Taibi A, Plumas-Marty B, Guevara-Espinoza A, Schöneck R, Pessoa H, Loyens M, Piras R, Aguirre T, Gras-Masse H, Bossus M
Centre d'Immunologie et de Biologie Parasitaire, Unité INSERM 167-CNRS 624, Institut Pasteur, Lille, France.
J Immunol. 1993 Sep 1;151(5):2676-89.
In the present study, we investigate the immunoprotective properties of trypomastigote excretory-secretory Ag (ESA) in experimental models. In the case of BALB/c mice, the immunization with ESA resulted in the reduction of parasitemia during acute infection and a significant level of protection in terms of mortality with more than 60% survival, whereas none of the mice in the control groups survived after 39 days postinfection. The same experiments performed in Fischer rats showed a high degree of protection against acute lethal infection with 100% survival, whereas 20 to 40% of rats in the control groups survived the acute phase of T. cruzi infection. Mouse and rat immune sera presented trypanolytic activity against Trypanosoma cruzi infective forms, and recognized two major parasite components of 85 and 24 kDa. The analysis of specific isotype profiles showed a predominance of IgG1, IgG2a, and IgG2b antibody responses. Rat antisera to ESA were then used to screen a trypomastigote cDNA library. Several clones were identified, all of which encoded for the 24-kDa protein. Using a mAb (Tcr7) produced against the native protein, the 31-kDa recombinant fusion protein was purified by affinity chromatography. The antisera to the recombinant protein used in IFA and immunoelectron microscopy showed that the localization of the 24-kDa protein differs among T. cruzi developmental stages. Protection experiments were performed in BALB/c mice using two synthetic peptides (20-40 and 109-124) derived from the primary sequence of the 24-kDa polypeptide. The results obtained clearly indicated that the peptide 109 to 124 containing a putative T cell epitope represents the most protective epitope, which induced 30 to 50% of protection against mortality during acute infection, whereas the percent survival in the control groups (OVA and 20-40 OVA peptide-immunized mice) was around 16%. Moreover, analysis of T cell proliferation in response to OVA-coupled peptides clearly indicated that only the 109 to 124 peptide had the capacity to induce the proliferation of T cells from peptide-immunized mice. Interestingly, only the 109 to 124-coupled peptide induced the proliferation of T cells from T. cruzi-infected mice.
在本研究中，我们在实验模型中研究了锥鞭毛体排泄-分泌抗原（ESA）的免疫保护特性。对于BALB/c小鼠，用ESA免疫可使急性感染期间的寄生虫血症减少，并在死亡率方面提供显著的保护，存活率超过60%，而对照组的小鼠在感染后39天无一存活。在Fischer大鼠中进行的相同实验显示，对急性致死性感染具有高度保护作用，存活率达100%，而对照组中有20%至40%的大鼠在克氏锥虫感染急性期存活。小鼠和大鼠免疫血清对克氏锥虫感染形式具有溶 trypanosome 活性，并识别出85 kDa和24 kDa的两种主要寄生虫成分。特异性同种型谱分析显示IgG1、IgG2a和IgG2b抗体反应占优势。然后用大鼠抗ESA血清筛选锥鞭毛体cDNA文库。鉴定出几个克隆，所有这些克隆都编码24 kDa蛋白。使用针对天然蛋白产生的单克隆抗体（Tcr7），通过亲和层析纯化了31 kDa重组融合蛋白。用于免疫荧光分析（IFA）和免疫电子显微镜的重组蛋白抗血清显示，24 kDa蛋白在克氏锥虫不同发育阶段的定位不同。使用源自24 kDa多肽一级序列的两种合成肽（20-40和109-124）在BALB/c小鼠中进行了保护实验。获得的结果清楚地表明，包含假定T细胞表位的109至124肽代表最具保护性的表位，在急性感染期间可诱导30%至50%的死亡率保护，而对照组（卵清蛋白和20-40卵清蛋白肽免疫小鼠）的存活率约为16%。此外，对卵清蛋白偶联肽刺激的T细胞增殖分析清楚地表明，只有109至124肽有能力诱导肽免疫小鼠的T细胞增殖。有趣的是，只有109至124偶联肽能诱导克氏锥虫感染小鼠的T细胞增殖。
