Role of HIV Env glycosylation in mucosal transmission

描述（由申请人提供）：对促进传播的病毒/宿主关系以及对感染后疾病进展率有贡献的病毒/宿主关系的精确理解对于设计有效的HIV-1疫苗至关重要。一个越来越被认为是至关重要但研究不足的参数是病毒环境糖基化不仅在优先传播中起作用，而且在病毒选择进入受体的能力和对中和抗体的易感性方面也起作用。此外，越来越多的数据表明，作为糖基化env蛋白的配体/受体的各种细胞表面分子，以及那些受糖基化或去糖基化病毒影响的细胞表面分子，可能有助于病毒产生的免疫反应的质量。这些问题只能通过适当的非人灵长类动物模型来客观地解决，例如猕猴感染了含有原发HIV-1分离株，特别是传播/创始病毒的env的shiv，以研究env糖基化在优先传播中的作用。因此，我们计划利用从赞比亚队列中获得的传播/创始人（T/F）病毒的HIV分子克隆来探索env聚糖的作用，其中进化支C病毒占主导地位。我们已经制作了一些独特的工具/试剂，并组建了一个非常有才华的研究团队，并概述了一系列系统的逻辑设计的研究，这些研究将首先关注T/F循环HIV株配对克隆中的聚糖的组成和排列，包括目标1中概述的n和o聚糖。基于糖基化特征，我们建议选择a)高糖基化，b)低水平env糖基化的递质病毒和c)作为对照，制备相应的具有复制能力的猿人免疫缺陷病毒（SHIV）进行进一步的研究，包括在原代细胞和细胞系中详细的体外复制动力学表征，聚糖含量，颗粒包膜糖蛋白含量，树突状细胞（DC）捕获。DC-T细胞转染，对IFN-α、抗-α4ß7的敏感性，以及针对特定凝集素样分子（Galectin-9、SIGLECs、MINCLE和CD200/200R）的阻断单抗。然后，我们将选择病毒在体内进行恒河猴的传播效率研究，主要基于可比较的体外复制，但不同的聚糖含量（目标2）。在特定目标3下，我们将定义Env糖基化的特征位点是否促进传播，使用基于“差”递质中枢的等基因SHIV，并使用重复多次低剂量阴道内（IVAG）攻击模型，仅突变那些与aim 2b中创建病毒对应的选定残基。这些研究结果将为今后合理设计HIV疫苗提供坚实的基础。

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.