New Hpv Therapeutic Vaccine Anti-tumor Effect And Mechanism Studies

The human papillomavirus (HPV) 6, 11, 16 and 18 are the common HPV types associated with various infections in humans. Among them, HPV-6 and HPV-11 belong to benign types and HPV-16 and HPV18, which are causative agents in the pathogenesis of cervical cancer or its precursor lesions, such as cervical intraepithelial neoplasia (CIN) besides condyloma acuminatum (CA), are considered as"high risk"or malignant HPV types. Some therapies such as interferon and antivirus drugs are used to treat the various infections of HPV with little effect. It is urgently needed that specific immunotherapy is developed to effectively clear HPV infection and prevent the infection from recurrent CA and canceration.Recently, the incidence of STDs (sexually transmitted diseases) is increasing with years. Genital transmission and infection have shown a rising trend. Insufficient activation of immune response against human papillomavirus (HPV), especially the cellular immune, will cause inability to clear the pathogen, leading to the chronicity of the HPV infection. High-risk HPV types, including type 16, are the most common causative pathogens of condyloma accuminatum, vaginal cancer and cervix cancer. HPV-DNA has been detected in almost 100% of the cervix lesions such as dysplasia, precancerosis and cancers. In recent years, HPV infection has become one of the most important healthy problems worldwide. Clearing the HPV infection, especially the high-risk HPV types infections, could not only eliminate the sexually transmission of HPV infection, but also reduce the incidence of associated cervical tumors of women.Clinic treatments widely used presently are limited to excision of the condyloma and specific/non-specific immunotherapy which may have a short-term effect but can not clear the pathogen and avoid the recurrence of HPV infection. With the development of research on HPV viral antigen, therapeutic vaccine has become a hot pot of the research on therapy of HPV and its associated tumors.Previous investigation reveals that specific cellular immunity mediated by CTLs plays a very important role on immune responses against viral infection and tumor. Through the T cells receptors (TCRs), CTLs can recognize the MHC class I-associated peptides complex on surface of the antigen presenting cells(APCs)such as malignant cells.As a result, CTLs are activated to kill the target cells by various mechanisms.This thereapeutic vaccine in the paper is a fused protein which link antigen E7 with HSP and HBcAg, and the protein developed a virus-like particle, which led to potent immunogenicity, enhangced cell immunity, and killed the cells infected with HPV, finally met the need of therapy. The paper intended to study the antitumor effect of VR111 compared with various components and to explore the immune mechamism of VR111.The present work comprises by two parts, in the first part, the antitumoractivity was studied in the mice with tumor. In this part, the antitumoractivity of VR111 was compared with various components. Furthermore, the antitumoractivity of coadministration of VR111 and cis-diamminedichloroplatinum was also studid. In the other part, the immune mechanism of VR111 was studied.In the first part, we observed survival rates in the study and measured the sizes of tumor. All the data were analyzed to access the antitumor effect of the vaccine in vivo. The results showed VR111 has an effective antitumor protection and this protection showed a good dose-response relationship. Antitumor effction could be showed in several respects: firstly, the vaccine could inhibite the growth of tumor, several repeated experiments showed a good reproducibility, inhibition rate could were more than 80%; secondly, the vaccine could raise the survival rate; thirdly, the vaccine could prolong live time of the mice with TC-1 tumor. Furthermore, the result proved coadministration of VR111 and cis-diamminedichloroplatinum could boost the antitumor effect, inhibition rate of tumor growth was 94.4% and survival rate was 90%. The coadministration showed an obvious synergism effect.The second part was divided into five subsections. The first subsection: the effect of the vaccine on lymphocyte immunity activity. In the subsection, 3H-TDR incorporation assay was used to study the splenic lymphocytes proliferation effect of the vaccine and other control components and ELLISA assay was used to study the effect on the externalization activity of different cytokine of splenic lymphocytes. The vaccine could promote the lymphosytes proliferation effect. Proliferation indexs after injection of different dose of vaccine were 10.8±2.2, 24.0±1.0, 31.8±3.7. The result showed a good dose-response relationship. Furthermore, fusion protein of HSP and E7 also showed a potent lymphosytes proliferation effect, the effects were not observed in other control components. The vaccine could boost the expression of Th1 cytokines, including IL-2 and IFN-γand had no effects on Th2 cytokines, including IL-4 and IL-10. E7 could enhance the expression of IFN-γ, but it had no effects on interleukin family cytokines, including IL-2, IL-4 and IL-10. The fusion protein of HSP and E7 also induced the expression of IL-2 and IFN-γand the induction was more potent than E7 protein. The results showed HSP could inhanc the activity of lymphocytes and induced the expression of Th1 cytokine. The mixture of HSP,E7 and HBcAg could induce the activity of lymphocytes.The second subsection: the effect of the vaccine on CD8+T lymphocyte immunity activity. In this subsection, flow cytometry was used to measure the amount of CD8+T lymphocyte after induction and the ELISPOT assay was used to determine the externalization of IFN-γof CD8+ T lymphocytes. The results showed the ratio of CD8+ T lymphocyte in the splenic lymphocyts reduced gradually with the increasment of dosage of VR111. There wre no obvious variances in HSP-E7 group. The vaccine could obviously promote the externalization of IFN-γof CD8+ T lymphocytes. The result showed a good dose-response relationship. Furthermore, aluminium adjuvant could promote the he externalization of IFN-γof CD8+ T lymphocytes.The third subsection: the role of CD4+ T lymphocytes and CD8+ T lymphocytes in antitumor effect in vivo. In this subsecton, anti-CD4 or anti-CD8 monoclonal antibody was used to deplet the CD4+ T lymphocytes and CD8+ T lymphocytes in mice. Flow cytometry was used to evaluat the Ab depletion efficiency. The result showed more than 90% of CD4+ T lymphocytes and CD8+ T lymphocytes have been depleted. In vivo studies using mice with targeted depleted of CD8+ or CD4+ lymphocyte subsets demonstrate that tumour regression following VR111 immunization is CD8-dependent and CD4-independent.The fourth subsection: the antitumor activity of cytotoxic T lymphocytes in vivo and in vitro. In this subsecton, 51Cr release assay was used to study the cytolytic activity of splennic lymphocytes targeted at TC-1 tumor cell and adoptive T cell transfer experiment were used to study the antitumor effect of cytotoxic T lymphocytes. The restimulated splenocytes from mice immunized with VR111 exibited a high cytolytic activity. The activity increased with the increasement of dosage immunized with VR111. The restimulated splenocytes from mice immunized with HSP-E7 exibited a lower cytolytic activity than VR111. In contrast, the restimulated splenocytes from mice immunized with E7, HBcAg-E7, or an admixture of HSP, HBcAg and E7 exhibited very low to no cytolytic activity. These results demonstrate that the covalent linkage between HSP, HBcAg and E7 is necessary for priming potent E7-specific CD8+ T cell responses. The result of adoptive T cell transfer experiment showed splenocytes from mice immunized with VR111 exibited a good antitumor activity than splenocytes from mice immunized with HSP-E7. The results indicated that tumour regression following VR111 immunization is CTL-dependent.The fifth subsection: the immunogenicity of VR111 in vivo. In this subsecton, ELISA was used to determine the concentration of antibody in serum. The results indicated that VR111 could induced a strong humoral immunity and produce a high titer of VR111-specific antibodies and the high level antibodies in vivo were maintained for a long period.In summarize, the major advancements are as follows:1) This novel HPV therapeutic vaccine showed a good antitumor effect which could be showed in several respects: firstly, the vaccine could inhibite notably the growth of tumor; secondly, the vaccine could raise the survival rate; thirdly, the vaccine could prolong live time of the mice with TC-1 tumor.2) Coadministration of VR111 and cis-diamminedichloroplatinum could boost the antitumor effect. The coadministration showed an obvious synergism effect .3) VR111 could enhance the cellular immuniologic response, and promote the proliferation and activity of splenic lymphocytes, including CD8+ T lymphocyte, differentiate into CTL.4) VR111 could enhance the humoral immuniologic response, which could be related to the prophylaxis of infection for HPV. It remained to be reasearched between humoral immuniologic response and antitumor effect.