Characterization and Generalization of a Novel Immunogenic Target on HIV-1 gp120

成功分离人类广泛中和抗体（bnAbs）和绘制分离的单克隆抗体（mAb）表位有助于揭示HIV-1包膜糖蛋白（Env）上的几个易感位点；然而，分离的bnab可能只占供体血清中和活性的一部分，表明HIV-1 Env的未知靶点。此外，大多数分离的bnab都显示出不同寻常的编码序列，这意味着潜在的诱导障碍。为了进一步探索通过疫苗接种诱导保护性抗体，我们建议鉴定和表征可能更可行的新功能抗体。先前，我们使用表面修饰的gp120稳定核心蛋白RSC3作为探针，鉴定出靶向gp120 cd4结合位点（CD4bs）的bnAb VRC01及其单克隆抗体。通过同样的探测方法，我们最近从一名同时产生vrc01类bnAb VRC-PG04的感染者身上发现了一种新的单抗VRC-PG05。迄今为止，VRC-PG05是针对HIV-1的最小体细胞突变中和单抗，其编码序列在人类抗体库中似乎并不罕见。VRC-PG05与gp120复合物的原子水平结构揭示了一个新的糖肽表位，该表位围绕gp120上的两个高度保守的聚糖N262和N448（基于HXB2编号）。去除任意一个糖基化位点导致VRC-PG05活性完全丧失，证实了N262/N448聚糖对单克隆抗体识别的重要性。基于糖肽表位的新颖性、与cd4bb定向的功能性单克隆抗体的共激发能力、相对“正常”的编码序列以及病毒中和功能，我们认为VRC-PG05及其表位是一个有吸引力的免疫靶点，因此建议将其发现推广到其他感染个体。我们假设：1)gp120上新发现的N262/N448位点具有免疫原性，并且hiv -1感染者体内经常会产生针对该位点的抗体；2)编码N262/N448聚糖位点的功能性抗体的基因序列不需要大多数已鉴定的HIV-1 bnAbs共有的不寻常特征。我们获得了带点突变的重组gp120蛋白，通过ELISA检测N262/N448的特异性。我们将这种单分子gp120 ELISA用于筛选85名HIV-1血清转化者的血浆样本，迄今为止鉴定出17名（20%）具有N262/N448特异性抗体。这些初步结果支持了我们关于新发现的N262/N448聚糖位点的免疫原性的第一个假设。从具有这种抗体特异性的个体中，我们建议分离出相应的单抗，确定单抗的抗病毒功能，并检查单抗编码序列是否显示出大多数已鉴定的单抗共有的任何不寻常的特征。如果成功，这个项目将产生新的收益

DESCRIPTION: Successes in isolating human broadly neutralizing antibodies (bnAbs) and mapping the isolated monoclonal antibody (mAb) epitopes have helped to reveal several sites of vulnerability on the HIV-1 envelope glycoprotein (Env); however, the isolated bnAbs may only account for part of the donor sera neutralizing activity, indicating unknown targets on HIV-1 Env. Additionally, most of the isolated bnAbs have demonstrated unusual coding sequences implicating potential elicitation barriers. To progress on our quest to elicit protective antibodie through vaccination, we propose to identify and characterize new functional antibodies that may be more feasible for elicitation. Using a resurfaced gp120 stabilized core protein RSC3 as a probe, we previously identified the bnAb VRC01 and its class of mAbs that target the CD4-binding site (CD4bs) of gp120. By the same probing method, we recently identified a new mAb, VRC-PG05, from an infected individual who also developed the VRC01-class bnAb VRC-PG04. To date VRC-PG05 is the least somatically mutated neutralizing mAb against HIV-1 and its coding sequences do not seem uncommon in the human antibody repertoire. Atomic-level structure of VRC-PG05 in complex with gp120 revealed a novel glycopeptide epitope centering on two highly conserved glycans at N262 and N448 (based on HXB2 numbering) on gp120. Removal of either glycosylation site resulted in complete loss of VRC-PG05 activity, confirming the importance of the N262/N448 glycans for the mAb recognition. Based on the novelty of the glycopeptide epitope, the ability for co-elicitation with the CD4bs-directed functional mAbs, the relatively "normal" coding sequences, as well as the virus neutralization function, we consider VRC-PG05 and its epitope an attractive immune target and thus propose to generalize its findings to additional infected individuals. We hypothesize that 1) the newly discovered N262/N448 glycan site on gp120 is immunogenic, and antibodies targeting this site are frequently elicited in HIV-1-infected individuals; and 2) genetic sequences that encode functional antibodies targeting the N262/N448 glycan site do not require unusual features shared by most of the identified HIV-1 bnAbs. We have derived recombinant gp120 proteins with point mutations to detect the N262/N448 specificity by ELISA. We applied this monomeric gp120 ELISA to screen plasma samples from 85 HIV-1 seroconverters, and so far identified 17 (20%) who possessed antibodies with the N262/N448 specificity. These preliminary results support our first hypothesis regarding the immunogenicity of the newly discovered N262/N448 glycan site. From individuals with this antibody specificity, we propose to isolate the corresponding mAbs, determine the mAb anti-viral functions, and examine if the mAb coding sequences display any unusual features shared by most of the identified bnAbs. If successful, this project will yield new functional anti-HIV-1 mAbs that may be more feasible for elicitation through vaccination.