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

 描述(申请人提供)：尽管对HIV-1的体液免疫反应在数量上很强，但预防措施很少见，因为在自然免疫或疫苗接种后，广泛的中和抗体并不常见。越来越多的证据表明，这反映了在B细胞发育或激活期间，在检查点施加的广泛中和反应所需的B细胞的反选择。这些观察预测，放松B细胞反选择应该会改变HIV特异性反应的质量，并建立具有预防潜力的谱系，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细胞的选择：首先是在免疫前B细胞发育的过渡阶段，然后是生发中心的活化B细胞之间。重要的是，在HIV-1包膜免疫之前用BLyS治疗小鼠可以增加小鼠对更广泛的中和活动的反应频率。总之，这些观察为我们详细研究过渡和/或生发中心检查点的宽松选择如何影响广泛中和反应的能力奠定了基础。在目标1中，我们将确定放宽BLyS的过渡性选择是否建立了更能广泛中和的前免疫谱系。我们将通过分析移行区、滤泡区和边缘区B细胞亚群的谱系组成和多样性来实现这一点，然后是单细胞 克隆和再表达抗体以评估HIV的中和能力。在目标2中，我们将使用类似的策略来确定过剩的BLyS是否改变生发中心的选择，并允许能够广泛中和反应的体细胞突变的生发中心B细胞持续存在并进入记忆B细胞池。我们将比较在Env免疫前用BLyS处理的小鼠的GC和Memory B细胞，或者在Env免疫后GC形成的早期阶段用外源BLyS处理的小鼠的GC和Memory B细胞。在目标3中，我们将把我们的发现扩展到非人类灵长类动物，通过确定恒河猴在BLyS治疗后是否表现出类似的TR和FO池的扩大，以及在接种Env疫苗之前BLyS治疗是否提高了中和抗体的广度。

 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.