权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular Approaches To Vaccine Development For Hepatitis C

丙型肝炎疫苗开发的分子方法

基本信息

批准号：
7734192
负责人：
T. Jake Liang
金额：
$ 50.67万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734192
关键词：
Adjuvant Animals Antibodies Antigens Architecture Arts Binding CD8B1 gene Caliber Cells Chronic Hepatitis Clinical Complex Cryoelectron Microscopy Cultured Cells Data Dengue Virus Depth Engineering Epitopes Flavivirus Genes Glycoproteins HCV Vaccine Health Hepatitis C Hepatitis C virus Human Immune response Immunity Immunization Infection Insecta Interferon Type II Laboratories Lymphocyte Modality Modeling Molecular Molecular Conformation Monoclonal Antibodies Mus Negative Staining Pan Genus Pan troglodytes Papio Pattern Peripheral Population Primary carcinoma of the liver cells Property Proteins Range Recombinants Serological Site Source Structural Protein Structure Subunit Vaccines System T-Lymphocyte T-Lymphocyte Epitopes Technology Treatment Protocols Viral Viral Proteins Viremia Virion Virus Assembly Virus-like particle Week density design dimer falls immunogenicity improved insight intrahepatic particle plasmid DNA reconstruction response size tissue culture vaccine development viral RNA

项目摘要

A system for efficient assembly of HCV structural proteins into HCV-like particles (HCV-LPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles have similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. The humoral and cellular immunogenicity of the HCV-LP had previously been demonstrated in the mouse and baboon models. In addition, we demonstrated the immunogenicity and induction of protective immunity by HCV-LP in chimpanzees. Chimpanzees, two in each group, were immunized with HCV-LP or HCV-LP adjuvant ASO1B. After four immunizations over an eight-month period, all animals developed strong HCV-specific cellular immune response including IFN-gamma and IL-2, CD4 and CD8 T-cell and proliferative lymphocyte responses against core, E1 and E2. The chimpanzees in both groups were challenged with a 100 CID50 of HCV CG1B inoculum. Upon challenge with HCV, one chimpanzee developed transient viremia with low HCV RNA titers (10E3-4 copiesml) in the third and fourth weeks post-challenge. The three other chimpanzees became infected with higher levels of viremia (10E4-5 copiesml) but their viral levels became unquantifiable (< 1000 copiesml) 10 weeks post-challenge. After HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral IFN-gamma T-cell and proliferative responses as well as the presence of intrahepatic T-cell response against the HCV structural proteins. These T-cell responses coincided with the fall in HCV RNA level. In comparison, four nave chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with viremia in the range of 10E5-6 copiesml. Our study suggests that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model. In an effort to improve and broaden the immunogenicity of HCV-LP, we have engineered T cell epitopes from the nonstructural genes into the HCV-LP. Our preliminary data showed that T cell response against the nonstructural epitopes carried by the chimeric HCV-LP were induced in mice. In addition, we are combining The HCV-LP approach with other modalities of immunization, such as plasmid DNA, in a prime-boost regimen. Understanding the structural features of HCV is crucial in designing immunogen that would induce protective immunity. The structural details of hepatitis C virus (HCV) have been elusive because of the lack of a robust tissue culture system for producing an adequate amount of virions from infectious sources for in-depth three-dimensional (3D) structural analysis. Using both negative-stain and cryo-electron microscopy (cryoEM), we show that HCV virions isolated from cell culture have a rather uniform size of 50 nm in diameter and that recombinantly expressed HCV-like particles (HCV-LPs) have similar morphologic, biophysical, and antigenic features. 3D reconstructions were obtained from HCV-LPs having the same size as the HCV virions in the presence and absence of monoclonal antibodies bound to the E1 glycoprotein. The 3D reconstruction of HCV-LP reveals a multilayered architecture, with smooth outer-layer densities arranged in a fishbone configuration. Reconstruction of the particles in complex with anti-E1 antibodies shows that sites of the E1 epitope are exposed and surround the 5-, 3-, and 2-fold axes. The binding pattern of the anti-E1 antibody and the fitting of the structure of the dengue virus E glycoprotein into our 3D reconstructions further suggest that the HCV-LP E1 and E2 proteins form a tetramer (or dimer of heterodimers) that corresponds morphologically and functionally to the flavivirus E homodimer. This first 3D structural analysis of HCV particles offers important insights into the elusive mechanisms of HCV assembly and maturation.

本实验室建立了一种在昆虫细胞中将HCV结构蛋白高效组装成HCV样颗粒（HCV-LP）的系统。这些非感染性HCV样颗粒具有与从HCV感染者分离的推定病毒体相似的形态学、血清学和生物物理学特性。与重组亚单位疫苗相反，HCV样颗粒的病毒蛋白可以天然的病毒体样构象存在，因此在引发保护性体液和细胞免疫应答方面可能是上级的。HCV-LP的体液和细胞免疫原性先前已在小鼠和狒狒模型中得到证实。此外，我们还证明了HCV-LP在黑猩猩中的免疫原性和诱导保护性免疫。用HCV-LP或HCV-LP佐剂ASO 1B免疫黑猩猩，每组2只。在8个月的时间内进行4次免疫接种后，所有动物都产生了强烈的HCV特异性细胞免疫应答，包括IFN-γ和IL-2、CD 4和CD 8 T细胞以及针对核心、E1和E2的增殖性淋巴细胞应答。两组中的黑猩猩都用100 CID 50的HCV CG 1B接种物进行了攻击。在用HCV攻击后，一只黑猩猩在攻击后第三和第四周发展为具有低HCV RNA滴度（10 E3 -4拷贝/ml）的短暂病毒血症。其他三只黑猩猩感染了较高水平的病毒血症（10 E4 -5拷贝/ml），但在攻击后10周，它们的病毒水平变得无法定量（< 1000拷贝/ml）。HCV攻击后，所有四只黑猩猩表现出外周IFN-γ T细胞和增殖反应的显着增加，以及肝内T细胞对HCV结构蛋白的反应的存在。这些T细胞反应与HCV RNA水平的下降相一致。相比之下，四只未受感染的黑猩猩感染了相同的HCV接种物，三只发生了持续感染，病毒血症在10 E5 -6 copiesml的范围内。我们的研究表明，HCV-LP免疫诱导强HCV特异性细胞免疫应答，并赋予部分保护对HCV的攻击在黑猩猩模型。为了改善和扩大HCV-LP的免疫原性，我们将非结构基因的T细胞表位工程化到HCV-LP中。我们的初步数据表明，嵌合HCV-LP携带的非结构表位的T细胞反应诱导小鼠。此外，我们正在将HCV-LP方法与其他免疫方式（如质粒DNA）结合起来，采用初免-加强方案。了解HCV的结构特征对于设计能够诱导保护性免疫的免疫原至关重要。丙型肝炎病毒（HCV）的结构细节一直难以捉摸，因为缺乏一个强大的组织培养系统，从感染源产生足够量的病毒粒子进行深入的三维（3D）结构分析。使用负染色和冷冻电子显微镜（cryoEM），我们表明，从细胞培养物中分离的HCV病毒粒子具有相当均匀的直径为50 nm的大小，重组表达的HCV样颗粒（HCV-LP）具有相似的形态，生物物理和抗原特征。在存在和不存在与E1糖蛋白结合的单克隆抗体的情况下，从具有与HCV病毒体相同大小的HCV-LP获得3D重建。HCV-LP的3D重建揭示了多层结构，具有以鱼骨状配置排列的平滑外层密度。与抗E1抗体复合的颗粒的重建显示E1表位的位点被暴露并且围绕5-、3-和2-倍轴。抗E1抗体的结合模式和登革病毒E糖蛋白的结构拟合到我们的3D重建进一步表明，HCV-LP E1和E2蛋白形成四聚体（或异源二聚体的二聚体），其在形态和功能上对应于黄病毒E同源二聚体。HCV颗粒的首次3D结构分析为HCV组装和成熟的难以捉摸的机制提供了重要的见解。