Evaluation Of Immune Responses Induced By A Novel Human Papillomavirus Type 16 E7 Peptide-based Vaccine With Candida Skin Test Reagent As An Adjuvant In C57BL/6 Mice

Objective:Persistent infection of high-risk human papillomavirus(HPV)is a necessary cause for the occurrence of high-grade cervical intraepithelial neoplasia(CIN),vulvar intraepithelial neoplasia(VIN)and vaginal intraepithelial neoplasia(VaIN),which may develop to cervical cancer,vulvar cancer and vaginal cancer respectively.Three licensed prophylactic vaccines(Cervarix,Gardasil 4 and Gardasil 9)have been demonstrated to be able to provide efficient protection against HPV infection.However,they have little therapeutic activity for patients with pre-established HPV infections or HPV-associated lesions.Therefore,it is urgent to develop therapeutic HPV vaccines designed for controlling HPV persistent infection or HPV-associated lesions.Cell mediated immune responses are crucial for the clearance of human papillomavirus(HPV)infection and HPV-associated lesions.Activated CD8+T cells are critical effector cells in recognizing and killing HPV-infected or HPV-transformed cells.CD4+T cells provide help for priming the generation and maintenance of CD8+T cells as well as for tumors immunity.An ideal therapeutic HPV peptide-based vaccine should induce both a robust CD8+T-cell response as well as a CD4+T-cell response for ensuring their efficiency.In this current study,we designed a novel HPV peptide-based vaccine which includes HPV16 E7 peptides and Candin as an adjuvant.The immune responses induced by the vaccine were comprehensively evaluated.The results may provide a Candidate adjuvant of human vaccine for inducing cell mediated immune responses,and lay foundation for the further research on novel tumor therapeutic vaccines,including cervical cancer.Methods:1.Acquisition of peptideHPV16 E7 protein was 98aa in length.Three peptides(HPV E7 1-35,HPV16 E736-70,HPV 16 E7 71-98)covering the HPV16 E7 protein were produced at the GL Biochem(Shanghai)Ltd.(Shanghai,China).The purity of the synthesized peptides was over 95%.2.Mice and immunizationSix to eight-week-old female C57BL/6 mice were randomly divided into four groups,four mice per group,1)Candin alone group,2)E7 peptide alone group,3)Vaccine group,4)PBS negative control group.All mice were injected corresponding reagents and were boosted for additional two times with the same amount of reagents,and each boost was in a 3-week interval.3.Analysis of splenocyte proliferation in vitroSplenocytes of each mice were divided into three groups,containing cells alone(negative control),cells with peptide,and cells with concanavalin(ConA).Using MTS measured splenocyte proliferation.The absorbance was measured at 490 nm using BioTek Synergy-2 Multi Plate Reader.1.Analysis of HPV16 E7-specific CD8+and CD4+T-cell responsesSplenocytes,which were harvested in the 8th week,were treated with phorbol 12-myri state 13-acetate(PMA),ionomycin,brofeldinA(BFA),Cytofix/Cytoperm Solution,fluorescent labeled antibodies,then detected the percentages of CD4,CD8 and IFN-'y by a FACS Fortessa flow cytometer.2.Analysis of Thl and Th2 cytokine in serumWe used CBA kit to treat serum which was harvested from mice in each group at 2 weeks after the 3rd immunization.Using FACS Fortessa flow cytometer detected the concentration of IFN-?,TNF-?,IL-2,IL-4 and IL-5.3.Analysis of Thl,Th2,Treg cellsUsing the Foxp3 staining buffer kit and fluorescent labeled antibodies treated splenocytes which were harvest in the 8th week,then detected CD4,CD25,FoxP3,T-bet and GATA3 by a FACS Fortessa flow cytometer.7.Evaluation of humoral-mediated immune responsesIn order to assess the humoral immune response induced by the vaccine,we determined the titer of anti-HPV E7 IgG antibody in serum collected from mice at weeks 3,6,and 8 by ELISA.8.Statistical analysisSPSS 19.0 software was used for statistical analyses.Statistical significance was determined with one-way analysis of variance(ANOVA)followed by the Dunnett's test for group comparisons.Differences were considered to be statistically significant when the P values were<0.05.Results:1.Splenocyte proliferation in vitroSignificantly increased proliferation was shown in groups immunized with the vaccine and peptides when compared with the control group immunized with PBS.No increase was seen with the Candin alone group.2.Analysis of HPV16 E7-specific CD8+and CD4+T-cell responsesThe percentages of IFN-? producing CD8+T cells and CD4+T cells in splenocytes were increased in the vaccine group,which indicated that the vaccine induced the generation and activation of HPV-specific CD8+T cells and CD4+T cells.Candin alone induced a significant increase in IFN-? producing CD4+T cells.3.Induction of Thl and Th2 cytokines in serumMice immunized with Candin alone generated statistically significant increased IFN-? compared to control group but not with peptide alone and vaccine.The increases in IL-2 and TNF-?in the vaccine group were more noticeably,they were not statistically significant.Immunization of mice with the vaccine elicited a statistically significant increase in IL-5 but the increases were not significant for Candin alone or peptide alone4.Analysis of Thl and Th2 cytokine in serumSplenocytes of immunized mice with the vaccine and Candin alone showed statistically significant increases in the percentages of CD4+T-bet+cells compared with the control.No significant difference in the levels of CD4+GATA3+ and CD4+CD25+FoxP3+ was shown in the immunized groups compared to the PBS control group.5.Evaluation of humoral-mediated immune responsesSuccessive immunization induced intensely increased antibody as the endpoint titer was 1:400 at week 6 and was over 1:12,800 at week 8 in group immunized with the vaccine.Conclusion1.Candin alone induced Thl responses.2.A novel human papillomavirus type 16 E7 peptide-based vaccine with Candida skin test reagent as an adjuvant induced significant HPV-specific CD8+T-cell response.3.The vaccine induced HPV-specific Thl responses.4.The vaccine induced HPV-specific humoral immune response.