Study On The Immune Effect Of AD-HPV16/18/58mE6E7 Trivalent Recombinant Adenovirus Vector Therapeutic Vaccine

Objective: The aim of this study was to reevaluate the cellular immune effect of the AD-HPV16/18/58mE6E7 trivalent recombinant adenoviral vector therapeutic vaccine constructed in our previous experiments on tumor-bearing mice expressing the HPV58E6E7 fusion gene.And continue to verify the immune effect of the new vaccine on tumor-bearing mice expressing HPV16,18E6 and/or E7 genes through animal experiments,and provide experimental basis and candidate vaccine for immunotherapy of cervical precancerous lesions and malignant tumors in the future.Methods:(1)To identify and validate mouse cervical cancer cell lines with stable expression of HPV16E6 and E7 proteins.Through literature review,it was learned that the lung epithelial cells of C57BL/6(H-2b)mice co-transformed by E6,E7 and ras genes of TC-1 line HPV16 of tumor cell lines could express HPV16E6 and E7 proteins stably.The cell line was constructed by professor T.C.Wu from John Hopkins university.The expression of HPV16E6 and E7 genes and proteins in this cell line was verified again by RT-PCR and Western Blot.(2)Tumorigenesis and validation of mice expressing HPV16E6 and E7 genes.C57BL/6 mice were subjected to subcutaneous tumorigenesis in the right backusing the TC-1 cell line after expanded culture and verification,and the expressions of HPV16E6 and E7 genes and proteins in tumor tissues of tumorigenic mice were verified by RT-PCR and WB.(3)A mouse cervical cancer cell line with stable expression of HPV18E6E7 fusion protein was constructed and verified.In combination with the previous experience of successfully constructing U14 cell lines(mouse cervical cancer cell line,HPV negative)with stable expression of HPV58E6E7 fusion protein,our research group successfully transfected U14 cells of cervical cancer in mice with a green fluorescent lentivirus system with stable expression of HPV18E6E7 fusion gene.After successfully expanded culture,the newly constructed mouse U14 cell line(named U14/LV-HPV18E6E7)expressing HPV18E7 fusion protein was verified by RT-PCR and Western Blot.(4)Tumorigenesis and validation of mice expressing HPV18E6E7 fusion gene.The expression of HPV18E6E7 fusion gene and protein in tumor tissues of tumorigenic mice was verified by RT-PCR and WB.(5)Immunization effect of new vaccine: a tumor-bearing mouse model was constructed by using mouse tumor-forming cell lines stably expressing HPV16E6 and E7 genes,HPV18 and 58E6E7 fusion genes,and the pre-established AD-HPV16/18/58mE6E7 trivalent recombination was constructed.Adenovirus vector therapeutic vaccine immunized mice,observed by subcutaneous xenograft morphology,xenograft growth,tumor inhibition rate and survival of mice;serum antibody ELISA,ELISPOT,specific CTL killing and other humoral and cellular immunity The effect of the novel vaccine against HPV type 16,18,58 tumors was examined and evaluated.Results:(1)The expression of HPV16E6 and HPV16E7 in mouse cervical cancer TC-1 cells was verified by RT-PCR and Western Blot.(2)The U14 cell line(U14/LV-HPV18E6E7)stably expressing the HPV18E6E7 fusion gene wassuccessfully constructed,and it was confirmed by RT-PCR and WB experiments that the cell line stably expressed the HPV18E6E7 fusion gene in vitro and in vivo.(3)The immune effect of the novel trivalent vaccine against adenovirus was verified by animal experiments.C57BL/6 mice immunized with the vaccine can inhibit the growth of cervical cancer cell U14 in mice expressing HPV58E6E7 fusion protein,HPV16E6 and E7 protein,and HPV18E6E7 fusion protein,respectively,to a certain extent,and can produce effective humoral and cellular immunity,protecting immune mice from the attack of transplanted tumor.(1)In the experimental study of anti-graft tumor protection,the vaccine has basically the same effect on HPV58,HPV16 and HPV18 tumor cells.The incubation period of subcutaneous tumor formation in mice of the vaccine group was longer than that of the control group,and the difference was statistically significant(P<0.05).The tumor growth rate of the vaccine group was significantly slower than that of the control group,the average tumor weight was significantly lower than that of the control group,and the survival period was longer than that of the control group,the difference was statistically significant(P<0.05).(2)Humoral immune serum specific antibody detection experiment(ELISA experiment): The humoral immunity of the vaccine against HPV58,HPV16 and HPV18 tumor cells is basically the same,and the immunized mice can induce E6 and E7,HPV16 for HPV58,respectively.The serum specific antibody of E7 and HPV18E7 epitope peptides had the highest antibody titer of1:25600,which was statistically significant compared with the control group(P<0.05).(3)In the ELISPOT study,the cellular immunity of the vaccine against HPV58,HPV16 and HPV18 tumor cells was basically the same.The specific secretion of IFN-? effector T cells induced by HPV58E6 and E7,HPV16E7 and HPV18E7 epitope polypeptides has been detected in this experiment,and thenumber of ELISPOT of mice in all the vaccine groups was significantly higher than that of the control group,with statistically significant differences(P<0.05).(4)In the experimental study of cytotoxic T cell killing,the cellular immunity of vaccines against HPV58,HPV16 and HPV18 tumor cells was basically the same.The CTL response of AD-HPV16/18/58mE6E7 adenovirus vector vaccine against U14/LV-HPV58E6E7 cells,TC-1 cells and U14/LV-HPV18E6E7 cells was up to 33.050%,30.160% and 30.890%,respectively.Conclusion: In this study,we successfully constructed a murine cervical cancer U14 cell line stably expressing HPV18E6E7 fusion protein,and systematically verified that the novel trivalent therapeutic adenovirus vector vaccine AD-HPV16/18/58mE6E7 can be used in HPV16,18,58 tumor mice.Induction of effective humoral and cellular immunity,confirmed the immune protection and immunotherapy of the vaccine,can be used as a vaccine candidate for HPV16,18,58 related cervical precancerous lesions and cervical cancer.In addition,immunological experiments were conducted to further verify the immunogenicity of HPV58E6 and E7 HLA-A2 restricted CTL epitope antigen polypeptides screened by our research group at the early stage,and HPV58E6 peptide was selected as the preferred epitope antigen polypeptide of HPV58 for subsequent experimental studies.