Animal Model for SIV Infection Control

描述(申请人提供)：我们开发了一种通过将SIVagm暴露于恒河猴(RMS)来控制SIV感染的模型。我们的初步结果表明，RMS的急性SIVagm感染的特征是：病毒复制水平高，急性粘膜CD4+T细胞急剧耗尽，这两者都与病原性SIV/HIV-1感染相似。令人惊讶的是，在慢性感染期间观察到SIVagm复制完全控制，导致在随访期间所有生物学参数恢复到基线。在这些精英控制者中，实验性的CD8细胞耗尽导致VLS的一过性增加。我们的假设是，细胞免疫反应负责控制SIVagm复制和RMS中疾病进展的缺乏。我们的总体目标是建立保护精英SIV控制器的相关性，并将这一知识扩展到致病性HIV-1感染。具体目的是：1.通过以下方法深入研究RMS受控SIVagm感染的发病机制：(A)确定RMS SIVagm感染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)研究受控SIVagm感染RMS过程中的“避难所沉默”；以及(D)确定整合SIVagm的稳定储存库。RMS将接种SIVagm，并在连续的时间点实施安乐死。将监测VLS、CD4+T细胞动力学、免疫激活、细胞增殖和凋亡。通过套式聚合酶链式反应、实时聚合酶链式反应和原位杂交，将确定初次感染期间使用SIVagm的组织定植以及稍后时间点的“避难所沉默”。通过研究从不同组织分离的静止的CD4+T细胞中具有复制能力的病毒，将检测到RMS中稳定整合的SIVagm储存库。从尸检组织中提取的纯化的静止的CD4+T细胞中的SIVagm DNA将通过实时荧光聚合酶链式反应进行定量。SIVagm感染的RMS控制器的CD8耗尽将补充SIVagm储存库研究的体外检测，并将确定导致血浆病毒血症反弹的组织储存库重新激活的顺序和时间。具体目的2.研究细胞免疫在RMS SIVagm复制调控中的作用。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CD8+和CD4+T细胞反应的动态、幅度、广度和质量。(Ii)在原发SIVagm感染期间耗尽RMS的免疫反应，并通过清除B细胞来维持CD8的消融。(Iii)通过使用在NHP中诱导免疫激活的药物Ontak，区分CD8耗尽后病毒反弹的机制是细胞免疫反应的相对贡献还是免疫激活。这些目标结合在一起，旨在彻底描述一种长期的非进行性SIV感染的特征，并确定这种高效控制病毒的机制。公共卫生相关性：该项目旨在开发一种控制SIV和艾滋病毒感染的动物模型。要了解精英控制者免疫保护的相关性，迫切需要这样的模型，但目前还没有这种模型。在我们的初步研究中，我们已经证明，感染了非洲绿猴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，从而在随访期间表现出完全的免疫恢复。然而，在体内，SIVagm感染控制器RMS中CD8细胞的耗尽导致病毒反弹，表明强烈的细胞免疫反应是病毒控制的原因。在这个新的模型中破译病毒控制的机制可能会对我们理解HIV的发病机制和设计控制HIV感染的新方法有很大帮助。

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.