为阐明机体在免疫应答过程中产生高亲和力和高体突变率保护性中和抗体的机理，我们提出在中国猕猴体内测试一个与现实世界紧密相关的抗原，埃博拉病毒的表面糖蛋白GP。我们要测试的假说是，保护性功能抗体的高亲和力和高体突变率是生发中心反应时间延长或者参与免疫应答的B细胞多次反应（即原参与免疫应答的B细胞重返生发中心）的结果。这两种机制互不排斥。我们提出三种免疫方案: 常规的混合多价免疫，续加免疫，参照生发中心反应时间的续加免疫。在每个免疫动物体内，我们会随时间追踪血清里的GP抗体滴度和中和抗体滴度，取淋巴结活检追踪生发中心形成的时间、数量、和大小，以及滤泡辅助T细胞的数量和功能。我们还会以单细胞克隆技术分离GP特异性的猴源单克隆抗体，并进一步分析这些单抗与抗原结合的亲和力，中和病毒的能力，以及它们的体突变率。依据这些数据，我们能直接检测生发中心的反应时间和次数是否与抗体的体突变率和病毒中和能力正相关。

To elucidate the mechanisms leading to antibodies with high affinity and high level of somatic hypermuation, we propose to test a “real-world” antigen, the Ebola glycoprotein (GP), with three immunization strategies in Chinese rhesus macaques. We will test the hypothesis that mechanisms favoring prolonged germinal center reactions and/or promoting B cells to re-enter germinal centers will lead to antibody responses with high affinity and increased somatic hypermutation. We propose three immunization strategies: 1) conventional multivalent immunization; 2) sequential immunization; 3) germinal-center-reaction-guided sequential immunization. From each immunized animal, we will follow the temporal changes in serum GP-binding antibody titer, virus-neutralizing antibody titer, number/size of germinal centers, and number/function of T follicular helper (Tfh) cells in lymph node biopsies. We will also isolate GP-specific macaque monoclonal antibodies (MAbs) by single B cell cloning and characterize the MAb binding affinity, neutralizing activity, and level of somatic hypermuation. With these data, we will specifically examine whether the duration and recurrence of germinal center reactions correlate with the level of MAb somatic hypermutation and cross neutralization function.