Development of prime-boost immunization schemes to elicit VRC01-class bNAbs in polyclonal human BCR backgrounds

摘要 VRC01类广泛中和抗体（bNAb）识别CD4-CD10内的保守表位。 HIV-1 Env.它们是已知最有效的bNAb之一，可以保护动物免受 实验性S/HIV感染，使其成为通过疫苗接种引发的高度有吸引力的抗体类型。他们 已从多名HIV-1感染受试者中分离出，但均来自相同的VH1 - 2等位基因（* 02） 和少量的轻链，所有这些轻链都表达5个氨基酸的CDRL 3。相对于成熟的，完全 突变形式的VRC01类抗体，其推断的种系形式不识别Env， 中和HIV-1这导致了先前的重组Env免疫原在免疫中无效的假设。 激活表达生殖系VRC 01 B类细胞受体（BCR）的幼稚B细胞，这可能部分解释了 为什么这些免疫原在疫苗研究中没有引起VRC01样抗体反应。我们报道了 设计结合种系VRC01类抗体的进化枝C衍生的Env蛋白（426c核心）， 在体内启动表达相应BCR的幼稚B细胞的扩增，但不足以诱导 这些BCR向其中和形式的成熟。为了引出任何一个 通过免疫的bNAb是由于存在于保守结构域的聚糖施加的空间限制。 Env的环D中的糖基化位点N276。这些聚糖限制了对生殖细胞识别的表位的接近， VRC01类抗体，但由于抗体经历体细胞超突变和基于亲和力的选择 它们"学习"如何绕过这个空间阻滞。我们的免疫策略成功地引发了VRC01类 因此，bNAbs将取决于我们引导种系VRC01类抗体沿着进化的能力。 特定的成熟途径来绕过N276聚糖施加的限制。在本项目中，我们建议 使用以前没有测试过的概念和试剂，努力克服这些主要障碍， 通过免疫产生VRC01类抗体。具体来说，我们建议使用抗独特型 针对种系VRC01类抗体的单克隆抗体（aiMAb），以特异性地增加 在用种系结合"426 c核心"免疫之前表达VRC01的B细胞的频率 免疫原，然后用选择VRC01类B细胞的基于Env的试剂加强免疫 可以绕过N276聚糖的限制。我们的研究将在一个迭代的 在表达VRC01类BCR的各种动物模型中， 多克隆人BCR库。

ABSTRACT VRC01-class broadly neutralizing antibodies (bNAbs) recognize a conserved epitope within the CD4- binding site of HIV-1 Env. They are among the most potent bNAbs known and protect animals from experimental S/HIV infection, making them a highly attractive type of antibody to elicit by vaccination. They have been isolated from multiple HIV-1-infected subjects, but are all derived from the same VH1-2 allele (*02) and a small number of light chains, all of which express a 5 amino-acid CDRL3. In contrast to the mature, fully mutated forms of VRC01-class antibodies, their inferred germline forms do not recognize Env and do not neutralize HIV-1. This led to the hypothesis that previous recombinant Env immunogens were ineffective in activating naïve B cells expressing germline VRC01-class B cell receptors (BCRs), which may, in part, explain why such immunogens have not elicited VRC01-like antibody responses in vaccine studies. We reported on the design of a clade C-derived Env protein (426c Core) that binds germline VRC01-class antibodies and initiates the expansion of naïve B cells expressing the corresponding BCRs in vivo, but is insufficient to induce the maturation of these BCRs towards their neutralizing forms. A major hurdle to overcome in order to elicit any bNAbs through immunization is due to steric restrictions imposed by glycans present at the conserved glycosylation site N276 in Loop D of Env. These glycans limit access to the epitope recognized by germline VRC01-class antibodies, but as the antibodies undergo somatic hypermutation and affinity-based selection they ‘learn’ how to bypass this steric block. The success of our immunization strategies to elicit VRC01-class bNAbs will therefore depend on our ability to guide the evolution of germline VRC01-class antibodies along particular maturation pathways to bypass the N276 glycan-imposed restrictions. In this Project we propose to use concepts and reagents, not tested previously, in an effort to overcome these major obstacles preventing the generation of VRC01-class antibodies by immunization. Specifically, we propose to use anti-idiotypic monoclonal antibodies (aiMAbs) against the germline VRC01-class antibodies to specifically increase the frequency of VRC01-expressing B cells prior to immunization with the germline-binding’ 426c Core immunogen, followed by booster immunizations with Env-based reagents that select for VRC01-class B cells that can bypass the restrictions imposed by the N276 glycans. Our studies will be performed in an iterative fashion in diverse animal models that express VRC01-class BCRs, including mice engineered to express a polyclonal human BCR repertoire.