Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi-infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.

中文翻译：

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Tc24在克氏锥虫不同生命阶段的定位和表达动力学。

Tc24-C4 是克氏锥虫的一种改良重组鞭毛钙结合蛋白，目前正在开发作为候选治疗亚单位疫苗，以预防或延缓慢性恰加斯心肌病的进展。当与 Toll 样受体激动剂联合使用时，Tc24-C4 免疫可减少动物模型中的寄生虫血症、心脏组织中的寄生虫以及心脏纤维化和炎症。为了支持对候选疫苗及其作用机制的进一步研究，产生了针对 Tc24-C4 的鼠单克隆抗体 (mAb)。在这里，我们报告了使用 mAb Tc24-C4/884 的新发现，该单克隆抗体通过 ELISA、蛋白质印迹和不同的成像技术检测 T. cruzi 中的 Tc24-WT 和 Tc24-C4 以及天然 Tc24。令人惊讶的是，在固定的克氏锥虫锥鞭毛体中通过 Tc24-C/884 检测 Tc24 需要对寄生虫进行透化，这表明 Tc24 并未暴露在克氏锥虫表面，使得抗体在 Tc24- 后诱导的保护中直接发挥作用。 C4 免疫的可能性较小。我们进一步观察到，用mAb Tc24-C4/884对克氏锥虫感染的细胞进行免疫染色后，当克氏锥虫锥鞭毛体进入宿主细胞并转化为无鞭毛体时，Tc24的表达显着降低。然而，在宿主细胞逃逸之前，Tc24 与寄生虫鞭毛生长相关，与重新转化为锥鞭毛体形式有关。这些观察结果是在疫苗免疫的潜在机制的背景下讨论的。