Novel vaccine against Norovirus

描述（由申请人提供）：辛辛那提儿童医院医疗中心（CCHMC）、弗吉尼亚理工学院和州立大学（弗吉尼亚理工大学）以及LigoCyte制药公司。根据RFA-AI-09-027，“生物防御食品和水传播疾病（R 01）的伙伴关系”，我们建议开发一种针对诺如病毒（NVs）的疫苗，这是NIAID B类食源性和水源性优先病原体之一，基于我们新发现的NVs亚病毒颗粒P颗粒。该P颗粒由病毒衣壳蛋白的突起（P）结构域的24个拷贝自发形成，呈八面体对称，具有真实的宿主受体结合性质，高度免疫原性，易于在E.大肠杆菌和酵母的表达，具有极高的产量和简单的纯化程序，并且在广泛的物理化学条件下高度稳定，使其成为用于粘膜免疫的优良亚单位疫苗。将进行代表不同早期至中期产品开发阶段的4个项目，以评价P颗粒疫苗。在第1阶段，我们将进行体外和小鼠免疫研究，并比较不同途径的P颗粒的不同疫苗接种方案（含/不含佐剂），以获得最大的安全性和有效性。我们还将使用新发现的猴杯状病毒（杜兰病毒）的细胞培养模型进行类似的研究，以验证P颗粒疫苗的有用性。在第2阶段，我们将表征代表不同基因型NV的NV的抗原变异，以基于确定的抗原类型（血清型）采用混合疫苗或共有疫苗方法开发广泛反应性疫苗。我们还将对目前占主导地位的GII-4病毒进行遗传和抗原分析，以解决有关NVs划时代进化的问题，为未来针对NVs的疫苗策略提供参考。在第3阶段，我们将使用我们实验室最近开发的人NV的无菌（Gn）猪攻毒模型，进一步表征候选疫苗在保护各种NV方面的安全性、免疫原性和广泛性。在最后阶段，我们将开发一种发酵生物反应器，用于规模化生产P颗粒，以便将来通过人类志愿者挑战研究和临床试验对疫苗进行评价。NV是遗传多样性的，并且没有合适的细胞培养物用于NV。我们的鸡尾酒/共识疫苗方法和通过替代“中和”试验和使用新发现的杜兰病毒和Gn猪模型评估疫苗的程序是高度创新的。我们相信，在本申请的研究结束时，将选择一组主要候选疫苗（疫苗株）用于未来的临床试验。 叙述/相关性：诺如病毒（NV）是急性胃肠炎的重要原因，影响发达国家和发展中国家所有年龄段的人。由于低感染剂量和高稳定性以及对消毒剂的抗性，NVs的广泛传播性质使得NVs容易成为生物恐怖袭击的媒介。在这项研究中，我们建议开发一种疫苗，针对NVs的基础上，我们新发现的亚病毒颗粒，P颗粒，NVs。该P颗粒由病毒衣壳蛋白的突起（P）结构域的24个拷贝自发形成，具有真实的宿主受体结合性质，高度免疫原性，易于在E.大肠杆菌和酵母菌，具有极高的产量和简单的纯化程序。它在广泛的物理化学条件下高度稳定，使其成为用于粘膜免疫的优良亚单位疫苗。已知NV在自然感染后会引起短暂的保护性免疫。在本研究中，我们将使用P颗粒疫苗的不同接种方案和免疫途径进行小鼠免疫研究，以获得最大的有效性和安全性。NV也是遗传多样性的，但我们最近的数据表明，由于人类组织血型抗原选择的趋同进化，NV的受体结合界面高度保守。我们将使用替代中和试验（受体阻断试验）对代表不同基因型的NV进行抗原表征，以基于定义的抗原类型（“血清型”）设计鸡尾酒/共有疫苗。NV仍然难以在体外培养。我们将利用我们新发现的恒河猴杯状病毒、杜兰病毒的细胞培养系统和人类NV的无菌（Gn）猪模型来证明概念并进一步评估疫苗在临床试验中未来开发的有用性。该项目将由一个研究小组进行，该研究小组由辛辛那提儿童医院医学中心（CCHMC）、弗吉尼亚理工学院和州立大学（弗吉尼亚理工大学）以及LigoCyte制药公司的分子病毒学、免疫学、疫苗开发等多学科专家组成。我们高度创新的独特P颗粒和鸡尾酒/共识疫苗的方法，以及通过替代“中和”试验和使用新发现的杜兰病毒和Gn猪模型评估疫苗的新程序，保证了我们将取得快速进展，通过I期临床试验达到下一个评估水平，以实现未来的商业化。

DESCRIPTION (provided by applicant): The Cincinnati Children's Hospital Medical Center (CCHMC), the Virginia Polytechnic Institute and State University (the Virginia Tech), and the LigoCyte Pharmaceuticals Inc. will team together for this application in response to RFA-AI-09-027, entitled "Partnerships for Biodefense Food- and Water-borne Diseases (R01). We propose to develop a vaccine against noroviruses (NVs), one of the NIAID Category B food- and water-borne priority pathogens, based on our newly discovered subviral particle, the P particle, of NVs. This P particle is spontaneously formed by 24 copies of the protrusion (P) domain of the viral capsid protein, in octahedral symmetry, with an authentic host receptor binding property, highly immunogenic, easily to produce in E. coli and yeast with extremely high yields and simple procedures for purification, and highly stable under a wide range of physic-chemical conditions, making it an excellent subunit vaccine for mucosal immunization. Four projects representing different early-to-middle product development stages will be performed to evaluate the P particle vaccine. In stage 1 we will perform in vitro and mouse immunization studies and compare different vaccination regimens of P particles by different routes with/without an adjuvant for a maximal safety and efficacy. We also will perform similar studies for proof-of-concept of the usefulness of P particle vaccine using the cell culture model of a newly discovered monkey calicivirus, the Tulane virus. In stage 2 we will characterize the antigenic variations of NVs representing different genotypes of NVs for developing a broadly reactive vaccine based on defined antigenic types (serotypes) with a cocktail or consensus vaccine approach. We also will perform genetic and antigenic analysis of the currently dominant GII-4 viruses to address a question about epochal evolution of NVs for future vaccine strategy against NVs. In stage 3 we will further characterize the safety, immunogenicity and broadness of the candidate vaccine in protection against various NVs using the gnotobiotic (Gn) pig challenge model of human NVs developed in our laboratories recently. In the last stage we will develop a fermentation bioreactor for scale up production of P particles for future evaluation of the vaccine by human volunteer challenge studies and clinical trials. NVs are genetically diverse and there is no suitable cell culture for NVs. Our cocktail/consensus vaccine approach and the procedures for evaluation of the vaccine by the surrogate "neutralization" assay and using the newly discovered Tulane virus and the Gn pig model are highly innovative. We are confident that a panel of lead candidates (vaccine strains) will be selected ready for future clinical trials by the conclusion of the studies in this application. Narrative/Relevance: Noroviruses (NVs) are an important cause of acute gastroenteritis, affecting people of all ages, in both developed and developing countries. The wide spread nature of NVs due to the low infection dose and the high stability and resistance to disinfectants makes NVs an easy agent for bioterrorist attack. In this study we propose to develop a vaccine against NVs based on our newly discovered subviral particle, the P particle, of NVs. This P particle is spontaneously formed by 24 copies of the protrusion (P) domain of the viral capsid protein, with an authentic host receptor binding property, highly immunogenic, ease to produce in E. coli and yeast with extremely high yields and simple procedures for purification. It is highly stable under a wide range of physic-chemical conditions making it an excellent subunit vaccine for mucosal immunization. NVs have been known to cause a brief protective immunity following a natural infection. In this study we will perform mouse immunization studies with different vaccination regimens and immunization routes of the P particle vaccine for the maximal efficacy and safety. NVs are also genetically diverse but our recent data showed that the receptor binding interfaces of NVs are highly conserved due to a convergent evolution selected by the human histo-blood group antigens. We will perform antigenic characterization of NVs representing different genotypes to design a cocktail/consensus vaccine based on defined antigenic types ("serotypes") using a surrogate neutralization assay (receptor blocking assay). NVs remain difficult to cultivate in vitro. We will take advantage of the cell culture system of our newly discovered rhesus monkey calicivirus, the Tulane virus, and the gnotobiotic (Gn) pig model of human NVs to prove concept and to further evaluate usefulness of the vaccine for future development in clinical trials. This project will be conducted by a research team with multidiscipline experts in molecular virology, immunology, vaccine development at the Cincinnati Children's Hospital Medical Center (CCHMC), the Virginia Polytechnic Institute and State University (Virginia Tech) and the LigoCyte Pharmaceuticals Inc. Our highly innovative approach of the unique P particles and the cocktail/consensus vaccine and the novel procedures for evaluation of the vaccine by the surrogate "neutralization" assay and using the newly discovered Tulane virus and the Gn pig model warrant that we will make a rapid progress leading to the next level of evaluation by the phase I clinical trials toward future commercialization.