宫颈癌是严重危害女性健康的疾病之一，而人乳头瘤病毒(HPV)的感染是导致宫颈癌的主要因素。虽然目前已经有三种预防性HPV疫苗批准上市，能够保护约90%的HPV感染引起的宫颈癌，但其对已经建立的HPV持续感染、癌前病变及癌症并没有治疗效果。因此，研发有效的治疗性HPV疫苗对于临床治疗HPV相关肿瘤具有重要意义。

       转化生长因子-β1（TGF-β1）是肿瘤微环境中关键的免疫抑制分子，其能够诱导初始T细胞分化为调节性T细胞（Treg），分化的Treg细胞通过多种机制显著抑制效应T细胞分化、增殖及功能。TGF-β1促进骨髓来源免疫抑制细胞（MDSCs）分化、增殖及向肿瘤迁移。此外，TGF-β1也可促肿瘤细胞增殖、促肿瘤血管生成、刺激细胞外基质表达等促进肿瘤的生长与转移。除了Treg细胞，肿瘤微环境中的免疫抑制细胞MDSCs、肿瘤相关巨噬细胞、以及成纤维细胞等都能够表达TGF-β1。因此，靶向TGF-β1进行干预，具有拮抗肿瘤免疫抑制微环境，促进抗肿瘤免疫的潜力。

        Toll-样受体(TLR) 3/9以及STING激动剂cGAMP显示了强效的诱导Th1/CTLs偏向的细胞免疫应答的能力，是有潜力的肿瘤疫苗佐剂；而纳米材料有助于抗原被DCs高效摄取、加工以及抗原向淋巴结迁移提高DCs激活效率，此外，纳米材料可提高抗原与核酸免疫刺激物的体内稳定性并促进两者向DCs的同时递送，利于高效激活免疫应答。因此，核酸佐剂与纳米材料的联合应用，具有诱导强的肿瘤抗原特异细胞免疫的潜力。

        目前肿瘤疫苗面临巨大挑战其中重要的原因是肿瘤抑制微环境对Th1/CTLs的抑制以及疫苗未诱导强的Th1/CTLs应答，因此本研究拟通过抗细胞因子主动免疫靶向调控TGF-β1以拮抗肿瘤免疫抑制微环境，以及应用新型核酸与纳米材料佐剂以刺激抗肿瘤免疫这两个方面，探讨提高治疗性疫苗抗肿瘤作用的有效策略。

       在靶向TGF-β1的研究中，基因重组获得的呈现了TGF-β1抗原肽的乙肝核心抗原（HBcAg）病毒样颗粒（VLPs）经预防性策略免疫小鼠，激发TGF-β1特异的抗体应答，然后皮下接种TC-1肿瘤细胞，当肿瘤长至5-6 mm大小时，以治疗性策略进行携带了HPV16 E7肽的HBcAg VLPs治疗性HPV疫苗的免疫干预。肿瘤大小监测显示，抗TGF-β1主动免疫显著增强了治疗性HPV疫苗接种对肿瘤生长的抑制；ELISA 表明IgG₁/IgG_2a的比例显著降低，IFN-γ水平升高而IL-4降低，提示Th1应答获得增强；ELISPOT 结果表明，分泌IFN-γ的淋巴细胞水平显著提高，相应地，流式分析显示小鼠CD4⁺IFN-γ⁺与CD8⁺IFN-γ⁺效应细胞数目显著增加，而CD4⁺Foxp3⁺ Treg细胞数目显著降低；同时，肿瘤组织血管生成及VEGF基因水平表达受到显著抑制；此外，Real time PCR检测表明IFN-γ、TNF-α及效应T细胞趋化因子CCL19基因表达水平上调，而MDSCs细胞趋化因子CXCL1基因水平下调；进一步的检测证实，TGF-β1信号通路激活关键分子pSmad3的水平降低。

       在核酸与纳米材料佐剂研究中，阳离子脂质体DOTAP联合核酸佐剂CpG、Poly I:C和cGAMP显著促进了治疗性VLPs疫苗激发的肿瘤生长抑制作用，提高了分泌IFN-γ的淋巴细胞、CD8⁺IFN-γ⁺、CD8⁺CD107a⁺、CD4⁺IFN-γ⁺效应T细胞、以及CD8⁺CD44⁺、CD4⁺CD44⁺ 免疫记忆T细胞水平，降低了免疫抑制的CD4⁺Foxp3⁺Treg细胞与CD11b⁺Gr-1⁺ MDSCs水平。进一步以 PLGA /DC-chol/HA新型纳米颗粒作为E7肽表位及核酸刺激物的递送载体，获得了类似的结果。体外实验显示，纳米颗粒与核酸佐剂尤其核酸佐剂联合应用有效增强了BMDC表面成熟标记CD40、CD80、CD83、CD86、MHC-Ⅰ分子的表达，同时，促进细胞因子TNF-α、IL-2和IL-12的分泌；在小鼠体内实验中，以预防性策略和治疗性策略免疫接种，纳米颗粒联合核酸佐剂尤其核酸佐剂组合显著抑制了小鼠肿瘤的生长，具有增强的CD8⁺IFN-γ⁺、CD8⁺CD107a⁺、CD4⁺IFN-γ⁺、CD8⁺CD44⁺、CD4⁺CD44⁺ T细胞水平，以及降低的CD11b⁺Gr-1⁺ MDSCs细胞水平，提示有效激发了CTLs/Th1偏向的抗肿瘤免疫应答。

       本研究结果强烈提示，抗细胞因子主动免疫靶向调控TGF-β1以及纳米材料联合核酸佐剂尤其是佐剂组合，显著增强了治疗性HPV疫苗激发的抗肿瘤效力与免疫应答，是有潜力的联合免疫策略，为治疗性肿瘤疫苗的研发提供了新的思路。

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 Th1/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 Th1/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 immunity.

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-γ 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-γ⁺ 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-γ and TNF-α 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-γ 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-α, 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/Th1 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.