梅毒疫苗新靶标-感染依赖性免疫优势抗原的筛选及在菌影递送多表位核酸疫苗中的应用

High prevalence of syphilis and its serious damage to the adult and newborn's health urgently need to develop a highly effective vaccine against Treponema pallidum. neither current recombinant protein vaccines from foreign labs nor Tp92 and Gpd DNA vaccines build by us ,which aimed to outer membrane protein (OMP) of T. pallidum , failed to induce complete protection when immunized with a single immunogen. which is maybe lacking of synergy between immunogens, incorrected natural protein conformation, lacking of a powerful antigen delivery system/adjuvant, ignoring the mucosal immunity , low density and instability of OMP. T. pallidum infection-dependent antigen is a kind of non-bacterial membrane antigen which is synthesized and secreted during infection，which play crucial roles in T. pallidum persistent infections and pathogenesis.Preliminary studies also confirmed some T.Pallidum infection-dependent antigens can induce protective immune response to T. pallidum infection and may become a new target for vaccine development. Base on this, we plan to do following studies: Firstly, analysis of whole genome ORFs of T. pallidum Nichols Standard strain，constructing high throughout fusion protein microarray to react with live T. pallidum organism infected or dead T.pallidum organism-immunized rabbit sera, by comparing their antigen profiles will greatly facilitate the identification of infection dependent antigens. Secondly, the immunodominant B cell antigens, which recognized by live T. pallidum infected rabbit sera with high frequency and intensity will use to stimulate urogenital tract infected rabbits splenocytes to screen immunodominant CD4+Th1 cell antigens.Thirdly, antigens with both B and CD4+Th1 cell immunogenicity will be further identified at epitope level, we plan to screen and identify immunodominant B, Th, or B-Th cell epitopes by immunological informatics,phage display technology and so on for constructing a concatemer composed of multiple immunodominant cell epitopes including outer membrane protein immundominant antigens and infection-dependent immunodomainant antigens. Then the concatemer will be use to construct a new type multi-epitope vaccine based on bacterial ghosts as a antigen delivery system which is able to induce high level mucosal and systemic Th1 bias humoral and cellar response and high effective protection .then detect the immune response and evaluation the protection efficiency in T.pallidum infection rabbit model. The expected results may beneficial in the design and development of an efficient human vaccine for the prevention of syphilis.

梅毒的高发和严重危害迫切需要研发有效的梅毒螺旋体（Tp）疫苗来预防。目前基于Tp膜蛋白展开的国外重组蛋白疫苗和我们首次构建的核酸疫苗均未能获完全保护，可能与免疫原单一、缺乏强效递送系统/佐剂、忽视黏膜免疫及Tp膜蛋白密度低，不稳定等有关。感染依赖性抗原是Tp在感染阶段合成分泌的非菌体抗原,前期研究也证实其与Tp保护性效应有关，可成为疫苗研制新靶标。为此本项目在前期研究基础上，从全基因组水平出发，拟构建Tp"高通量融合蛋白微阵列"与活Tp感染或灭活Tp免疫的兔血清反应，分析两组血清抗体反应谱差异，全面筛选感染依赖性抗原；流式细胞术、ELISA进一步筛选获得免疫优势B细胞、CD4+Th1细胞抗原；再采用免疫信息学、噬菌体展示等技术，筛选鉴定优势B/Th/B-Th细胞表位，构建联合膜蛋白和感染依赖性抗原的菌影递送多表位核酸疫苗，观察动物免疫保护性和探讨免疫保护机制，为研制新型高效Tp疫苗提供依据

梅毒的高发和严重危害迫切需要阐明梅毒螺旋体（Tp）的致病机制、研发有效的Tp疫苗和筛选新的有效的诊断抗原来预防和加强临床诊断。目前梅毒的致病机制尚未明了，而基于Tp膜蛋白展开的国外重组蛋白疫苗和我们首次构建的核酸疫苗均未能获完全保护。感染依赖性抗原是Tp在感染阶段合成分泌的非菌体抗原,前期研究也证实其与Tp保护性效应有关，可成为疫苗研制新靶标。本项目在前期研究基础上，构建了Tp“高通量融合蛋白微阵列”与活Tp感染或灭活Tp免疫的兔血清反应，分析两组血清抗体反应谱差异，全面筛选出感染依赖性抗原蛋白8个；通过流式细胞术、ELISA进一步筛选获得免疫优势B细胞、CD4+Th1细胞抗原2个（Tp0971,Tp0134）；构建了联合膜蛋白Tp92,TpGpd和感染依赖性抗原Tp0971,Tp0134的菌影递送多价/多表位核酸疫苗，观察动物免疫保护性和探讨免疫保护机制，为研制新型高效Tp疫苗提供依据。同时本研究围绕外膜蛋白 Tp92 和人单核细胞之间开展了相互作用的相关研究，发现Tp外膜蛋白 Tp92 能经 THP-1 细胞表面的 CD14 and/orTLR2，诱 导 人 单 核 细 胞 发 生 由.pro-caspase-1.介 导 的 焦 亡 和 由RIPK1/caspase-8/caspase-3 介导的凋亡，降低固有免疫应答系统清除Tp的能力。发现 Tp92 能通过识别 THP-1 细胞表面的 CD14 and/or TLR2，诱导 IL-8的分泌，这种分泌能受到核转录因子 NF-κB 的调控，而细胞因子 TNF-α，IL-1β，IL-6，IL-10，IL-18，MCP-1 的分泌水平未见明显升高。初步证实外膜蛋白 Tp92 在Tp早期感染宿主过程中能诱导人单核细胞死亡及 IL-8 的分泌，在应对宿主固有免疫应答系统识别和清除过程中发挥着重要作用。对微阵列筛选出的几个Tp感染依赖性抗原（Tp0971、Tp0470、Tp0768、Tp0134、Tp0462、Tp0663）进行了基于Tp感染依赖性诊断抗原的ELISA法与现有临床血清学诊断金标准方法TPPA、一种市售的临床商品化梅毒初筛ELISA试剂盒以及一种商品化RPR试剂盒的临床诊断价值的筛选比较评价。发现几个筛选出的感染依赖性抗原均具有一定临床诊断价值；Tp0971具有应用于梅毒治疗临床判愈的潜在价值。