Construction And Immunological Characteristics Of The Recombinant Literia Monocytogenes Expressing Codon-Optimized E7Gene Of HPV16

Cervical cancer is the main cause of high mortality in women in developing countries and serious impact on worldwide women’s health. Persistent infection with human papillomavirus (HPV) is the most important etiological factor in cervical cancer, over99%of cervical carcinomas harboring HPV DNA. The HPV E6and E7proteins can interact with each other and are believed to sustain the malignant state by perturbing the cell cycle regulatory proteins, thereby preventing cell cycle arrest and apoptosis. Thus, both proteins can be ideal targets for immunotherapy. Now, HPV prophylactic vaccines were produced and can hold promise to reduce the worldwide incidence of cervical cancer. However, limitations in current HPV vaccine strategies make the development of HPV therapeutic vaccines for the treatment of HPV-associated lesions particularly important.Listeria monocytogenes is a facultative intracellular bacterium that enters professional antigen presenting cells, presents passenger antigens to the major histocompatibility complex class Ⅰ and Ⅱ pathways, then elicits of CD4+and CD8+T-cell-mediated immune responses. It was demonstrated that attenuated L.monocytogenes as a novel live vaccine vector, could induce strong protective immune responses, and shows effective antitumor immunotherapeutics.In this study, using LM4as a delivering vector, two attenuated L.monocytogenes strains LM4△hly::E7-1and LM4△hly::E7-3expressing codon-optimized HPV16E7gene were successfully constructed. The growth characteristics, stability, hemolysis activity and immune reactivity of recombinant strains were further analyzed. At the same time, the safety, therapeutic efficacy and anti-tumor mechanism were studied in a tumor-burdened murine model.1. Construction of recombinant attenuated Listeria monocytogenes strains expressing codon-optimized of HPV16E7geneFirstly, codons in HPV16E7were replaced by Listeria monocytogenes preferent ones and the optimized E7was named E7-1, based on this, amino acids24and26of E7-1protein were replaced by glycine(G) respectively, the obtained gene was named E7-3. Then using LM4as parental strain, codon-optimized of HPV16E7genes were inserted downstream of Phly promoter-signal sequence by the method of homologous recombination, and successfully constructed two attenuated L.monocytogenes strains LM4△hly::E7-1and LM4△hly::E7-3. After analyzing the extracted secretion proteins of these two strains in Western blot assay, it was shown that66kDa fusion protein was detected using an anti-E7monoclonal antibody, and indicated that the expressed proteins had good immune reactivity. Furthermore, the result of hemolytic test verified that the recombinant bacteria still kept the ability of expressing LLO, but the hemolysis titers of LM4△hly::E7-1and LM4△hly::E7-3were lower than that of LM4. Moreover, results of stability test indicated that E7-1and E7-3genes were stable in the genome of two recombinant strains. The growth curves of both recombinant strains were no significant difference comparing with LM4, accounted for that E7-1or E7-3gene inserted into the LM4genome had no affect to the growth of bacteria. The successfull constructed of LM4△hly::E7-1and LM4△hly::E7-3laid a solid foundation for further research.2. The therapeutic efficacy evaluation of LM4△hly::E7-1and LM4△hly::E7-3in a tumor-burdened murine modelFirstly, the safety of LM4△hly::E7-1and LM4△hly::E7-3was evaluated. After mice infected with two recombinant strains respectively, the bacteria load in spleens and livers were determined at different time points, the dynamic experiments indicated that the bacteria completely removed on day7. Histological sections revealed that a small amount of lymphocytes infiltration existed in the livers of LM4△hly::E7-1or LM4△hly::E7-3immunized mice, and neutrophilic infiltration focal liver was visible around the central vein. Similarily, the spleens had no obvious pathological changes. However, the liver cells of LM4△hly control mice had light granular degeneration and vesicular degeneration, splenic white pulp inside structure of splenic corpuscle increased. All those results verified that the recombinant bacteria LM4△hly::E7-1and LM4△hly::E7-3were high safe.A tumor-bearing model was set up in C57BL/6mice, then the therapeutic effects of recombinant bacteria LM4△hly::E7-1and LM4△hly::E7-3were determined after primary and secondary immunization via subcutaneoccs route. The results showed that the tumor of LM4△hly group and PBS group gradually grew as time went by, and all the mice of control groups died up to36days. But for the vaccination groups, part of the mice tumor completely eliminated and others grew very slowly. LM4△hly::E7-1group showed more significant therapeutic effect than LM4△hly::E7group. In addition, sandwich ELISA assay results showed that levels of IFN-y was higher than IL-4, indicated that immune responses elicited by recombinant bacteria were biased towards Thl-type. LM4△hly::E7-1group could induced higher levels of IFN-y than LM4△hly::E7group (P<0.05), while LM4△hly::E7-3group and LM4△hly::E7group had no significant difference. The results of lymphocyte proliferation assay showed that with the stimulation of E749.57peptide, LM4△hly::E7-1group could induced significant lymphocyte proliferation comparing with LM4Ahly::E7group (P<0.05) in ex-vivo, while LM4△hly::E7-3group and LM4△hly::E7group had no significant difference. What is more, the vaccine strains could induce the strong E7specific CTL activity in tumor-burdened mice, the CTL effect of LM4△hly::E7-1group was higher than those of other two immunization groups, while the PBS group and LM4△hly group did not produce E7specific CTL activity.