Animal Model for SIV Infection Control

描述（由申请人提供）：我们通过将恒河猴（RMs）暴露于SIVagm，建立了一个控制SIV感染的模型。我们的初步结果表明，rm急性SIVagm感染的特征是：高水平的病毒复制和急剧的急性粘膜CD4+ t细胞耗竭，两者与致病性SIV/HIV-1感染相似。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CD8细胞耗竭导致VLs的短暂增加。我们的假设是，细胞免疫反应负责控制SIVagm复制和RMs疾病进展的缺乏。我们的总体目标是建立精英SIV控制者保护的相关性，并将这一知识扩展到致病性HIV-1感染。具体目标是：通过(a)确定RMs感染SIVagm的病毒学和免疫学参数，(b)确定RMs组织中SIVagm感染的时间和程度，(c)研究RMs控制SIVagm感染期间的“避难所沉默”，以及(d)确定集成SIVagm的稳定储存库，深入研究RMs控制SIVagm感染的发病机制。rm将接种SIVagm，并在顺序的时间点实施安乐死。监测VLs、CD4+ t细胞动力学、免疫激活、细胞增殖和凋亡。SIVagm在初次感染期间的组织定植以及随后时间点的“避难所沉默”将通过巢式PCR、实时PCR和原位杂交来确定。通过研究从不同组织分离的静止CD4+ T细胞中的复制能力病毒，可以检测到RMs中稳定的集成SIVagm储库。SIVagm DNA的定量将通过实时聚合酶链反应（real-time PCR）在尸检组织中检测纯化的静息CD4+ t细胞。SIVagm感染的RMs控制器的CD8耗损将补充SIVagm储库研究的体外试验，并将确定负责血浆病毒血症反弹的组织储库的重新激活的顺序和时间。具体目标2。目的：研究细胞免疫在RMs中SIVagm复制控制中的作用。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评估，确定血液和组织中CD8+和CD4+ T细胞反应的动态，大小，广度和质量。（ii）在原发性SIVagm-感染期间，RMs中的免疫应答被耗尽，并通过消除B细胞维持CD8消融。（iii）通过使用一种在NHPs中诱导免疫激活的药物Ontak，区分细胞免疫应答与免疫激活作为CD8耗损后病毒反弹机制的相对贡献。综上所述，这些目标旨在彻底表征长期非进展性SIV感染，并确定这种高效控制病毒的机制。公共卫生相关性：该项目旨在开发控制SIV和HIV感染的动物模型。这种模型对于理解精英控制器中免疫保护的相关关系是非常必要的，但目前还没有。在我们的初步研究中，我们已经表明，感染了来自非洲绿猴的SIV的恒河猴（RMs）在急性感染期间复制了高水平的病毒，但后来有能力完全控制病毒，因此在随访期间显示完全恢复免疫力。然而，在sivagm感染的对照RMs中，CD8细胞的体内消耗导致病毒反弹，这表明强大的细胞免疫反应是病毒控制的原因。在这个新模型中破译病毒控制的机制可能有助于我们理解HIV的发病机制和设计控制HIV感染的新方法。

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.