Interplay between antibody affinity maturation and HIV evolution in a single host

 描述(申请人提供)：快速进化的病原体，如人类免疫缺陷病毒(HIV)，逃避大多数疫苗诱导的抗体提供的免疫防御。所提出的诱导广谱中和抗体(BNAbs)的策略需要对疫苗接种或感染的免疫反应的进化有更深入的了解。在感染艾滋病毒的个体中，病毒和B细胞一起进化，形成了病毒和抗体的“军备竞赛”。这项提案的目的是分析CH505和CH0694等捐赠者的军备竞赛，这些捐赠者已经开发出广泛的抗体，这将为免疫原设计提供信息。 捐赠者CH505产生了一系列抗体，称为CH103，它与gp120 CD4结合位点相互作用。对前体(UCA)和中间FEB的晶体结构的分析，以及与各种HIV包膜gp120的结合测量表明，在这一谱系的进化过程中，可变轻链(VL)和重链(VH)结构域的相对取向发生了变化，以消除与gp120的不利接触。由于VH-VL移位将改变抗体与gp120相互作用的方式，因此将对VH-VL界面变化和gp120亲和力的变化进行更深入的分析。Gp120与UCA或中间体Fab I3.2的络合物的晶体结构将被确定，其VH-VL取向与成熟的bNab的VH-VL取向不同，后者已与gp120络合。作为协作，还将对自由Fab和Fab/gp120络合物进行分子动力学(MD)模拟，并结合额外的结合实验。来自同一捐赠者的DH2 35谱系的抗体引发了病毒逃逸突变，从而改善了与CH103谱系抗体的结合，因此可能加速了CH103谱系的亲和力成熟。将确定DH2 35谱系成员Fabs的晶体结构，以了解DH2 35抗体尽管表位重叠，但中和宽度有限，与CH103 bNAbs有何不同，并重建这两个谱系与病毒之间的相互作用。 除了快速变异外，HIV还使用重糖基化来逃避免疫系统。与多糖结合的bNAbs已经被分离出来，这表明病毒碳水化合物可以作为疫苗靶标。从供者CH0694中鉴定出一种早期的bNab，称为DH175，并被证明是“类似PGT的”。虽然许多PGT bNAb识别N332糖基化，但它们实际的表位不同于它们识别的多糖和gp120肽。为了了解抗体亲和力的成熟和病毒在这一谱系中的进化，将确定DH175/gp120复合体和前体抗体的游离Fabs的晶体结构。通过在Fabs和/或gp120包膜中引入突变并确定亲和力和中和性质，将检验从晶体结构推导出的关于亲和力成熟阶段的假设。如果合适的话，还将进行MD模拟，作为协作。这些数据将确定赋予糖依赖抗体广泛中和特征的机制(S)。

 DESCRIPTION (provided by applicant): Rapidly evolving pathogens, such as the human immunodeficiency virus (HIV), escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies (bnAbs) require a deeper understanding of evolution of the immune response to vaccination or infection. In HIV infected individuals, viruses and B-cells evolve together, creating a virus-antibody "arms race". The objective of this proposal is to analyze the arms race in donors such as CH505 and CH0694, who have developed antibodies of significant breadth, which would be informative for immunogen design. Donor CH505 produced a lineage of antibodies, called CH103, which interact with the gp120 CD4 binding site. Analysis of crystal structures of progenitor (UCA) and intermediate Fabs, along with binding measurements to various HIV envelope gp120s, indicated that there was a shift in the relative orientation of the variable light-chain (VL) and heavy-chain (VH) domains during evolution in this lineage to relieve unfavorable contacts with gp120. Because a VH-VL shift would change the way the antibodies interact with gp120, a deeper analysis of VH-VL interface changes and changes in affinity for gp120 will be performed. Crystal structures will be determined of gp120 in complex with the UCA or intermediate Fab I3.2, whose VH-VL orientations differ from that of the mature bnAb, already solved in complex with gp120. Molecular dynamics (MD) simulations will also be done, as a collaboration, of free Fabs and Fab/gp120 complexes, and coupled with additional binding experiments. Antibodies from the DH235 lineage, from the same donor, triggered virus escape mutations that improved binding to CH103 lineage antibodies and therefore could have accelerated affinity maturation in the CH103 lineage. Crystal structures of Fabs of DH235 lineage members will be determined to understand how the DH235 antibodies, which have limited neutralization breadth, differ from CH103 bnAbs despite overlapping epitopes, and to reconstruct the interplay among the two lineages and virus. In addition to rapid mutation, HIV also uses heavy glycosylation to evade the immune system. BnAbs that bind to glycans have been isolated, suggesting that viral carbohydrates can serve as vaccine targets. An early bnAb, called DH175, was identified from donor CH0694 and shown to be "PGT-like". While many PGT bnAbs recognize N332 glycosylation, their actual epitopes differ in the glycans and gp120 peptides they recognize. To understand antibody affinity maturation and virus evolution in this lineage, crystal structures of the DH175/gp120 complex and of the free Fabs of precursor antibodies will be determined. Hypotheses concerning stages of affinity maturation deduced from the crystal structures will be tested by introducing mutations into the Fabs and/or gp120 envelope and determining affinities and neutralization properties. MD simulations will also be performed, as a collaboration, if appropriate. These data will identify mechanism(s) that confer broadly neutralizing characteristics on glycan-dependent antibodies.