Animal Model for SIV Infection Control

描述（由申请人提供）：我们通过将恒河猴（RM）暴露于SIVagm，开发了一种受控的SIV感染模型。我们的初步结果表明，急性SIVagm感染的RM的特点是：高水平的病毒复制，和戏剧性的急性粘膜CD 4 + T细胞耗竭，都类似于致病性SIV/HIV-1感染。令人惊讶的是，在慢性感染期间观察到SIVagm复制的完全控制，导致在随访期间所有生物学参数恢复到基线。这些精英控制者的实验性CD 8细胞耗竭导致VL短暂增加。我们的假设是，细胞免疫反应是负责控制SIVagm复制和缺乏疾病进展的RM。我们的总体目标是建立保护精英SIV控制者的相关因素，并将这一知识扩展到致病性HIV-1感染。具体目标是：具体目标1。通过以下方法彻底研究RM中控制性SIVagm感染的发病机制：（a）定义RM中SIVagm感染的病毒学和免疫学参数，（B）表征RM组织中SIVagm感染的时间和程度，（c）研究RM控制性SIVagm感染期间的“保护区沉默”，以及（d）确定整合SIVagm的稳定储库。RM将接种SIVagm，并在连续时间点处以安乐死。将监测VL、CD 4 + T细胞动力学、免疫活化、细胞增殖和凋亡。将通过巢式PCR、实时PCR和原位杂交来确定初次感染期间SIVagm的组织定殖以及随后时间点的“避难所沉默”。RM中稳定整合的SIVagm储库将通过研究从不同组织分离的静息CD 4 + T细胞中的复制能力病毒来检测。将通过实时PCR对尸检时检查的组织中纯化的静息CD 4 + T细胞中的SIVagm DNA进行定量。SIVagm感染的RM控制者的CD 8耗竭将补充用于研究SIVagm储库的体外测定，并将确定负责血浆病毒血症反弹的组织储库再活化的顺序和时间。具体目标2。目的研究细胞免疫在RM中控制SIVagm复制中的作用。我们将：（i）通过Elispot、多功能细胞内细胞因子染色（ICS）以及颗粒酶和穿孔素表达的评估来确定血液和组织中CD 8+和CD 4 + T细胞应答的动力学、幅度、广度和质量。(ii)在原发性SIVagm感染期间耗尽RM中的免疫应答，并通过消除B细胞维持CD 8消融。(iii)通过使用Ontak（一种在NHP中诱导免疫激活的药物），区分细胞免疫应答与免疫激活的相对贡献（作为CD 8耗竭后病毒反弹的机制）。结合起来，这些目标旨在彻底表征长期非进展性SIV感染，并确定负责这种高效控制病毒的机制。公共卫生相关性：该项目旨在开发一种用于控制SIV和HIV感染的动物模型。这样的模型是迫切需要了解精英控制器的免疫保护的相关性，它还没有。在我们的初步研究中，我们已经表明，感染了来自非洲绿色猴的SIV的恒河猴（RM）在急性感染期间将病毒复制到高水平，但后来有能力完全控制病毒，因此在随访期间显示出完全恢复的免疫力。然而，在SIVagm感染的控制RM中，体内CD 8细胞的耗竭导致病毒反弹，表明强细胞免疫应答是病毒控制的原因。在这个新的模型中破译病毒控制的机制可能会大大有助于我们理解艾滋病毒的发病机制和设计新的方法来控制艾滋病毒感染。

DESCRIPTION (provided by applicant): We developed a model of controlled SIV infection by exposing rhesus macaques (RMs) to SIVagm. Our preliminary results showed that acute SIVagm infection in RMs is characterized by: high levels of viral replication, and dramatic acute mucosal CD4+ T-cell depletion, both similar to pathogenic SIV/HIV-1 infections. Surprisingly, a complete control of SIVagm replication was observed during chronic infection, resulting in the return to baseline of all the biological parameters during the follow-up. Experimental CD8 cell depletion in these elite controllers resulted in transient increases in VLs. Our hypothesis is that cellular immune responses are responsible for the control of SIVagm replication and lack of disease progression in RMs. Our overall objective is to establish the correlates of protection of elite SIV controllers and to expand this knowledge to pathogenic HIV-1 infection. The specific aims are: Specific Aim 1. To thoroughly study the pathogenesis of controlled SIVagm infection in RMs by: (a) defining the virological and immunological parameters of SIVagm infection of RMs, (b) characterizing the timing and extent of SIVagm infection in tissues of RMs, (c) studying "sanctuary silencing" during controlled SIVagm infection of RMs, and (d) determining the stable reservoirs of integrated SIVagm. RMs will be inoculated with SIVagm and euthanized at sequential time points. VLs, CD4+ T-cell dynamics, immune activation, cell proliferation and apoptosis will be monitored. Tissue colonization with SIVagm during the primary infection as well as "sanctuary silencing" at later time points will be determined by nested PCR, real-time PCR and in situ hybridization. Stable integrated SIVagm reservoirs in RMs will be detected by studying the replication-competent virus in resting CD4+ T- cells isolated from different tissues. Quantification of SIVagm DNA in purified resting CD4+ T-cells from tissues examined at necropsy will be done by real-time PCR. CD8 depletion of SIVagm-infected RMs controllers will complement the in vitro assays for the study of SIVagm reservoirs and will determine the order and timing of reactivation of tissue reservoirs responsible for the rebound of plasma viremia. Specific Aim 2. To study the role of cellular immunity in control of SIVagm replication in RMs. We will: (i) Determine the dynamics, magnitude, breadth and quality of CD8+ and CD4+ T cell responses in blood and tissues by Elispot, polyfunctional intracellular cytokine staining (ICS), and assessment of granzyme and perforin expression. (ii) deplete immune responses in RMs during primary SIVagm- infection and maintain the CD8 ablation through elimination of B cells. (iii) Differentiate between the relative contribution of cellular immune responses versus immune activation as the mechanism of viral rebound after CD8 depletion, by using Ontak, a drug that induces immune activation in NHPs. Combined, these aims are designed to thoroughly characterize a long-term nonprogressive SIV infection and to determine the mechanisms responsible for this highly efficient control of virus. PUBLIC HEALTH RELEVANCE: This project is intended to develop an animal model for the control of SIV and HIV infections. Such a model is badly needed to understand the correlates of immune protection in elite controllers and it is not yet available. In our preliminary studies, we have shown that rhesus macaques (RMs) infected with SIV from African green monkeys have replicated the virus to high levels during acute infection but later have the ability to completely control the virus and consequently show complete restoration of immunity during the follow-up. However, in vivo depletion of CD8 cells in SIVagm-infected controller RMs resulted in the rebound of virus showing that strong cellular immune responses are responsible for viral control. Deciphering the mechanisms of viral control in this new model may significantly contribute to our understanding of HIV pathogenesis and design of new approaches for controlling HIV infection.