Norovirus P Particle, A Multifunctional Platform For Vaccine Development

诺如病毒粒子，疫苗开发的多功能平台

• 批准号: 8190929
• 负责人: Ming Tan
• 金额: $ 22.94万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8190929
• 关键词: Animals; Antibody Formation; Antigen Presentation; Antigens; Attention; Blocking Antibodies; Capsid; Chimera organism; Cloning; Communicable Diseases; Data; Distal; Epitopes; Escherichia coli; Figs - dietary; Flu virus; Goals; Human; Immune; Immune response; Immunization; Immunology; Individual; Infection; Laboratories; Lead; Medical center; Modeling; Molecular Biology; Molecular Cloning; Mus; Norovirus; Pediatric Hospitals; Peptides; Prevention; Procedures; Process; Production; Proteins; Recombinant Proteins; Records; Research; Rotavirus; Rotavirus Infections; Science; Site; Speed; Stimulus; Structure; Subunit Vaccines; Surface; Surface Antigens; T-Lymphocyte Epitopes; Technology; Testing; Tetanus; Trefoil Motif; Vaccinated; Vaccination; Vaccines; Virus; Virus Diseases; Work; Yeasts; base; commercialization; cost; flu; immunogenic; improved; influenzavirus; monomer; mouse model; nanoparticle; neutralizing antibody; novel; novel vaccines; particle; protective efficacy; receptor; research study; structural biology; user-friendly; vaccine candidate; vaccine development; vector; virology

DESCRIPTION (provided by applicant): This R21 application proposes to develop the norovirus (NV) P particle into a vaccine platform to fulfill the need of control and prevention of infectious diseases. This NV P particle is formed by 24 copies of the protruding (P) domain of NV capsid which has an ideal size for a subunit vaccine. The P particles can be readily produced in E. coli with simple purification procedures and low cost. The P particles are highly stable and immunogenic. In this study we will take advantage of the 3 surface loops of each P domain for foreign antigens presentation by insertion of the antigens into the surface loops. We have proven this concept by insertion of various small epitopes and large antigens on one of these loops, including the M2e epitope of influenza virus and rotavirus (RV) surface antigen VP8s. The P particle-M2e and -VP8 chimeras induced high titers of neutralizing antibodies against replication of influenza virus and RV and protected vaccinated mice from infection of the two viruses, respectively, in mouse challenge models. In addition, the resulting antibodies blocked NV-receptor attachment. In this study we will improve the P particle vaccine platform for broader applications by systematically assessing the capability and capacity of the other surface loops of the P particle to present foreign antigens and by construction of new user-friendly P particle vectors. In addition, we will perform challenge study using mouse model to improve the immune enhancement and production efficiency of the P particle platform through 1) increase of copy number of an inserted antigen, 2) addition of immunogenic stimulus molecules to the antigens, and 3) addition of a purification tag to the chimera. This study will gather further evidence on the usefulness of the P particle platform for antigen presentation. The proposed research herein that base logically on our prior work combines the strengths of three labs of Drs. Tan, Jiang and McNeal at the Cincinnati Children's Hospital Medical Center with expertises in molecular biology, structural biology, and virology, immunology and vaccine technology. With the large body of promising preliminary data, our track-records on research and the collaborative spirit between our research teams, we are confident that we will fully the proposed research to develop the NV P particle platform for presenting diverse foreign antigens and move the science forward. PUBLIC HEALTH RELEVANCE: In this application we proposed to develop our recently discovered subviral particle, the norovirus (NV) P particle, into a vaccine platform for antigen presentation and thus for novel vaccine development. The P particle is composed of 24 copies of NV protruding (P) domains. Each P domain in the P particle has three exposed loops on its distal surface and these loop have been shown to be excellent sites for foreign antigen presentation. Antigens can be inserted onto one to three surface loops of the P domain by molecular cloning. The resulting recombinant protein will spontaneously form chimeric P particle that contain at least 24 copies of the inserted antigen on the surface of the P particle. The P particles can be easily produced in E. coli, are highly stable and highly immunogenic and, therefore, are an ideal vaccine platform. We have accumulated a large amount of data in insertion and presentation of several protein and peptide antigens on the P particle, including the M2e epitope of influenza virus and the surface antigen VP8 of rotavirus (RV). In this application we will gather further evidence on usefulness of P particle as a platform for vaccine development against various infectious diseases. Two lines of experiments will be performed to fulfill our goals. In Aim 1 the capability and capacity of individual surface loops for small-to-large antigen insertions will be systematically assessed for a maximal application of the P particle platform. To facilitate these experiments, user-friendly P particle vectors with a convenient cloning cassette in each loop will be generated. Our goal is to maximize the usefulness of the P particle vaccine platform. In Aim 2 the usefulness of the P particle vaccine platform will be further evaluated by mouse challenge studies on a human flu virus and a mouse RV. Particular attention will be paid to the improvement of immune enhancement by increasing the copy number of an antigen per P particle. This will be achieved through an insertion of a repeated M2e epitopes in individual loops of the P particle. We also will examine whether further immune enhancement can be reached by an extra antigenic stimulus molecule to be inserted to the P particles-M2e and -VP8 chimeras. Through these studies we wish to further build up confidence on our research which may help to speed up the process of commercialization of the P particle vaccine platform for a broad application by many laboratories around world.

描述（由申请人提供）：此R21申请建议将诺如病毒（NV）P粒子开发到疫苗平台中，以满足控制和预防传染病的需求。该NV P粒子由NV Capsid的突出（P）结构域的24份副本形成，该副本具有亚基疫苗的理想大小。可以以简单的纯化程序和低成本的大肠杆菌轻松生产P颗粒。 P颗粒高度稳定且免疫原性。在这项研究中，我们将通过将抗原插入表面环中的每个P结构域的3个表面环进行外抗原表现。我们通过在其中一个环上插入各种小表位和大型抗原，包括流感病毒和轮状病毒（RV）表面抗原VP8的M2E表位，证明了这一概念。 P粒子-M2E和-VP8嵌合体在小鼠挑战模型中分别诱导了针对流感病毒和RV复制的抗体中和抗体的高滴度。另外，所得抗体阻断了NV受体附着。在这项研究中，我们将通过系统地评估P颗粒的其他表面环的能力和能力来提高P颗粒疫苗平台，并通过构建新的用户友好型P粒子向量。此外，我们还将使用小鼠模型进行挑战研究，以通过1）增加插入的抗原的拷贝数增加P颗粒平台的免疫增强和生产效率，2）在抗原中添加免疫原性刺激分子，以及3）3）在嵌合体中添加纯化标签。这项研究将收集有关P颗粒平台对抗原呈递的有用性的进一步证据。本文提出的研究以逻辑为基础，以我们先前的工作为基础，结合了三个博士实验室的优势。辛辛那提儿童医院医疗中心的谭，江和麦克尼尔具有分子生物学，结构生物学和病毒学，免疫学和疫苗技术方面的专业知识。凭借大量有希望的初步数据，我们的研究和研究团队之间的协作精神赛道，我们有信心我们将充分进行拟议的研究来开发NV P粒子平台，以展示多样化的外国抗原并推动科学的前进。 公共卫生相关性：在此应用中，我们提议将我们最近发现的亚病毒病毒（NV）P粒子开发成抗原呈递的疫苗平台，从而用于新型疫苗发育。 P粒子由NV突出（P）域的24个拷贝组成。 P粒子中的每个P结构域在其远端表面都有三个暴露环，并且这些环被证明是外抗原呈递的极好位点。可以通过分子克隆将抗原插入P结构域的一到三个表面环上。所得的重组蛋白将自发形成嵌合P颗粒，该嵌合颗粒至少包含24份在P颗粒表面上插入的抗原。 P颗粒可以轻松地在大肠杆菌中产生，高度稳定且高度免疫原性，因此是理想的疫苗平台。我们已经在P颗粒上插入几种蛋白质和肽抗原的插入和表示，包括流感病毒的M2E表位和轮状病毒（RV）的表面抗原VP8。在此应用中，我们将收集有关P颗粒作为针对各种传染病的疫苗开发平台的进一步证据。将进行两条实验以实现我们的目标。在AIM 1中，将系统地评估单个表面回路对小型抗原插入的能力和能力，以最大程度地评估P粒子平台的最大应用。为了促进这些实验，将生成每个循环中具有方便的克隆盒的用户友好的P粒子向量。我们的目标是最大化P颗粒疫苗平台的实用性。在AIM 2中，P颗粒疫苗平台的有用性将通过小鼠挑战对人流感病毒和小鼠RV的挑战研究进一步评估。通过增加每个P颗粒的抗原的拷贝数来提高免疫增强的改善。这将通过将重复的M2E表位插入P颗粒的单个环中插入来实现。我们还将检查是否可以通过额外的抗原刺激分子来实现进一步的免疫增强，以插入P颗粒-M2E和-VP8嵌合体。通过这些研究，我们希望进一步增强对我们的研究的信心，这可能有助于加快p粒子疫苗平台的商业化过程，以促进世界各地许多实验室的广泛应用。