Mechanistic studies of BLyS-mediated modulation in HIV-1 Env-specific antibody responses

 描述（由申请方提供）：尽管对HIV-1的体液免疫应答在数量上很强，但由于自然免疫或疫苗接种后不常见广泛中和抗体，因此预防性治疗很少见。越来越多的证据表明，这反映了B细胞发育或活化期间施加的检查点处的广泛中和应答所需的B细胞的反选择。这些观察结果预测，放松B细胞反选择应该改变艾滋病毒特异性反应的质量，并建立具有预防潜力的剧目，但这一预测尚未得到直接检验。细胞因子BLyS在两个检查点调节B细胞选择：首先在过渡到成熟的免疫前B细胞发育步骤;然后在生发中心的活化B细胞之间。重要的是，在HIV-1包膜免疫之前用BLyS处理小鼠产生了具有更广泛中和活性的小鼠应答频率增加。总之，这些观察结果使我们能够详细研究过渡和/或生殖中心检查点的放松选择如何影响广泛中和反应的能力。在目标1中，我们将确定是否放松过渡选择BLyS建立一个免疫前库更能够广泛的中和。我们将通过分析移行、滤泡和边缘区B细胞亚群的库组成和多样性，然后分析单细胞 克隆和重新表达抗体，以评估HIV中和能力。在目标2中，我们将使用类似的策略来确定过剩BLyS是否改变生发中心选择，并允许能够广泛中和应答的体细胞突变的生发中心B细胞持续存在并进入记忆B细胞库。我们将比较来自在用Env免疫之前用BLyS处理的小鼠的GC和记忆B细胞，或来自在Env免疫后GC形成的早期阶段用外源性BLyS处理的小鼠的GC和记忆B细胞。在目标3中，我们将通过确定在BLyS处理后恒河猴是否显示TR和FO池的类似扩大，以及在Env疫苗接种前BLyS处理是否改善中和抗体宽度，将我们的发现扩展到非人灵长类动物。

 DESCRIPTION (provided by applicant): Despite quantitatively robust humoral immune responses to HIV-1, prophylaxis is rare because broadly neutralizing antibodies are uncommon after natural immunization or vaccination. Increasing evidence suggests this reflects the counterselection of B cells needed for broadly neutralizing responses at checkpoints imposed during B cell development or activation. These observations predict that relaxing B cell counterselection should alter the quality of HIV-specific responses and establish repertoires with prophylactic potential, but this prediction has not been directly tested. The cytokine BLyS modulates B cell selection at two checkpoints: first at the transitional to mature preimmune B cell developmental step; and then among activated B cells in the germinal center. Importantly, treating mice with BLyS prior to HIV-1 envelope immunization yields an increased frequency of mice responding with broader neutralizing activity. Together, these observations position us for detailed study of how relaxed selection at the transitional and/or germinal center checkpoints impacts the ability to mount broadly neutralizing responses. In Aim 1, we will determine whether relaxing transitional selection with BLyS establishes a preimmune repertoire more capable of broad neutralization. We will accomplish this through analyses of repertoire composition and diversity in the transitional, follicular and marginal zone B cell subsets, followed by single-cell cloning and re-expression of antibodies to assess HIV neutralizing capacity. In Aim 2, we will use a similar strategy to determine whether surplus BLyS alters germinal center selection, and allows somatically mutated germinal center B cells capable of broadly neutralizing responses to persist and enter memory B cell pools. We will compare GC and memory B cells from mice treated with BLyS prior to immunization with Env, or from mice treated with exogenous BLyS in the early stages of GC formation after Env immunization. In Aim 3, we will extend our findings to nonhuman primates, by determining whether rhesus macaques show similar enlargement of TR and FO pools after BLyS treatment, and whether BLyS treatment prior to Env vaccination improves neutralizing antibody breadth.