HPV6 Therapeutic Vaccine For The Treatment Of Genital Warts

Genital warts (GW) are benign epithelial tumours caused by infections with human papillomavirus (HPV). Clinically apparent genital warts affect about 1% (approximately 1.4 million) of the sexually active population (15-49 years) in the United States. In China genital warts have been the second most common type of sexually transmitted diseases (STD). It has been reported that low-risk HPV types 6 and 11 are found in more than 90% of genital warts. As Genital warts commonly recur after treatment, it will bring patients psychological stress and high cost of treatments. There are several typical treatments that can be used to treat genital warts such as drug therapy(including imiquimod, podophyllotoxin and trichloroacetic acid) and physical ablation (including cryotherapy, excision, electrosurgery and laser surgery). Although they may be very effective for individual, they are difficult for them to clear the HPV infection. So novel kinds of treatment need to be developed.The HPV therapeutic vaccine can induce a strong HPV specific immune response and the clearance of HPV infection.The body can develop HPV-specific T remember cell and induce a sustainable anti-HPV effect.Therefore HPV therapeutic vaccines have a promising furture in the therapy of genital warts. Therapeutic HPV vaccine strategies have focused primarily on stimulating the production and activation of T cells by targeting E6 and/or E7 proteins. At the same time, HPV L2 protein was proved to be able to induce cross-neutralising antibody. So These three proteins were chosen as the target for genital warts therapeutic vaccine.The design of this study was based on that:The former research of our group has proved that the recombinant protein HPV6BL2AE7E6 was a promising candidate therapeutic vaccine for the treatment of genital warts. In this study high expression level of the protein will be achieved through codon optimization. In addition, we set three kinds of immune procedure that is, four dosages of the vaccine will be given with intervals of day 1,3,7 respectively, named 1-2-3-4,1-4-7-10,1-8-15-22. In every immune procedure,we design five groups to test the immune response elicit by protein plus differernt adjuvants such as CpG and Poly(I:C). After the evaluating of HPV-specific cellular immune responses and humoral responses by ELISPOT and ELISA, the immuned mice will be challenged with B16-HPV6E7 tumor cells to see wether they will be protectd from tumor.The main results are as follows:1. The genes of recombinant protein were optimized and high expression level of the protein was obtained in E.coli.2. The recombinant protein was expressed, identified and purified.3. The results of the evaluation for immune response showed us that the response to E6 was significantly stronger than that to E7. The protein plus with CpG could elicte the strongest cellular and humoral response in 1-2-3-4 immune procedure. The immune response was significantly enhanced when protein was plus with Poly(I:C).4. The HPV6E7 stably expressing tumor cell line was constructed and identified and the HPV6 mice tumor model was established.5. The mice immuned with different procedure were challenged with E7-expressed B16 cells. Data showed that sixty days after the challenge,the group of protein plus CpG in 1-2-3-4 could protect 50% mice from the tumor cells. According to the statistic analysis of volume of tumor, protein plus CpG or Poly(I:C) in the three procedures could all inhibit the growth of tumor significantly.In conclusion,we expressed the protein and evaluated the differences of immune response elicted by the protein plus with CpG or Poly(I:C) in three kinds of immune procedure both in vivo and in vitro. Data showed that HPV6BL2△E7E6 recombinant protein was an ideal candidate of therapeutic vaccine for the treatment of genital warts.