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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。诺如病毒是流行性非细菌性胃肠炎（肠道流感）的最常见原因。诺瓦克病毒（NV）是这组病毒的原型株。由于无法在细胞培养中培养病毒和缺乏动物模型，了解其分子发病机制的进展需要在人类中进行许多实验。用于研究的病毒的主要来源是从人类志愿者获得的粪便样本，这些志愿者实验性地感染了由NIH提供的NV（NIH-8 fIIa）。使用这些样品对基因组进行测序，并在杆状病毒表达系统中表达第二个开放阅读框（ORF），在该系统中产生重组的非感染性病毒样颗粒（rNV-VLP），其是不含病毒核酸的蛋白质壳。该小组最近的一项研究表明，rNV-VLP制剂（原型疫苗），当口服给人类志愿者时，似乎是安全的，并产生IgG免疫应答。目前尚不清楚这种免疫反应是否能预防疾病。本方案旨在开发病毒攻毒模型，以检测疫苗有效性。先前使用NV攻毒池进行的人体攻毒研究显示，在成年人群中的感染率约为80%。随后对攻毒材料的研究表明，分泌状态是感染的重要预测因素。缺乏H型-1寡糖的表达与NV攻击后的感染保护相关，而在具有H型-1寡糖表达的那些中感染发生率>97%。由于原始NV攻毒样本池不再可用，我们使用从约14年前感染NV（NIH-8 fIIa）的健康受试者中获得的储存粪便标本制备新的攻毒样本池（滤液）。这是在GMP条件下进行的。新挑战合并液（批次42399）的病毒抗原和RT-PCR分析（病毒-RNA滴度）与NIH-8 fIIa NV批次的残留样品比较良好。采用细胞培养、RT-PCR和体内试验检测了批次42399的等分试样，未检出外源因子。此外，根据21 CFR 610.11，在动物中检测了批次42399的等分试样，并通过了一般安全性检测。最后，联系了粪便捐献者，临床实验室检查包括艾滋病毒、肝炎、梅毒以及肝功能检查均正常。该受试者的结核病PPD试验结果也为阴性，并且健康状况良好。本研究在DMID/NIAID/NIH提交的IND申请的支持下进行。本研究的目的是确定新NV攻毒样本池的安全性、感染性和临床发作率。健康分泌状态阳性志愿者将接受NV（批次42399）激发，该剂量接近先前激发研究中使用的剂量。预计大约三分之二的受感染者会出现症状。随后，将用较低剂量对较小的受试者组进行攻毒，以确定疫苗模型中后续研究的人感染剂量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.