Rotavirus: Studies of Intestinal Tropism and Innate and Heterotypic Immunity

轮状病毒：肠道趋向性以及先天性和异型免疫的研究

• 批准号: 8993853
• 负责人: Harry Bernard Greenberg
• 金额: --
• 依托单位: VETERANS ADMIN PALO ALTO HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8993853
• 关键词: Acute; Address; Adult; Antibodies; Antibody-mediated protection; Antigens; Antiviral Agents; Antiviral Response; Apoptotic; Attenuated; B-Lymphocytes; Binding; Biological Models; Cells; Cessation of life; Child; Cytometry; Development; Diarrhea; Disease; Elderly; Enteral; Epithelial Cells; Epithelium; Epitopes; Gastrointestinal tract structure; Generations; HIV/HCV; Hepatitis C Vaccine; Immune; Immunity; Immunization; Immunocompromised Host; Immunoglobulins; Individual; Infant; Infection; Influenza; Interferons; Intestines; Knowledge; Licensing; Life; Mammals; Measurement; Mediating; Membrane Proteins; Microfluidics; Molecular; Mono-S; Monoclonal Antibodies; Morbidity - disease rate; Mus; Natural Immunity; Nature; Pathogenesis; Pattern; Phosphorylation; Play; Population; Proteins; Role; Rotavirus; Rotavirus Infections; Rotavirus Vaccines; Rotavirus disease; STAT1 gene; STAT3 gene; Serotyping; Services; Signal Transduction; Small Intestines; Sorting - Cell Movement; Specificity; Surface; System; Technology; Testing; Tissues; Transcript; Tropism; Vaccines; Veterans; Villous; Viral; Virus; Virus Activation; arm; attenuation; base; biodefense; cell type; combinatorial; in vivo; influenza virus vaccine; member; microbial; microbial host; mortality; mouse model; neutralizing antibody; novel; pathogen; prevent; public health relevance; response; single cell analysis; transcription factor

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.

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。这些病毒也会导致健康成年人、老年人和免疫功能低下的人患上腹泻病。RV主要在小肠的成熟绒毛尖端细胞中复制。轮状病毒以宿主特异性方式感染大多数哺乳动物;通常，在一种哺乳动物中引起感染的RV毒株不会在另一种物种中引起疾病。存在的几个同源的小鼠RV菌株，是小鼠的天然病原体，使小鼠模型的一个非常听话的实验系统，研究微生物的宿主范围限制，发病机制，和免疫，特别是因为这些因素涉及到粘膜感染。最近的研究表明，以物种特异性方式抑制先天免疫是宿主范围限制的关键决定因素。自然RV感染有效地诱导异型（以及同型）保护症状的再感染，尽管存在很大的血清型多样性之间循环RV菌株。B细胞和RV特异性抗体介导对症状性再感染的免疫。导致广泛交叉反应性免疫的机制和宿主范围限制的机制基础都不清楚。 为了解决这两个未知数，我们提出了两个具体目标：1）在体内表征同源鼠和异源猴RV感染对STAT 1和STAT 3转录因子的影响，以进一步阐明RV宿主范围限制和先天免疫的机制。我们的假设是，同源RV在肠道中成功复制，这是由于它们能够抑制STAT 1依赖的先天性抗病毒反应，这是通过病毒激活STAT 3实现的。我们建议使用novl质量细胞术和微流控qRT-PCR技术在蛋白质和转录水平上表征单个肠细胞中对感染的STAT 1/3依赖性先天信号传导反应。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细胞反应。异型（血清型交叉反应性）免疫在预防RV疾病中起关键作用，并且也是几种已建立的（例如，减毒活流感疫苗）和实验性（例如，HIV和HCV）疫苗。我们将在单个IG分子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菌株可以被“中和”的个人免疫球蛋白分子直接在两个“特异性”RV表面蛋白。为了验证这一假设，我们将使用一种新的系统，从免疫小鼠中快速克隆和表达数百至数千种RV特异性功能活性单克隆抗体（mAb）。我们假设，具有异型特异性（以及同型反应性）的单个mAb在单一天然抗原暴露后诱导，并且这些“广泛反应性”的IG分子负责交叉保护性免疫的发展。