The Effects Of Tumor Microenvironment Regulation By Active Immunization Against TGF-β1 And Application Of Nucleic Acid And Nanoparticle Adjuvants On A Therapeutic HPV Vaccine

Cervical cancer is one of the serious diseases that endangers women’s health,and the main cause of it is human papillomavirus(HPV)infection.Although there are three commercial prophylactic HPV vaccines,which can protect about 90%of cervical cancer caused by HPV infection,its have no treatment effects on the persistent infection of HPV,Squamous intraepithelial neoplasia and cancer.Therefore,the development of effective therapeutic HPV vaccine has great significance for the clinical treatment of HPV related tumors.TGF-β1 is a key immunosuppressive molecule in tumor microenvironment and can induce the differentiation of naive T cell into regulatory T cell(Treg)which significantly inhibit the differentiation,proliferation and function of T cell through various mechanisms.TGF-β1 promotes the differentiation,proliferation and migration of marrow derived immunosuppressive cells(MDSCs).In addition,TGF-β1 can promote the growth and metastasis of tumor by promoting tumor cell proliferation,angiogenesis and stimulating the expression of extracellular matrix.Except for Treg cells,the immunosuppressive cells in tumor microenvironment including MDSCs,tumor associated macrophages(TAM),and fibroblasts can also secrete TGF-β1.Therefore,active immune intervention of TGF-β1 has a promising potential to antagonistic tumor immunosuppressive microenvironment and promote anti-tumor immunity.Toll-like receptor(TLR)3/9 and STING agonist cGAMP as the potential vaccine adjuvant shows a strong ability to induce Thl/CTLs biased cellular immune response.Nanoparticles can improve the efficiency of antigen uptake and processing by DCs,and enhance the migration of antigen to lymph nodes.In addition,nanoparticals can improve the in vivo stability of antigens and nucleic acid adjuvants and promote their simultaneous delivery to DCs,which is conducive to the efficient activation of immune responses.Therefore,the combined application of nucleic acid adjuvants and nanoparticle has the potential to induce strong antigen-specific anti-tumor cellular immunity.Due to the tumor immunosuppressive microenvironment and low efficacy of Thl/CTLs response,this study aims to explore the effective strategies of enhancing the anti-tumor effect of therapeutic vaccine through targeting TGF-β1 by active immunity to antagonize the tumor immunesuppressive microenvironment and the application of novel nucleic acid and nanoparticle adjuvants to stimulate anti-tumor immunty.In the study of targeting TGF-β1,mice were immunized with the recombinant hepatitis B core antigen(HBcAg)virus like particles(VLPs)presenting TGF-β1 epitope by prophylactic strategy to induce TGF-β1 specific antibody response,and then subcutaneously inoculated with TC-1 tumor cells.When the tumor reached 5-6 mm,mice were immunized with the HBcAg VLPs therapeutic HPV vaccine which carrying HPV16 E7 peptide by therapeutic strategy.The results showed that active immune regulation of TGF-β1 could significantly enhance the ability of tumor inhibiton,decrease the proportion of IgG1/IgG2a and the expression level of IL-4,and increase the level of IFN-y which induced by therapeutic HPV vaccine inoculation.ELISPOT showed that the level of lymphocytes secreting IFN-γ was significantly increased.Accordingly,flow cytometry showed that the number of CD4+IFN-γ+ and CD8+IFN-y+ T cells were significantly increased,while the number of CD4+ Foxp3+ Treg cells were significantly decreased.At the same time,angiogenesis and VEGF gene expression in tumor tissue were significantly inhibited,and the gene expression level of IFN-y and TNF-a and the effector T cell chemokine CCL19 was up-regulated,while the MDSCs chemokine CXCL1 was down-regulated detected by Real time PCR.Moreover,pSmad3，the key molecule of TGF-β1 signaling pathway was decreased.In the study of nucleic acid and nanopartical adjuvants,cationic liposome DOTAP combined with nucleic acid adjuvant CpG,Poly I:C and cGAMP significantly enhanced the inhibitory ability of tumor growth induced by therapeutic VLPs vaccine,and increased the level of lymphocytes secreting IFN-y and CD8+IFN-γ+,CD8+CD107a+,CD4+IFN-γ+ effector T cells and CD8+CD44+ CD4+CD44+ memory T cells,and decreased the level of CD4+Foxp3+Treg and CD11b+Gr-1+ MDSCs immunesuppresive cells.PLGA/DC-chol/HA nanoparticles were further used as delivery carriers of E7 peptide epitope and nucleic acid stimulator,and similar results were obtained.In vitro experiments showed that the combination of nanoparticles and nucleic acid adjuvants,especially combined application of nucleic acid adjuvants,could effectively enhance the expression of CD40,CD80,CD83.CD86,MHC-I,and promote BMDCs to secrete the proinflammatory cytokines TNF-a,IL-2 and IL-12.In vivo experiments,the combination of nanoparticles and nucleic acid adjuvants,especially combined application of nucleic acid adjuvants,significantly inhibited the tumor growth in both preventive and therapeutic strategies,and increased the levels of CD8+IFN-γ+、CD8+CD107a+、CD4+IFN-γ+.CD8+CD44+,CD4+CD44+ T cells and decreased the level of CD11b+Gr-1＋MDSCs cells which suggested that CTLs/Thl biased anti-tumor immune response can be effectively stimulated.The results strongly suggested that active immune regulation of TGF-β1 and the combination of nanopaticles and nucleic acid adjuvants,especially adjuvants combination,significantly enhance the anti-tumor efficacy and immune response induced by therapeutic HPV vaccine.It is a potential combined immunization strategy and provides new ideas for the development of therapeutic tumor vaccine.