Identification of the Initial Targets of Transmission

SIV/SHIV在恒河猴中的阴道和直肠传播模型为研究SIV/SHIV在恒河猴中的传播提供了潜在的见解。 在性行为期间粘膜暴露于HIV后建立系统性病毒获得的机制 传输感染的靶细胞的性质和阴道攻击后的传播部位仍然存在 只有部分定义，甚至更少的是已知的直肠传播的早期事件。来深入了解 感染的最初目标，我们已经开发了一种单轮双报告系统，可以特异性地 鉴定被挑战接种物感染的细胞。此外，我们最近报道，猕猴阴道 用双报告载体和可复制的SIV的混合物进行挑战使我们能够鉴定小的， 攻毒后48小时SIV复制的早期病灶。如《2000年世界卫生组织报告》所载初步数据所示， 应用中，我们也已经能够适应单轮双报告系统，以确定第一个细胞 在无创伤直肠攻击后感染有趣的是，我们对阴道和直肠早期传播的研究 用M-嗜性JRFL和T-嗜性SIVmac 239假型化的单轮双报告基因48小时 攻击后的细胞都显示出对相同的早期靶细胞的相似偏好，其中大多数细胞被感染 是Th 17细胞，未成熟DC是少数群体。这一观察表明，现有的 启动粘膜获取的靶细胞可能有限。为了扩展这些见解，我们将研究如何 不同的包膜蛋白和SIV蛋白Vpx影响早期向性。我们知道，后来在 发病机制和一般免疫激活，多种细胞类型可以感染和感染后耗尽 成为系统性的。因此，我们将通过检测SIV/SHIV靶细胞的早期变化来确定SIV/SHIV靶细胞的早期变化。 病毒传播的动力学，因为最初的感染灶从2天扩大到4天。最后，早在48小时内 在用SIVmac 239阴道攻击后，我们观察到宿主对粘膜感染的反应的证据，包括 凋亡的感染细胞、裂解的感染细胞和吞噬的感染细胞。同样，初步RNA-Seq 分析揭示了与48小时感染灶相关的基因表达的变化。通过组合 组织切片的显微镜分析和检测与宿主基因表达变化相关的细胞 使用荧光抗体和RNA探针，我们将能够可视化宿主对病毒的第一波反应， 病毒我们将确定谁，在哪里，什么时候，哪些细胞被感染，哪些细胞产生病毒 特定的警报，以及哪些细胞响应这些警报。总的来说，完成这些研究将 导致我们对阴道和直肠最早期事件级联的理解大大增加， 包括感染细胞的位置和表型以及先天宿主对感染的反应 在感染的最初几天内。更好地了解粘膜传播的最早期事件， 推动艾滋病预防科学工作的潜力。细胞类型和位置的知识， 最早的传播目标将揭示干预措施必须针对哪些地方，以产生最大影响。

Vaginal and rectal transmission models of SIV/SHIV in rhesus macaques offer potential insights into the mechanisms by which systemic viral acquisition is established after mucosal exposure to HIV during sexual transmission. The nature of target cells infected and the sites of transmission after vaginal challenge remain only partially defined, and even less is known about early events of rectal transmission. To gain insights into the initial targets of infection, we have developed a single-round dual reporter system that can specifically identify the cells infected by the challenge inoculum. Further, we have recently reported that macaque vaginal challenge with a mixture of the dual reporter vector and replication competent SIV enables us to identify small, early foci of SIV replication 48 hours post-challenge. As illustrated in preliminary data included in the application, we have also been able to adapt the single-round dual reporter system to identify the first cells infected after atraumatic rectal challenge. Interestingly, our studies of vaginal and rectal early transmission with the a single-round dual reporter pseudotyped with M-tropic JRFL and the T-tropic SIVmac239 48 hours post-challenge all show a similar preference for the same early target cells, with the majority of cells infected being Th17 cells, and the immature DC being a minor population. This observation suggests that the available target cells to initiate mucosal acquisition may be limited. To extend these insights, we will examine how different envelope proteins and the SIV protein Vpx influence early tropism. We know that later during pathogenesis and general immune activation, multiple cell types can be infected and depleted after infection becomes systemic. Therefore, we will determine early changes in the SIV/SHIV target cells by examining the kinetics of viral spread as the initial foci of infection expands from 2 to 4 days. Finally, as early as 48 hours post-vaginal challenge with SIVmac239, we observe evidence of host responses to mucosal infection including apoptotic infected cells, lysed infected cells, and phagocytosed infected cells. Likewise, preliminary RNA-Seq analysis revealed changes in gene expression associated with the 48 hour foci of infection. By combining microscopic analyses of tissue sections and detecting the cells associated with host gene expression changes using fluorescent antibodies and RNA probes, we will be able to visualize the first wave of host responses to the virus. We will define the who, where, and when of which cells are infected, which cells are generating virus specific alarms, and which cells are responding to these alarms. Collectively, completion of these studies will result in a great increase in our understanding of the cascade of the earliest events of vaginal and rectal transmission including the location and phenotype of infected cells and the innate host responses to infection within the first few days of infection. A better understanding of the earliest events of mucosal transmission has the potential to advance efforts in HIV prevention science. Knowledge of the cell type and location of the earliest targets of transmission will reveal where the intervention must be targeted for maximal impact.