Construction Of HPV58mE6E7 Fusion Gene Vaccine And Detection Of Its Immunogenicity And Antitumor Response In Mice

Cervical carcinoma is one of the common gynecological malignancies, which incidence is the second in the female malignant tumor. According to statistics, there are about 50 million new cases of cerical carcinoma in worldwide annually, including 80% of cases will take place in developing countries. Our new cases a year, accounting for 13.15 million, approximately 28.8% of total, new cases of cerical carcinoma in the world Nearly 10 years, epidemiological data based with large cases shows:HPV DNA exists in 99% of cerical carcinoma cases. HPV infection is the primary factor which results in cervical carcinoma and CIN. Almost all cerical carcinoma cells exist HPV DNA and protein. HPV transformation protein can be used as one kind of antigen to stimulate the immune response against HPV. People hope through HPV vaccine to prevent and treat cerical carcinoma. No cross immunoprotection between various HPV genotypes, pertinence is necessary to develop HPV vaccine. Without detailed epidemiology reports about guangxi HPV infection, developing HPV vaccine is blind. To enable pertinence of Guangxi HPV vaccine, we researched the region of HPV genotype in Guangxi and provided a credible information as a strong basis of Guangxi HPV vaccine developmentWe adopted PCR and sequencing methods to test 135 cases of diagnosed cervical carcinoma tissue DNA. The results showed:98.52% of 133 cases cervical carcinoma patients infected HPV. Patients infected HPV16,18,31,33,35,45,52,58,59 types were 76,17,125 0,0,1,2,63,5, respectively. Infection rates of the 9 types are 93.98%, 57.14%,12.78%,0,0,0.75%,1.50%,47.37% and 3.76%, respectively. Type 16,18 and 58 rank 1,2,3. Quantitative detection by real-time Q-PCR, average virus load of types HPV 16,18 and 58 are 7.89 x 109,9.55 x 103 and 2.38 x 10, respectively. Virus load of type HPV 16 is obviously higher than types 18 and 58 (P<0.05), and the difference of virus load of types HPV18 and 58 was no significant values (P>0.05).Because infection rate of type HPV58 in guangxi is very high, developing type HPV58 vaccine is very necessary. As transformation protein of type HPV58, E6 and E7 gene can integrate HPV DNA in HPV58(+) tumor cells chromosomes and continuously express E6 and E7 protein, which make host cells transform to malignant direction. However, E6 and E7 protein as persistent viral antigens, it can stimulate body to produce strong CTL to HPV58. Therefore, HPV58 E6, E7 as two ideal tumor antigens, are suitable for immunotherapy and prevention for HPV58 (+) cerical carcinoma and CIN, but the prerequisite is that you must eliminate transformation activity of E6 and E7 gene to ensure the safety of vaccine. Before constructing the vaccine, we designed six mutations in the transformation sites of E6 and E7 gene to eliminate transformation active while retaining immunogenicity. Having removed HPV58mE6E7 fusion gene transformation, human IgG Fc was fused together with HPV58mE6E7 to form HPV58mE6E7-Fc, which plus signal peptide in the front and membrane anchor protein GPI in the end. GP can anchor and express the tumor antigen in the cell membrane. Finally, sig-HPV58mE6E7Fc-GPI was inserted eukaryotic expression vector PVAX1-IRES-GM/ B7 which can express GM-CSF and B7.1 fusion protein. PVAX1-HPV58mE6E7FcGB vaccine can express the fusion antigen and GM-CSF and B7.1 synchronously, which can enhance the collaborative immune action of the vaccine. Then, we constructed mice melanoma cell line B16-HPV58E6E7, which can stablely express HPV58E6E7 fusion gene. HPV58E6E7-GST fusion protein was be obtained by the way of prokaryotic expression and purify.We immuned C57BL/6 mice with PVAX1-HPV58mE6E7FcGB vaccine by intramuscular injection plus local electrical pulses Results indicated that the immuned mice were protected from HPV58E6E7 (+) tumor cells attack. mice subcutaneous tumor formation time in vaccine group was later than in control group (P＜0.05). Tumor of vaccine group mice growed significantly slower than in control group, so average tumor weigh less than in control group (P< 0.05). This resulted survival time in vaccine group was longer than control group (P<0.05). Results also confirmed that the vaccine immuned mice can induce specific serum antibody, which can reach maximal 1:25600, compared with the control group (P＜0.05). ELISPOT can detect specificial T cells of vaccine immuned mice which activated by HPV58E6E7-GST recombinant protein antigen Numbers of spots in vaccine group are higher than in single antigen group (P<0.05), indicating that HPV58mE6E7 fusion with adjuvants as human IgG Fc, GM-CSF and B7.1 can obviously increase immunogenicity of HPV58mE6E7and enhance its immunical effect., We also detected special CTL response of the mice immuned by PVAX1-HPV58mE6E7FcGB vaccine by LDH way and obtained the highest data of 45%. Thest datas proved that the mice immuned by the vaccine r can produce effective humoral immune and cellullar immunity and protect the mice against B16-HPV58E6E7 cells(1-2 x 105)attack.To improve the effect of HPV58 vaccine, the following vaccine named PVAX1-HPV58mE6E7Fc-hIL12 has been structured and succeed in expressing in eukaryotic cell.In brief, we constructed PVAX1-HPV58mE6E7FcGB gene vaccine on the basis of Guangxi HPV epidemiology investigation. This vaccine can produce effective humoral and cellular immunity in the immumed mice and be used to treat HPV58 (+) cerical carcinoma and CIN as a candidate vaccine. It can show some clinical value by removing the tumor cells infected by type HPV58 and preventing the development of the disease.