Cloning, characterization and in vivo testing of mucosally transmitted SIV

项目概要/摘要 HIV/SIV疫苗研究中最重要的知识空白之一涉及以下的分子特性： 负责粘膜传播的病毒和导致 病毒感染。本提案通过利用我们的 实验室最近发现了一种新的策略，用于识别粘膜传播的HIV-1和SIV病毒 基因组（Keele，PNAS 2008; Keele，J Exp Med 2009）。这种基于单基因组扩增的策略， 在随机病毒进化模型的背景下对血浆病毒RNA进行测序和分析， 这些病毒实际上负责传播和生产性感染。这种创新反过来 首次实现了对实际传播/创始SIV的分子克隆和生物学分析 病毒在这个应用中，我们建议将这种新方法扩展到SIV-猕猴感染模型， 为了验证以下假设：全长SIVsmE 660和SIVmac 251病毒基因组的分子克隆 可以识别对应于传播/创始病毒的病毒，将显示其具有传染性和复制性 主管，并将重演病原体感染印度恒河猴。这种克隆代表了新的 用于解释粘膜SIV最早期病毒-宿主细胞相互作用的分子试剂 传播，并可提供新的分子定义的病毒攻击株的传播，致病 疫苗研究。项目的具体目标分为R21（目标#1-3）和R33（目标#4-5）。 阶段服从进行/不进行决定。目的是：1）识别传播/创始人SIVsmE 660和 SIVmac 251病毒负责建立生产性临床感染后，低剂量粘膜（ir和 IVag）接种; 2）分子克隆全长传播/创始人SIVsmE 660和SIVmac 251前病毒 分子克隆SIVsmE 660和SIVmac 251病毒株的生物学特性 与SIVsmE 660和SIVmac 251病毒分离株在体外的复制效率相比， 4）测定克隆的大肠杆菌的感染性、复制动力学和致病性 低剂量粘膜接种后印度恒河猴中的SIVsmE 660和SIVmac 251病毒株; 5)为了表征病毒在传播和感染之间的多样化和适应性的分子途径， 建立设定点病毒血症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揭示粘膜传播的分子基础， SIVsmE 660和SIVmac 251，鉴定保护性疫苗引发的免疫应答的新的潜在靶点，和 提供急需的遗传多样性、粘膜传播的SIV毒株的分子克隆， 传播、发病机理和疫苗研究。

Project Summary/Abstract 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.