3MATV3 inhibitory effect on TC-1 advanced subcutaneous tumor.

Selecting 20 healthy female C57 mice, subcutaneously inoculating TC-1 cells on the buttocks, 1x 10 ⁵ cells per mouse, randomly dividing the 12 th day of subcutaneous tumor inoculation into two groups, namely MATV0 and MATV3, intramuscularly injecting 1x 10 ¹¹ vp of virus, inoculating the same dose of virus on the 17 th day of subcutaneous tumor inoculation booster needle, observing and measuring the size of the TC-1 subcutaneous tumor of the mice on every other day, and drawing a tumor growth curve. As shown in FIG. 16, MATV3 can effectively inhibit the growth of TC-1 late stage tumor with a tumor inhibition rate of 81.8%.

3.4MATV3 induce specific cellular immune responses in TC-1 subcutaneous tumor mice.

Mice from groups MATV0 and MATV3 were sacrificed on day 31 with subcutaneous tumor inoculation as in 6.3 above to establish a TC-1 advanced stage tumor model, tumors from mice were dissected, spleen lymphocytes were isolated, and activation of spleen lymphocytes was tested by Elispot assay and multicolor flow. As shown in FIG. 17, MATV3 vaccine was effective in activating IFN-y production of lymphocytes against HPV 16E 7 and HPV 18E 7, as compared to control MATV 0. The results of polychromatic flow are shown in figure 18 to show that MATV3 vaccine is also effective in activating CTL and Th2 cells in mice and that activated cd8+ cells are also significantly increased.

4. Summary of the immune response of the recombinant adenovirus vector MATV3 in mice.

The MATV3 bivalent vaccine intramuscular injection of the adenovirus vector into mice can effectively cause lymphocyte-specific CTL and TH cell activation, can effectively inhibit the growth of TC-1 subcutaneous tumor, and can generate effective memory protection effect. However, in vivo experiments in mice have found that activation of specific immune responses against HPV 18E 7 is somewhat worse, probably related to fewer HPV 18E 7 epitopes of lymphocytes in mice, but the responses in humans remain viable.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Claims

1. A fusion protein, which is a fusion protein with an amino acid sequence of SEQ ID No. 2.

2. A biological material associated with the fusion protein of claim 1, which is any one of the following B1) to B8):

B1 A nucleic acid molecule encoding the fusion protein of claim 1;

B2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1),

B4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

B8 A recombinant microorganism comprising the recombinant vector of B4);

wherein the vector comprises an adenovirus vector;

Wherein the recombinant microorganism comprises HEK293A cells.

3. The biological material according to claim 2, wherein the nucleic acid molecule is a nucleic acid molecule as shown in 1), 2) or 3):

3) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the protein of claim 1.

4. Use of the fusion protein of claim 1 for the preparation of a medicament for the treatment of HPV 16/18.

5. Use of the biomaterial of claim 2 for the manufacture of a medicament for the treatment of HPV 16/18.

6. A primer pair for amplifying a fragment of a nucleic acid molecule encoding the fusion protein of claim 1.

7. The primer pair of claim 6, wherein the amplification primer pair is a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO. 4.

8. A method for preparing a biological material according to claim 2, comprising the steps of (1) cloning HPV16/18 sequences and (2) constructing a novel plasmid by homologous recombination of the sequences of step 1.

9. A method for preparing a medicament for treating HPV16/18, which is characterized in that the medicament is obtained by cloning, breeding and purifying the biological material according to claim 2.

10. A medicament for the treatment of HPV16/18, comprising the fusion protein of claim 1 or the biomaterial of claim 2.