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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。诺如病毒是引起流行性非细菌性肠胃炎（肠道流感）的最常见原因。诺瓦克病毒（NV）是这类病毒的原型株。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体中进行许多实验。用于研究的主要病毒来源是从美国国立卫生研究院（NIH- 8fiia）提供的实验性感染NV的人类志愿者的粪便样本中获得的。利用这些样本对基因组进行测序，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传染性病毒样颗粒（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）在口服给人类志愿者时，似乎是安全的，并产生了IgG免疫反应。目前尚不清楚这种免疫反应是否能预防疾病。该方案旨在建立病毒攻击模型，以测试疫苗效力。以前的人类感染研究是用NV感染池进行的，结果显示，在未选择的成年人群中，感染率约为80%。随后对挑战材料的研究表明，分泌物状态是感染的重要预测因子。缺乏H型1寡糖的表达与NV攻击后的感染保护有关，而H型1寡糖表达者的感染发生率为97%。由于原来的NV攻击池不再可用，我们使用从大约14年前感染NV （NIH-8fIIa）的健康受试者获得的储存粪便标本来产生新的攻击池（滤液）。在GMP条件下进行。新攻毒池（编号42399）的病毒抗原和RT-PCR分析（病毒rna滴度）与NIH-8fIIa NV批次的残留样品比较良好。使用细胞培养、RT-PCR和体内试验对批次42399的等份进行了测试，未检测到外来因子。此外，Lot 42399的等份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，与粪便供者取得联系，临床实验室检查包括HIV、肝炎、梅毒和肝功能检查均正常。该受试者肺结核PPD检测呈阴性，健康状况良好。本研究是在DMID/NIAID/NIH提交的IND申请的支持下进行的。本研究的目的是建立新的NV攻击池的安全性、感染性和临床发作率。健康的分泌物状态阳性的志愿者将接受NV （Lot 42399）剂量的挑战，该剂量与之前的挑战研究中使用的剂量相近。大约三分之二的感染者预计会出现症状。随后，较小的受试者群体将接受较低剂量的挑战，以确定50%的人类感染剂量，以便在疫苗模型中进行后续研究。

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.