Dissecting the mechanisms of HIV resistance in vivo to broadly neutralizing antibodies

摘要 HIV-1 包膜糖蛋白 (Envs) 介导病毒进入宿主细胞，是中和病毒的唯一目标 抗体。广泛中和抗体 (bnAbs) 靶向 HIV-1 包膜上高度保守的位点并中和 来自不同分支的各种不同菌株。尽管如此，bnAb 免疫疗法旨在抑制 HIV-1 复制有时会导致产生 bnAb 抗性 HIV-1 毒株，并且 HIV-1 毒株具有预 现有的 bnAb 耐药性可以通过治疗前的预筛选来识别。因此，了解底层 bnAb 耐药机制对于 bnAb 未来在免疫治疗和预防中的应用至关重要。 导致多种 bnAb 耐药的机制和促进 bnAb 逃逸的间接机制 不同的群体有特殊的公共卫生问题。 我们的研究旨在提供有关不同水平 bnAb 耐药性的重要见解。在具体目标 1 中，我们 将研究对多种 bnAb 具有耐药性的 HIV-1 病毒反弹株的直接耐药机制。我们会 筛选 bnAb 疗法临床研究的样本，识别表现出最高水平的 HIV-1 毒株的 Env 对几种 bnAb 的耐药程度，研究 Env 序列、功能、糖基化模式并确定 原子级分辨率的抗性环境结构。我们的综合方法将提供独特的配置文件 选定的多 bnAb 抗性 Env，整合了导致 bnAb 抗性的所有潜在机制。在 一个平行的方向，我们将研究反弹的 HIV-1 毒株通过细胞间传播传播的能力， 它允许在不同组的 bnAb 存在的情况下进行有效的病毒复制。我们将检验假设 尽管参与者血清中 bnAb 水平较高，但对 bnAb 敏感的 HIV-1 菌株仍能复制 RV397试验可以通过细胞间传播有效传播。此外，我们还将研究分子 表现出细胞间传输效率和 bnAb 耐药性增加的菌株机制。具体来说 目标 2 我们将定义最佳 bnAb 组合以克服 bnAb 耐药性并使用抗体酵母展示 技术对 bnAb 进行生物工程改造，提高对 bnAb 敏感且耐药的 HIV-1 的亲和力 菌株。这种方法将使我们能够确认 HIV-1 对 bnAb 的耐药机制并测试 假设 bnAb 的特定变化可以改善 bnAb 广度并允许靶向耐药子集 HIV-1 毒株。 总的来说，我们的研究将为多重bnAb耐药的HIV-1 Envs提供高分辨率和全面的观点， 体内 HIV-1 耐药性的替代途径，以及克服 bnAb 耐药性的潜在方法。我们的 结果将为制定 HIV-1 免疫治疗和预防新策略奠定坚实的基础 努力。

ABSTRACT HIV-1 envelope glycoproteins (Envs) mediate viral entry into host cells and are the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) target highly conserved sites on HIV-1 Envs and neutralize a wide range of diverse strains from different clades. Nevertheless, bnAb immunotherapy aiming to suppress HIV-1 replication sometimes leads to development of bnAb-resistant HIV-1 strains, and HIV-1 strains with pre- existing bnAb resistance can be identified by prescreening before treatment. Thus, understanding the underlying mechanisms of bnAb resistance are critical for the future application of bnAbs for immunotherapy and prevention. Mechanisms that lead to multi-bnAbs resistance and indirect mechanisms that facilitate escape of bnAbs from different groups are of particular public health concern. Our study is structured to provide important insights into bnAb resistance at different levels. In Specific Aim 1 we will study direct resistance mechanisms of rebounded HIV-1 strains that are resistant to multiple bnAbs. We will screen samples from clinical studies of bnAb therapy, identify Envs of HIV-1 strains that exhibit the highest degree of resistance to several bnAbs, study Env sequence, function, glycosylation patterns and determine the structures of resistant Envs at atomic level resolution. Our comprehensive approach will provide unique profiles of selected multi-bnAb resistant Envs that integrate all potential mechanisms contributing to bnAb resistance. In a parallel direction, we will study the ability of rebounded HIV-1 strains to spread through cell-cell transmission, which allows efficient viral replication in the presence of different groups of bnAbs. We will test the hypothesis that bnAb-sensitive HIV-1 strains that replicate despite high levels of bnAb in the serum of participants from the RV397 trial can efficiently spread by cell-cell transmission. Additionally, we will investigate the molecular mechanisms of strains that exhibit increased cell-cell transmission efficiency and bnAb resistance. In Specific Aim 2 we will define optimal bnAb combinations to overcome bnAb resistance and use antibody yeast display technology to bioengineer recombinant bnAbs with improved affinity against bnAb-sensitive and resistant HIV-1 strains. This approach will allow us to confirm mechanisms of HIV-1 resistance to bnAbs and to test the hypothesis that specific changes in bnAbs can improve bnAb breadth and allow targeting of a subset of resistant HIV-1 strains. Overall, our study will provide high-resolution and comprehensive view on multi-bnAb resistant HIV-1 Envs, on alternative pathways of HIV-1 resistance in vivo, and on potential approaches to overcome bnAb resistance. Our results will form a strong basis for the development of new strategies for HIV-1 immunotherapy and prevention efforts.