沙眼衣原体感染依赖性免疫优势抗原的筛选及保护性初步评价

Chlamydia trachomatis urogenital tract infections can lead to severe chronic complications. Developing effective and safe subunit vaccines are considered the most effective means for preventing chlamydial infection and diseases. Chlamydia infection-phase dependent antigens are newly synthesized by replicating chlamydia organisms during infection, which play crucial roles in chlamydia persistent infections and pathogenesis. We showed previously that mice intranasally immunized with live organisms induce robust protective immunity to urogenital tract infection, which were highly correlate with humoral and cellular immune response to chlamydia infection-phase dependent antigens. So we hypothesize that these in vivo expressed immunodominant antigens should be promising chlamydial subunit vaccine candidates. Base on this, we plan to do following studies: Firstly, analysis of Chlamydia trachomatis serovar D whole genome ORFs, constructing high throughout fusion protein microarray to react with live chlamydial organism infected or dead organism-immunized mice sera, by comparing their antigen profiles will greatly facilitate the identification of infection-phase dependent antigens. Secondly, the immunodominant B cell antigens, which recognized by live chlamydia infected mice sera with high frequency and intensity will use to stimulate urogenital tract infected mice splenocytes to screen immunodominant CD4+Th1 cell antigens. Thirdly, antigens with both B and CD4+Th1 cell immunogenicity will be further identified for the antibody reactions specify and the distribution pattern in vitro and in vivo. Finally, confirmed immunodominant infection-phase dependent antigens will use to construct polyvalent vaccine, then detect the immune response and evaluation the protection efficiency in urogenital tract infection mice model. The expected results may beneficial in the design and development of an efficient human vaccine for the prevention of chlamydial sexually transmitted infections.

沙眼衣原体（Chlamydia trachomatis，Ct）生殖道感染防治形势日趋严峻，迫切需要研发新型有效的Ct疫苗以预防和控制Ct感染。感染依赖性抗原是衣原体在胞内感染增殖阶段合成并分泌的抗原。前期研究证实Ct活EB（原体）感染诱导的保护性效应与感染依赖性抗原有关，该类抗原很可能成为Ct疫苗研制的新靶标。本课题组在掌握了国际先进的疫苗候选抗原核心筛选技术及前期研究的基础上，从Ct全基因组水平出发，拟构建Ct"高通量融合蛋白微阵列"，与活EB感染或灭活EB免疫的鼠血清反应，通过比较分析两组血清抗体反应谱的差异，广泛全面筛选Ct感染依赖性抗原；流式细胞术、酶联免疫吸附试验进一步筛选获得免疫优势的B细胞、CD4+Th1细胞抗原；再经间接免疫荧光、免疫组织化学等方法鉴定后，构建新型多价Ct疫苗，动物模型初步评价疫苗的免疫应答水平及保护性效果，为研制新型、有效、特异的Ct疫苗提供实验依据。

沙眼衣原体（Chlamydia trachomatis，Ct）生殖道感染日趋严峻，目前仍缺乏有效疫苗。亚单位疫苗是衣原体疫苗发展的必然趋势。本项目首先从Ct全基因组水平出发，采用pGEX-6p原核表达系统，高效克隆表达Ct可溶性GST融合蛋白，制备“高通量融合蛋白微阵列”，再分别与活EB感染或灭活EB免疫的鼠血清反应，通过比较分析两组血清抗体反应谱的差异，广泛全面筛选Ct感染依赖性及免疫优势的B细胞抗原，体外刺激生殖道衣原体感染小鼠的脾细胞，进一步筛选能诱导脾细胞产生高水平IFN-γ和低水平IL-17细胞因子的免疫优势T细胞抗原；其次，大规模纯化各种优势抗原在鼠衣原体（MoPn）的同源物，分别与佐剂CpG和IFA混匀后肌注途径免疫Balb/c小鼠，再采用MoPn EB阴道感染小鼠，比较不同抗原免疫对小鼠下生殖道衣原体清除率和上生殖道病变的影响，筛选潜在有效的Ct亚单位疫苗候选抗原；最后，以IL-2作为免疫佐剂，用包含多种优势抗原的多亚单位联合疫苗滴鼻免疫小鼠，观察联合免疫的对小鼠生殖道衣原体感染的保护性效果。研究结果表明：1. Ct低剂量活EB滴鼻感染或高剂量UV-EB肌肉接种后，诱导小鼠产生的抗体效价两组之间无显著性差异（ｐ＝0.56）；2.克隆表达的Ct全基因组908个ORFs编码的抗原，仅有60个抗原可被至少一份血清识别，其中30个抗原只被活EB滴鼻感染组小鼠血清识别，18个抗原能被两组血清识别或仅被其中一组血清识别且识别频率≥50%，为免疫优势抗原；3.5种免疫优势抗原（OmcB，ArtJ，MIP，Inc及HP）可体外刺激感染鼠脾细胞产生高IFN-γ、低IL-17水平的CD4+ Th1型细胞免疫应答，5种抗原经纯化后肌肉接种免疫小鼠，仅MIP蛋白诱导小鼠产生了较强的保护性免疫应答，表现为下生殖道脱落上皮细胞中的衣原体含量显著减少，清除速率加快；上生殖道的输卵管积水程度和炎症反应减轻，发病率下降；4.以IL-2为佐剂，采用MIP、Inc或OmcB抗原构建的多亚单位联合疫苗，滴鼻免疫小鼠诱导产生的抗衣原体感染保护性效果优于MIP单一亚单位疫苗。该研究项目极大的促进了新型Ct疫苗研究的发展并可为其它重要病原菌的新型疫苗研制提供了参考依据。