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: 8264954
• 负责人: Ming Tan
• 金额: $ 19.13万
• 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/8264954
• 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; public health relevance; 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（申请人提供）：本次R21申请拟将诺如病毒（NV）P颗粒开发为疫苗平台，以满足传染病控制和预防的需要。该NV P颗粒由NV衣壳的突出（P）结构域的24个拷贝形成，其具有用于亚单位疫苗的理想尺寸。在E.纯化步骤简单，成本低。P颗粒是高度稳定和免疫原性的。在这项研究中，我们将利用每个P结构域的3个表面环，通过将抗原插入表面环来呈递外源抗原。我们已经通过在这些环之一上插入各种小表位和大抗原（包括流感病毒的M2 e表位和轮状病毒（RV）表面抗原VP 8）来证明这一概念。在小鼠攻毒模型中，P颗粒-M2 e和-VP 8嵌合体分别诱导针对流感病毒和RV复制的高滴度中和抗体，并保护接种疫苗的小鼠免受两种病毒的感染。此外，所得抗体阻断NV-受体附着。在这项研究中，我们将通过系统地评估P颗粒的其他表面环呈递外源抗原的能力和容量，并通过构建新的用户友好的P颗粒载体，来改进P颗粒疫苗平台，以获得更广泛的应用。此外，我们将使用小鼠模型进行攻击研究，以通过1）增加插入抗原的拷贝数，2）向抗原添加免疫原性刺激分子，和3）向嵌合体添加纯化标签来提高P颗粒平台的免疫增强和生产效率。本研究将收集关于P颗粒平台用于抗原呈递的有用性的进一步证据。本文提出的研究在逻辑上基于我们先前的工作，结合了辛辛那提儿童医院医学中心Tan、Jiang和McNeal博士三个实验室的优势，具有分子生物学、结构生物学、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，我们的研究记录以及我们研究团队之间的合作精神，我们相信我们将充分开展拟议的研究，开发用于呈递多种外源抗原的NV P颗粒平台，并推动科学向前发展。 公共卫生关系：在本申请中，我们提出将我们最近发现的亚病毒颗粒，诺如病毒（NV）P颗粒，开发成用于抗原呈递的疫苗平台，从而用于新型疫苗开发。P颗粒由24个拷贝的NV突出（P）结构域组成。P颗粒中的每个P结构域在其远端表面上具有三个暴露的环，并且这些环已被证明是外源抗原呈递的极好位点。抗原可以通过分子克隆插入到P结构域的一个至三个表面环上。所得重组蛋白将自发形成嵌合P颗粒，其在P颗粒表面上含有至少24个拷贝的插入抗原。在E.大肠杆菌中表达，是高度稳定和高度免疫原性的，因此是理想的疫苗平台。我们已经积累了大量的数据，在插入和呈递的几个蛋白质和肽抗原的P颗粒，包括流感病毒的M2 e表位和轮状病毒（RV）的表面抗原VP 8。在本申请中，我们将收集P颗粒作为针对各种传染病的疫苗开发平台的有用性的进一步证据。为了实现我们的目标，我们将进行两个实验。在目标1中，将系统地评估单个表面环用于小到大抗原插入的能力和容量，以最大限度地应用P颗粒平台。为了促进这些实验，将生成每个环中具有方便的克隆盒的用户友好的P颗粒载体。我们的目标是最大限度地发挥P颗粒疫苗平台的作用。在目的2中，将通过对人流感病毒和小鼠RV的小鼠攻击研究进一步评价P颗粒疫苗平台的有用性。将特别注意通过增加每个P颗粒的抗原拷贝数来改善免疫增强。这将通过在P颗粒的各个环中插入重复的M2 e表位来实现。我们还将研究是否可以通过插入P颗粒-M2 e和-VP 8嵌合体的额外抗原刺激分子来达到进一步的免疫增强。通过这些研究，我们希望进一步建立对我们的研究的信心，这可能有助于加快P颗粒疫苗平台的商业化进程，以供世界各地许多实验室广泛应用。