Transformation activity and antigenicity of the human papillomavirus type 58 E6E7 fusion gene mutant

Abstract

To develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity. The E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay. Sequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks in the nude mice of experimental and negative control groups, but among the 10 mice of positive control group tumor was formed in 6 mice. Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine, the antibody titer was 25 600, and specific immunity spots were 218.8 ± 34.4, significantly higher than that in the control group. The fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity. HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.