Project-002

摘要 循环中的(第二级)HIV-1变异株对抗体介导的中和具有高度抵抗力，使 有效的疫苗设计是一个重大挑战。令人鼓舞的是，我们之前的HIVRAD计划中的工作 证明了能够交叉中和多个异源第2层病毒的抗体反应是 靶向N-糖链缺失启动和异种糖链修复三聚体-脂质体的诱导 助推器。从这些研究中分离出两种具有广谱中和活性的单抗 与天然三聚体形成的复合体的高分辨结构表明，有一株mAb靶向 保守的CD4结合位点(CD4bs)和另一个靶向gp41：gp120界面区域， 证实了这一结果。在当前的应用程序中，我们将通过评估响应来加强这些工作 由有序的、新颖的、基于三聚体的免疫原接种到印度来源的恒河猴体内而引发。 除了使用基于PF蛋白的三聚体外，我们还将测试基于给药的三聚体平台 项目1中所述的信使核糖核酸脂质纳米粒。 在项目2中，我们将描述在印度进行评估的疫苗方案所引起的B细胞反应 用快速、高通量单抗(MAb)分离核心B的恒河猴 确定目标表位，并指导加强免疫原的选择，如果需要，还可选择三聚体 通过消除或掩蔽不需要的非中和免疫显性反应来重新设计 随后的免疫研究。我们将与Core C的调查人员互动，他们也将生成 免疫显性抗体反应的负染电子显微镜信息 (NsEM)用于评估疫苗诱导的血清反应的方法。使用环境特定的单抗， 我们将使用下一代进一步确定环境特异性抗体谱系如何随着时间的推移而演变 测序(NGS)和免疫球蛋白发现，这是一种针对猕猴使用而优化的计算工具。我们会 生成猕猴种系VDJ等位基因的个性化数据库，以进行精确的基因分配， 对于正确的关于抗体亲和力成熟和SHM水平的结论是必要的。我们将进一步使用 IgDiscover结果和新的单倍型模块用于调查某些等位基因或组合是否 等位基因易诱发幼年猕猴成体的中和抗体反应。我们 将调查随着时间的推移中和和非中和抗体谱系的扩展水平 问疫苗诱导的中和抗体谱系是否在长期免疫隔间中持续存在 作为记忆B细胞和浆细胞，这对疫苗的长期保护作用至关重要。

Summary Circulating (tier 2) HIV-1 variants are highly resistant to antibody-mediated neutralization, making broadly effective vaccine design a major challenge. Encouragingly, work in our previous HIVRAD program demonstrated that Ab responses capable of cross-neutralizing multiple heterologous tier 2 viruses were elicited by targeted N-glycan deletion priming and heterologous glycan restorative Env trimer-liposome boosting. The isolation of two monoclonal antibodies with broadly neutralizing activity from these studies and high-resolution structures in complex with native-like trimers revealed that one mAb targeted the conserved CD4 binding site (CD4bs) and the other targeted the gp41:gp120 interface region, substantiated this result. In the current application, we will build on these efforts by evaluating responses elicited by well-ordered, novel, trimer-based immunogens inoculated into Indian origin rhesus macaques. In addition to the use pf protein-based trimers, we will test trimer platforms based on administration of mRNA lipid nanoparticles as described in Project 1. In Project 2, we will characterize B cell responses elicited by vaccine regimens evaluated in Indian origin rhesus macaques in Core B by rapid, high-throughput monoclonal antibody (mAb) isolation to define the targeted epitopes and guide both the choice of boosting immunogens and, if needed, trimer redesign by eliminating or masking unwanted non-neutralizing immunodominant responses for subsequent immunization studies. We will interact with the investigators in Core C, who also will generate information about immunodominant Ab responses through their negative stain electron microscopy (nsEM)-based method for evaluation of vaccine-induced serum responses. Using the Env-specific mAbs, we will further determine how Env-specific antibody lineages evolve over time using Next Generation Sequencing (NGS) and IgDiscover, a computational tool optimized for use in rhesus macaques. We will generate individualized databases of macaque germline VDJ alleles for precise gene assignments, which is necessary for correct conclusions about Ab affinity maturation and SHM levels. We will further use the IgDiscover results and the new haplotype module to investigate if certain alleles, or combinations of alleles, predispose to elicitation of neutralizing Ab responses in either adult of juvenile macaques. We will investigate the level of expansion of neutralizing and non-neutralizing Ab lineages over time and ask if vaccine-induced neutralizing Ab lineages persist in long-lived immune compartments such as memory B cells and plasma cells, which are critical for the long-term protective effects of vaccines.