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HIV-1的唯一靶点。 抗体的广泛中和抗体（bnAb）靶向HIV-1 Env上的高度保守位点，并中和 来自不同进化枝的多种多样的菌株。然而，旨在抑制 HIV-1复制有时会导致产生抗bnAb的HIV-1毒株，而具有前 现有的bnAb抗性可以通过治疗前的预筛选来鉴定。因此，了解底层 bnAb抗性的机制对于bnAb用于免疫治疗和预防的未来应用至关重要。 导致多bnAbs耐药的机制和促进bnAbs逃逸的间接机制 不同的群体特别受到公众健康关注。 我们的研究旨在为不同水平的bnAb耐药性提供重要见解。具体目标1， 将研究对多种bnAb具有耐药性的HIV-1病毒株反弹的直接耐药机制。我们将 从bnAb治疗的临床研究中筛选样本，鉴定表现出最高水平的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Envs结构。我们全面的方法将提供独特的配置文件 选择的多重bnAb抗性Env，其整合了有助于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菌株通过细胞间传播的能力， 其允许在不同组bnAb存在下的有效病毒复制。我们将检验这个假设 尽管来自美国的参与者血清中bnAb水平很高，但bnAb敏感的HIV-1菌株仍在复制， RV 397试验可通过细胞间传播有效传播。此外，我们将研究分子 这是菌株表现出增加的细胞-细胞传递效率和bnAb抗性的机制。在特定 目的2：确定克服bnAb耐药性的最佳bnAb组合，并利用酵母展示抗体 生物工程重组bnAb技术，提高对bnAb敏感性和耐药性HIV-1的亲和力 菌株这种方法将使我们能够确认HIV-1对bnAb的耐药性机制，并测试HIV-1对bnAb的耐药性。 假设bnAb中的特异性变化可以改善bnAb宽度并允许靶向耐药的亚群， HIV-1病毒株。 总的来说，我们的研究将提供关于多重bnAb耐药HIV-1 Envs的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 研究结果将为开发HIV-1免疫治疗和预防的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.