Cloning, characterization and in vivo testing of mucosally transmitted SIV

描述（由申请人提供）：HIV/SIV疫苗研究中最重要的知识空白之一涉及负责粘膜传播的病毒的分子特性以及导致生产性病毒感染的初始病毒-宿主细胞相互作用。本提案通过利用我们实验室最近发现的用于鉴定粘膜传播的HIV-1和SIV病毒基因组的新策略来解决该优先领域（Keele，PNAS 2008; Keele，J Exp Med 2009）。该策略基于随机病毒进化模型背景下的血浆病毒RNA的单基因组扩增、测序和分析，鉴定了实际负责传播和生产性感染的那些病毒。这一创新，反过来，使第一次有可能的分子克隆和生物学分析的实际传播/创始人SIV病毒。在本申请中，我们建议将这种新方法扩展到SIV猕猴感染模型，并测试以下假设：可以识别与传播/创始病毒对应的全长SIVsmE 660和SIVmac 251病毒基因组的分子克隆，将被证明具有传染性和复制能力，并将重演印度恒河猴的病原性感染。这样的克隆代表了新的分子试剂，用其来破译负责粘膜SIV传播的最早的病毒-宿主细胞相互作用，并且可以为传播、发病机理和疫苗研究提供新的分子定义的病毒攻击株。该项目的具体目标分为R21（目标#1-3）和R33（目标#4-5）阶段，可根据进行/不进行决定。目标是：1）鉴定负责在低剂量粘膜免疫后建立生产性临床感染的传播/创始SIVsmE 660和SIVmac 251病毒。（2）分子克隆全长传播/创始SIVsmE 660和SIVmac 251前病毒基因组; 3）对分子克隆进行生物学表征-衍生的SIVsmE 660和SIVmac 251病毒株与SIVsmE 660和SIVmac 251病毒分离株在体外的复制效率，细胞嗜性，和包膜表型; 4）确定低剂量粘膜接种后克隆的SIVsmE 660和SIVmac 251病毒株在印度恒河猴中的感染性、复制动力学和致病性; 5）表征病毒多样性的分子途径以及在传播和建立设定点病毒血症之间的适应性，作为同源和异源疫苗攻击研究的前奏。这些研究的结果有望阐明SIVsmE 660和SIVmac 251粘膜传播的分子基础，确定新的潜在靶点，为保护性疫苗引起的免疫反应，并提供急需的遗传多样性，粘膜传播的SIV株的传播，发病机制和疫苗研究的分子克隆。 公共卫生相关性：艾滋病毒/艾滋病疫苗研究中最重要的知识差距之一涉及HIV-1的分子特性，这些特性是性传播的原因，也是导致产生性病毒感染的最初病毒-宿主细胞相互作用的原因。本建议利用我们实验室最近发现的一种新的方法，用于识别传播的HIV-1病毒，并将这种新技术应用于发现和表征传播的猿猴免疫缺陷病毒（SIV）。这些结果将揭示SIV在恒河猴模型中粘膜传播的分子基础，并有望帮助识别新的疫苗靶点和策略。

DESCRIPTION (provided by applicant): One of the most important knowledge gaps in HIV/SIV vaccine research relates to the molecular properties of viruses that are responsible for mucosal transmission and the initial virus-host cell interactions that lead to productive viral infection. The present proposal addresses this priority area by taking advantage of our laboratory's recent discovery of a novel strategy for identifying mucosally transmitted HIV-1 and SIV viral genomes (Keele, PNAS 2008; Keele, J Exp Med 2009). This strategy, based on single genome amplification, sequencing and analysis of plasma viral RNA within the context of a model of random virus evolution, identifies those viruses that are actually responsible for transmission and productive infection. This innovation, in turn, makes possible for the first time the molecular cloning and biological analysis of actual transmitted/founder SIV viruses. In this application, we propose to extend this new approach to the SIV-macaque infection model and to test the following hypothesis: Molecular clones of full-length SIVsmE660 and SIVmac251 viral genomes corresponding to transmitted/founder viruses can be identified, will be shown to be infectious and replication competent, and will recapitulate pathogenic infection in Indian rhesus macaques. Such clones represent novel molecular reagents with which to decipher the earliest virus-host cell interactions responsible for mucosal SIV transmission and can provide new molecularly-defined virus challenge strains for transmission, pathogenesis and vaccine research. Specific aims of the project are organized into R21 (Aims #1-3) and R33 (Aims #4-5) phases amenable to a go/no-go decision. Aims are: 1) To identify transmitted/founder SIVsmE660 and SIVmac251 viruses responsible for establishing productive clinical infection following low-dose mucosal (ir and ivag) inoculation; 2) To molecularly clone full-length transmitted/founder SIVsmE660 and SIVmac251 proviral genomes; 3) To biologically characterize molecular clone-derived SIVsmE660 and SIVmac251 virus strains compared with SIVsmE660 and SIVmac251 virus isolates in vitro with respect to replication efficiency, cell tropism, and envelope phenotype; 4) To determine infectivity, replication kinetics, and pathogenicity of cloned SIVsmE660 and SIVmac251 virus strains in Indian rhesus macaques following low-dose mucosal inoculation; 5) To characterize molecular pathways of virus diversification and adaptation between transmission and the establishment of set-point viremia as a prelude to homologous and heterologous vaccine-challenge studies. Results from these studies promise to shed new light on the molecular basis of mucosal transmission by SIVsmE660 and SIVmac251, identify new potential targets for protective vaccine-elicited immune responses, and provide much needed molecular clones of genetically-diverse, mucosally-transmitted SIV strains for transmission, pathogenesis and vaccine research. PUBLIC HEALTH RELEVANCE: One of the most important knowledge gaps in HIV/AIDS vaccine research relates to the molecular properties of HIV-1 that are responsible for sexual transmission and the initial virus-host cell interactions that lead to productive viral infection. The present proposal takes advantage of our laboratory's recent discovery of a novel method for identifying transmitted HIV-1 viruses and applies this new technology to the discovery and characterization of transmitted simian immunodeficiency viruses (SIV). These results will shed new light on the molecular basis of mucosal transmission by SIV in the rhesus macaque model and promise to aide in the identification of new vaccine targets and strategies.