Rotavirus: Studies of Intestinal Tropism and Innate and Heterotypic Immunity

描述(由申请人提供)： 轮状病毒(RV)是全球幼儿严重腹泻的最重要原因。这些病毒还会在健康的成年人、老年人和免疫系统受损的人群中引起腹泻疾病。轮状病毒主要在小肠的成熟绒毛顶端细胞中复制。轮状病毒以宿主特有的方式感染大多数哺乳动物；一般来说，在一种哺乳动物身上引起感染的轮状病毒株不会在另一种动物中引起疾病。几个同源的小鼠轮状病毒毒株的存在使小鼠模型成为研究微生物宿主范围限制、发病机制和免疫的一个高度易处理的实验系统，特别是当这些因素与粘膜感染有关时。最近的研究表明，以物种特有的方式抑制天然免疫是限制寄主范围的关键决定因素。自然轮状病毒感染有效地诱导异型(以及同型)保护免受症状性再次感染，尽管循环RV毒株之间存在巨大的血清型差异。B细胞和RV特异性抗体介导对症状性再感染的免疫。导致广泛交叉反应免疫的机制和限制寄主范围的机制都不清楚。为了解决这两个未知问题，我们提出了两个具体的目标：1)在体内研究同源小鼠和异种猴轮状病毒感染对STAT1和STAT3转录因子的影响，以进一步揭示RV宿主范围限制和先天免疫的机制。我们的假设是，同源RV由于能够抑制STAT1依赖的先天抗病毒反应而在肠道中成功复制，这是通过病毒激活STAT3实现的。我们建议使用新的质量细胞术和微流控QRT-PCR技术在单个肠道细胞的蛋白质和转录水平上表征依赖STAT1/3的先天信号对感染的反应。2)在克隆免疫球蛋白(Ig)水平上确定RV感染后异型保护性免疫的分子基础。我们将使用一种新的小鼠模型系统，非常适合研究克隆B细胞的反应。异型(血清交叉反应)免疫在预防轮状病毒疾病中起着关键作用，也是几种已建立的(例如，减毒活流感疫苗)和试验(例如，艾滋病毒和丙型肝炎)疫苗的一个非常可取的特征。我们将在单个Ig分子的水平上定义诱导异型中和反应的性质和条件。我们的假设是，血清型不同的RV毒株可以被针对两种“血清型特异性”RV表面蛋白中的任何一种的单个LG分子“中和”。为了验证这一假设，我们将使用一种新的系统来快速克隆和表达成百上千个来自免疫小鼠的RV特异性功能活性单抗(MAbs)。我们假设，具有异型特异性(以及同型反应性)的单个单抗是在单一天然抗原暴露后诱导的，这些“广泛反应”的Ig分子负责交叉保护免疫的发展。

DESCRIPTION (provided by applicant): Rotaviruses (RV) are the most important cause of severe diarrhea in young children worldwide. These viruses also cause diarrheal disease in healthy adults, the elderly, and the immune compromised. RV replicates primarily in the mature villous tip cells of the small intestine. Rotaviruses infect most mammals in a host-specific fashion; in general, RV strains that cause infection in one mammal do not cause disease in another species. The existence of several homologous murine RV strains that are natural pathogens of mice make the mouse model a highly tractable experimental system to study microbial host range restriction, pathogenesis, and immunity, especially as these factors relate to mucosal infections. Recent studies demonstrate that inhibition of innate immunity in a species-specific manner is a critical determinant of host range restriction. Natural RV infection effectively induces heterotypic (as well as homotypic) protection from symptomatic re-infection despite the existence of great serotype diversity among circulating RV strains. B cells and RV-specific antibodies mediate immunity to symptomatic re-infection. Neither the mechanism that results in broadly cross-reactive immunity nor the mechanistic basis for host range restriction is known. To address these two unknowns, we propose two Specific Aims: 1) Characterize in vivo the effects of homologous murine and heterologous simian RV infection on the STAT1 and STAT3 transcription factors to further unravel the mechanisms underlying RV host range restriction and innate immunity. Our hypothesis is that homologous RVs replicate successfully in the gut due to their ability to inhibit STAT1-dependent innate antiviral responses, and this is achieved by viral activation of STAT3. We propose to characterize STAT1/3- dependent innate signaling responses to infection at the protein and transcript level in individual intestinal cells using novl mass cytometry and microfluidic qRT-PCR technologies. 2) Determine, at the clonal immunoglobulin (Ig) level, the molecular basis for heterotypic protective immunity following RV infection. We will use a novel mouse model system ideally suited for the study of the clonal B cell response. Heterotypic (serotype cross-reactive) immunity plays a critical role in preventing RV disease and also is a highly desirable feature of several established (e.g., live attenuated influenza vaccine) and experimental (e.g., HIV and HCV) vaccines. We will define, at the level of individual Ig molecules, the nature and conditions for induction of heterotypic neutralizing reactivity. Our hypothesis is that serotypically diverse RV strains can be "neutralized" by individual lg molecules directed at either of the two "serotype-specific" RV surface proteins. To test this hypothesis, we will use a novel system to rapidly clone and express hundreds to thousands of RV-specific functionally active monoclonal antibodies (mAbs) from immunized mice. We hypothesize that individual mAbs with heterotypic specificity (as well with homotypic reactivity) are induced following a single natural antigen exposure and that these "broadly reactive" Ig molecules are responsible for the development of cross-protective immunity.