Cruzipain Induces Both Mucosal and Systemic Protection against Trypanosoma cruzi in Mice

Cruzipain, the major cysteinyl proteinase of Trypanosoma cruzi, is expressed by all developmental forms and strains of the parasite and stimulates potent humoral and cellular immune responses during infection in both humans and mice. This information suggested that cruzipain could be used to develop an effective T. cruzi vaccine. To study whether cruzipain-specific T cells could inhibit T. cruzi intracellular replication, we generated cruzipain-reactive CD4(+) Th1 cell lines. These T cells produced large amounts of gamma interferon when cocultured with infected macrophages, resulting in NO production and decreased intracellular parasite replication. To study the protective effects in vivo of cruzipain-specific Th1 responses against systemic T. cruzi challenges, we immunized mice with recombinant cruzipain plus interleukin 12 (IL-12) and a neutralizing anti-IL-4 MAb. These immunized mice developed potent cruzipain-specific memory Th1 cell responses and were significantly protected against normally lethal systemic T. cruzi challenges. Although cruzipain-specific Th1 responses were associated with T. cruzi protective immunity in vitro and in vivo, adoptive transfer of cruzipain-specific Th1 cells alone did not protect BALB/c histocompatible mice, indicating that additional immune mechanisms are important for cruzipain-specific immunity. To study whether cruzipain could induce mucosal immune responses relevant for vaccine development, we prepared recombinant attenuated Salmonella enterica serovar Typhimurium vaccines expressing cruzipain. BALB/c mice immunized with salmonella expressing cruzipain were significantly protected against T. cruzi mucosal infection. Overall, these data indicate that cruzipain is an important T. cruzi vaccine candidate and that protective T. cruzi vaccines will need to induce more than CD4(+) Th1 cells alone.

克鲁兹因（Cruzipain）是克氏锥虫（Trypanosoma cruzi）的主要半胱氨酸蛋白酶，该寄生虫的所有发育阶段与菌株均可表达该蛋白，且在人类与小鼠感染期间可激发强效的体液与细胞免疫应答。上述研究背景提示，克鲁兹因可用于开发高效的克氏锥虫疫苗。为探究克鲁兹因特异性T细胞能否抑制克氏锥虫的胞内增殖，我们构建了克鲁兹因反应性CD4阳性（CD4(+)）辅助性T细胞1型（Th1）细胞系。该类T细胞与受感染巨噬细胞共培养时可分泌大量γ干扰素（gamma interferon），进而诱导一氧化氮（NO）的产生并降低胞内寄生虫的增殖水平。为探究克鲁兹因特异性Th1应答在体内对抗全身性克氏锥虫攻击的保护效应，我们使用重组克鲁兹因联合白细胞介素12（IL-12）与中和性抗IL-4单克隆抗体（MAb）免疫小鼠。经免疫的小鼠可产生强效的克鲁兹因特异性记忆性Th1细胞应答，且可显著抵抗通常具有致死性的全身性克氏锥虫攻击。尽管克鲁兹因特异性Th1应答在体外与体内均与克氏锥虫保护性免疫相关，但仅过继转移克鲁兹因特异性Th1细胞无法保护BALB/c组织相容性相合小鼠，这表明额外的免疫机制对于克鲁兹因特异性免疫至关重要。为探究克鲁兹因能否诱导与疫苗开发相关的黏膜免疫应答，我们构建了表达克鲁兹因的减毒重组肠炎沙门氏菌鼠伤寒血清型（Salmonella enterica serovar Typhimurium）疫苗。经表达克鲁兹因的沙门氏菌免疫的BALB/c小鼠可显著抵抗克氏锥虫的黏膜感染。综上，上述实验数据表明，克鲁兹因是一款重要的克氏锥虫候选疫苗，且开发保护性克氏锥虫疫苗仅诱导CD4阳性（CD4(+)）Th1细胞是不够的。