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和导致产生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一种识别粘膜传播HIV-1和SIV病毒基因组的新策略来解决这一优先领域（Keele, PNAS 2008; Keele, J Exp Med 2009）。这一策略基于在随机病毒进化模型的背景下对血浆病毒RNA进行单基因组扩增、测序和分析，确定了那些实际负责传播和生产感染的病毒。这一创新，反过来，使分子克隆和实际传播/创始人SIV病毒的生物学分析首次成为可能。在这项应用中，我们建议将这种新方法扩展到siv -猕猴感染模型中，并验证以下假设：可以鉴定出与传播/建立者病毒相对应的全长SIVsmE660和SIVmac251病毒基因组的分子克隆，将被证明具有传染性和复制能力，并将概括印度恒河猴的致病性感染。这些克隆代表了一种新的分子试剂，可以用来破译负责粘膜SIV传播的最早的病毒-宿主细胞相互作用，并可以为传播、发病机制和疫苗研究提供新的分子定义的病毒攻击株。项目的具体目标被组织到R21（目标#1-3）和R33（目标#4-5）阶段，这些阶段可以做出做/不做的决定。目的是：1)鉴定低剂量粘膜（ir和ivag）接种后产生临床感染的传播/创始SIVsmE660和SIVmac251病毒；2)分子克隆全长传播/建立SIVsmE660和SIVmac251原病毒基因组；3)比较分子克隆衍生的SIVsmE660和SIVmac251病毒株与SIVsmE660和SIVmac251病毒分离株在体外复制效率、细胞趋向性和包膜表型方面的生物学特性；4)测定克隆SIVsmE660和SIVmac251病毒株在印度恒河猴低剂量粘膜接种后的传染性、复制动力学和致病性；5)表征病毒在传播和建立设定点病毒血症之间的多样化和适应性的分子途径，作为同源和异源疫苗攻击研究的前奏。这些研究结果有望揭示SIVsmE660和SIVmac251粘膜传播的分子基础，确定保护性疫苗引发免疫反应的新潜在靶点，并为SIV传播、发病机制和疫苗研究提供急需的具有遗传多样性的粘膜传播SIV菌株的分子克隆。

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.