Role of HIV Env glycosylation in mucosal transmission

描述(由申请人提供)：对促进传播的病毒/宿主关系以及感染后导致疾病进展速度的病毒/宿主关系的深入了解，对于设计有效的艾滋病毒-1疫苗至关重要。一个越来越被认为是关键但研究不足的参数是，病毒膜糖基化不仅在优先传播中发挥作用，而且在病毒选择进入受体的能力以及对中和抗体的敏感性方面也发挥着作用。此外，目前正在积累的数据表明，作为糖基化env蛋白的配体/受体的各种细胞表面分子，以及那些受糖基化与去糖化病毒影响的分子，可能有助于提高病毒产生的免疫反应的质量。这些问题只能使用适当的非人类灵长类动物模型来客观地解决，例如感染SHIV的猕猴，该模型包含来自HIV-1初级分离株的包膜病毒，特别是来自传播/创始病毒的包膜病毒，以研究包膜糖基化在优先传播中的作用。因此，我们计划使用从传播/创始(T/F)病毒获得的一组HIV分子克隆来探索环境多糖的作用，这些克隆来自赞比亚的一个队列-那里C分支病毒占主导地位。我们已经开发了几种独特的工具/试剂，并组建了一支才华横溢的研究团队，并概述了一系列系统的逻辑设计的研究，这些研究首先将重点放在目标1中概述的T/F循环HIV毒株包括N-和O-糖链的成对克隆中的糖链的组成和排列。根据糖链图谱，我们建议选择a)高度糖基化的、b)低水平的环境糖基化病毒和c)作为对照的方正病毒，以制备相应的具有复制能力的猿人免疫缺陷病毒(Shiv)，用于进一步的深入研究，包括详细的原代细胞和细胞系复制动力学、糖蛋白含量、颗粒包膜糖蛋白含量、树突状细胞(DC)捕获、DC-T细胞感染、对干扰素-α的敏感性、抗α4？7和针对特定凝集素样分子(Galectin-9、SIGLEC、MINCLE和CD200/200R)的阻断单抗。然后，我们将选择病毒进行体内测试，以进行猕猴体内传播效率研究，主要基于可比较的体外复制但不同的多糖含量(目标2)。在特定的目标3下，我们将定义是否是环境蛋白糖基化的标志性位点(S)促进传播，使用基于“差”转运体骨干的等基因SIV，并使用重复的多次低剂量阴道内挑战模型，仅突变与目标2b的创始人病毒相对应的选择残基。这些研究结果将为未来合理的HIV疫苗设计提供坚实的基础。

DESCRIPTION (provided by applicant): A refined understanding of virus/host relationships that promote transmission and those that contribute to the rate of disease progression following infection is critical for the design of effective vaccines against HIV-1. One parameter that is increasingly recognized as being critical and understudied is the role of viral env glycosylation not only in preferential transmission but also in the ability of the virus to select receptors for entry and for its susceptibility to neutralizing antibodies. In addition, data is now accumulating o show that a variety of cell surface molecules that serve as ligands/receptors for the glycosylated env proteins and those that are influenced by glycosylated versus de-glycosylated viruses, could contribute to the quality of immune response the virus engenders. These issues can only be objectively addressed using appropriate nonhuman primate models such as macaques infected with SHIVs that contain the env from primary HIV-1 isolates, particularly from transmitted/founder viruses to study the roles of env glycosylation in preferential transmission. Therefore, we plan to explore the role of env glycans using sets of HIV molecular clones obtained from transmitted/founder (T/F) viruses from a Zambian cohort-where clade C viruses predominate. We have generated several unique tools/reagents and assembled a highly talented team of Investigators and a series of systematic logically designed studies are outlined which will first focus on the composition and arrangement of glycans in paired clones from T/F circulating HIV strains including N-and O-glycans as outlined in aim 1. Based on glycan profiles, we proposed to select a) a highly glycosylated, b) low level of env glycosylated viruses from transmitter and c) as control, a founder virus to prepare corresponding replication competent Simian Human Immunodeficiency viruses (SHIV) for further advanced studies, which include detailed in vitro characterization of replication kinetics in primary cells and cell lines, glycan content, particle envelope glycoprotein content, dendritic cell (DC) capture, DC-T cell trans-infection, sensitivity to IFN-α, anti-α4ß7 and blocking mAbs against select lectin-like molecules (Galectin-9, SIGLECs, MINCLE, and CD200/200R). Then we will select the viruses to test in vivo for transmission efficiency studies in rhesus macaques based primarily on comparable in vitro replication but differing glycan content (aim 2). Under specific aim 3, we will define whethe it is the signature site(s) of Env glycosylation that facilitates transmission, using isogenic SHIV based on the backbone of the "poor" transmitter and mutating only those select residues corresponding to the founder virus from aim 2b, using a repeated multiple-low-dose Intra-Vaginal (IVAG) challenge model. Results of these studies will provide a strong foundation for future rational HIV vaccine design.