EVALUATION OF TWO NEW CHALLENGE POOLS OF NORWALK VIRUS INOCULA IN HUMAN SUBJE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。诺如病毒是引起流行性非细菌性胃肠炎(肠道流感)的最常见原因。诺沃克病毒(Norwalk Virus，NV)是这组病毒的原型株。由于无法在细胞培养中培养病毒，也缺乏动物模型，要想了解其分子发病机制，需要在人类身上进行许多实验。用于研究的病毒的主要来源是从实验中感染了NIH(NIH-8FIIa)提供的NV的人类志愿者那里获得的粪便样本。利用这些样本对基因组进行了测序，并在杆状病毒表达系统中表达了第二个开放阅读框架(ORF)，在那里它产生了重组的、非传染性的病毒样颗粒(RNV-VLP)，这些颗粒是不含病毒核酸的蛋白质外壳。该小组最近的一项研究表明，RNV-VLP制剂(一种原型疫苗)，当口服给人类志愿者时，似乎是安全的，并产生免疫球蛋白G免疫反应。目前尚不清楚这种免疫反应是否对疾病具有保护作用。该方案旨在开发病毒挑战模型，以测试疫苗的效力。以前用NV挑战池进行的人类挑战研究表明，在未选定的成年人群中，感染率约为80%。随后对挑战材料的研究表明，分泌者状态是感染的重要预测因素。缺乏H-1型寡糖的表达与NV攻击后对感染的保护有关，而在表达H-1型寡糖的患者中，感染的发生率为97%。由于原来的NV挑战池不再可用，我们使用从大约14年前感染NV(NIH-8FIIa)的健康受试者那里获得的储存粪便样本来产生新的挑战池(滤液)。这是在GMP条件下完成的。新的攻击池(批次42399)的病毒抗原和逆转录聚合酶链式反应分析(病毒-核糖核酸滴度)与NIH-8FIIa NV批次的残留样本进行了很好的比较。用细胞培养、逆转录聚合酶链式反应和体内试验检测了42399批次的等量样品，没有检测到不定性物。此外，批次42399的等量在动物身上进行了测试，并根据21 CFR 610.11通过了一般安全测试。最后，联系了粪便捐赠者，临床实验室测试包括艾滋病毒、肝炎和梅毒以及肝功能测试都是正常的。这名受试者的PPD结核病检测也呈阴性，并继续保持着极好的健康状况。这项研究是在DMID/NIAID/NIH提交的IND申请的支持下进行的。这项研究的目标是建立新的NV挑战池的安全性、传染性和临床发病率。健康的分泌者状态阳性的志愿者将接受NV(批次42399)的挑战，剂量与先前挑战研究中使用的剂量大致相同。预计大约三分之二的感染者会出现症状。随后，较小的受试者群体将接受较低剂量的挑战，以确定用于疫苗模型后续研究的人类感染剂量的50%。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Noroviruses are the most common cause of epidemic, non-bacterial gastroenteritis (intestinal flu). Norwalk virus (NV) is the prototype strain of this group of viruses. Due to the inability to grow the virus in cell culture and the lack of an animal model, progress in understanding its molecular pathogenesis has required that many experiments be done in humans. The principal source of virus for research has been stool samples obtained from human volunteers who were experimentally infected with NV supplied by the NIH (NIH-8fIIa). Using these samples the genome was sequenced and the second open reading frame (ORF) was expressed in the baculovirus expression system where it produced recombinant, non-infectious virus-like particles (rNV-VLPs) which are protein shells that do not contain viral nucleic acids. A recent study by this group demonstrated that a rNV-VLP preparation (a prototype vaccine), when given by mouth to human volunteers, appeared safe and produced an IgG immune response. It remains unknown whether this immune response is protective against disease. This protocol is designed to develop the virus challenge model to test vaccine efficacy. Previous human challenge studies performed with a NV challenge pool an showed infection rate of ~80% in an unselected adult populations. Subsequent studies of challenge material showed that secretor status was an important predictor of infection. Lack of expression of a H type-1 oligosaccharide was associated with protection from infection following NV challenge whereas infection occurred in >97% among those with H type-1 oligosaccharide expression. As the original NV challenge pool was no longer available, we used stored stool specimens obtained from an otherwise healthy subject infected approximately 14 years ago with NV (NIH-8fIIa) to produce a new challenge pool (filtrate). This was done under GMP conditions. The viral antigen and RT-PCR analysis (viral-RNA titer) of the new challenge pool (Lot 42399) compared well with a residual sample of the NIH-8fIIa NV lot. Aliquots of Lot 42399 were tested using cell culture, RT-PCR, and in vivo tests and no adventitious agents were detected. In addition, aliquots of Lot 42399 were tested in animals and passed tests for General Safety according to 21 CFR 610.11. Finally, the stool donor was contacted and clinical laboratory tests including HIV, hepatitis panel, and syphilis as well as liver function tests were all normal. The subject also had a negative PPD test for tuberculosis and continues in excellent health. This study is conducted under the auspices of an IND application submitted by the DMID/NIAID/NIH. The goal of this study is to establish the safety and infectivity and clinical attack rate of the new NV challenge pool. Healthy secretor status positive volunteers will be challenged with a dose of NV (Lot 42399) that approximates the dose used in previous challenge studies. Approximately two-thirds of infected subjects are expected to become symptomatic. Subsequently, smaller groups of subjects will be challenged with lower dosages to define the Human Infectious Dose 50% for subsequent studies in the vaccine model.