Development Of HPV Therapeutic DNA Vaccine And Animal Experiment

Human papillomavirus (HPV) infection is a significant risk for cervical carcinoma. Many studies have demonstrated that HPV is one of the independent risk factors in oral cancer. HPV DNA can be detected in25%HNSCC cases. The most frequently detected type is HPV16. Due to the different prevalence of HPV infection in OSCC in different places, this study first investigated the epidemiology of HPV infection in OSCC in Wuhan city.Researchers have proved that prophylactic vaccines could prevent HPV infection in those people who never infected with HPV. But they failed in clearing the established infection. Hence, development of therapeutic vaccine is necessary for treating HPV-associated cancers. In this study, we established two DNA vaccines, pCTLA4-E7E6and pE7E6, within the codon optimized genes. The effect of these two DNA vaccines has been investigated.This research included three parts:Part1, Prevalence of HPV infection in OSCC:a case-control study in Wuhan, ChinaSpecimens were collected from both tumor tissues of case patients and normal control patients who were seen for3rd molar removal between2009and2013. All tissue samples were collected and stored at-80℃immediately until use. DNA extraction of samples was accomplished with QIAamp Extraction Kit within48h after specimens were obtained. Samples were analyzed by PCR performed with GP5+/GP6+primers (5’-TTTGTTACTGTGGTAGATACYAC-3’/5’-GAAAAATAAACTGTAAATCATAT TC-3T) that targeting conserved sequences within the capsid gene LI. The positive PCR products were then sequenced by Sangon Biotech. Analyses were conducted with SPSS20.0for Mac. Conclusions:The overall prevalence rate of HPV infection was higher in case patients than in control subjects (27.5%versus2.9%, respectively).Part2, Construction and detection of HPV therapeutic DNA vaccine1. Construction of HPV therapeutic vaccineTo reduce transformation activity, amino acids C24and E26of HPV16E7were modified to disrupt the L-X-C-X-E (where X is any amino acid) motif, through which E7binds to pRb. In addition, C91of the E7region were modified to reduce transformation activity. In the HPV16E6region, amino acids C63and C106were modified to disrupt the C-X-X-C zinc finger binding motifs and protect p53from degradation. All wild type amino acids were mutated to glycine. Codons of the modified E7E6fusion gene were further optimized with humanized codons to enhance expression and immunogenicity in humans. The designed fusion gene fragment was synthesized by Invitrogen Corporation and cloned to pVAX1vector (Invitrogen, USA) by Kpn1and NotI restriction sites. The resulting plasmid was named pE7E6.The pCTLA4-E7E6vaccine was constructed by cloning the E7E6fragment into pGJAP/VAX1at Kpnl and Notl sites. The resulting plasmid contains the signal peptide and extracellular domains of CTLA-4, the hinge and Fc region of human IgG1, and the E7E6fusion gene. Corresponding plasmids carrying wild-type (wt) E7E6were also constructed and named pwCTLA4-E7E6and pwE7E6. Two plasmids expressing either wild-type E7or E6were constructed and named pwE7or pwE6, respectively. All plasmids were verified by DNA sequencing. Conclusions:plasmid pCTLA4-E7E6, pE7E6, pwCTLA4-E7E6, pwE7E6, pwE7, pwE6were successfully constructed.2. Detection of the mechanism of plasmid HPV vaccineThe constructed plasmid HPV vaccines were transfected into293cells by using Lipofectamine2000. The total proteins were collected to analyze the expression of protein E7, E6, p53and protein Rb. Also, the proteins in the supernatant were collected to analyze the concentration of the fusion protein. The western blot method was adopted to detect those proteins. Then, FACS method was adopted to analyze the binding ability of fusion protein in the supernatant to DC2.4cells. Conclusions:the target proteins can be expressed correctly. No degradation of p53or Rb were observed. The binding assay showed the fusion protein secreted in the supernatant could bind the DC2.4cells successfully.Part3, Animal experiment Plasmid pCTLA4-E7E6, pE7E6, and pVAX1for in vivo application were vacuum dried and redissolved in normal saline (NS) to obtain the final DNA concentration at1μg/μl.1, Therapeutic immunizationThirty C57BL/6mice were randomly divided into four groups. TC-1cells (2×105) in100μL of PBS were subcutaneously injected into the left flank of all mice. Group pCTLA4-E7E6(n=8) or Group pE7E6(n=7) was intramuscularly immunized with100μg of pCTLA4-E7E6or pE7E6on the left leg on day10, respectively. Group pVAX1(n=8) and Group PBS (n=7) were respectively immunized with pVAX1and PBS as controls on day10. All animals were boosted on day17. Tumor size was recorded twice or thrice a week. On day38, all mice were sacrificed, and spleens were harvested for CTL assay. Serum samples were collected on days0,24, and38. Results: pCTLA4-E7E6induced stronger immune responses than pE7E6in therapeutic immunization.2, Preventive immunizationA total of32C57BL/6mice were randomly divided into four groups. Group pCTLA4-E7E6(n=8) or Group pE7E6(n=8) group was intramuscularly immunized with100μg of pCTLA4-E7E6or pE7E6on the left leg on days0and14, respectively. Group pVAX1(n=8) and Group PBS (n=8) were respectively immunized with pVAX1and PBS as controls. On day28, the mice were challenged with6×104TC-1tumor cells in100μL of PBS. Tumor size was recorded twice or thrice a week. The serum samples were collected on days0,14,28, and60. The spleens were immediately harvested after being sacrificed on day80. pCTLA4-E7E6induced stronger immune responses than pE7E6in preventive immunization.