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.

我们的实验室已经开发了一种将HCV结构蛋白有效组装成昆虫细胞中HCV样颗粒（HCV-LP）的系统。这些非感染的HCV样颗粒具有与从HCV感染的人分离的推定病毒体相似的形态，血清学和生物物理特性。与重组亚基疫苗相反，HCV样颗粒的病毒蛋白可以以天然的病毒粒子样构象表示，因此在引发保护性的体液和细胞免疫反应方面可能是优越的。 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了证明。此外，我们证明了黑猩猩中HCV-LP对保护性免疫的免疫原性和诱导。黑猩猩每组两者用HCV-LP或HCV-LP辅助ASO1B免疫。在八个月内进行了四次免疫接种后，所有动物都会出现强大的HCV特异性细胞免疫反应，包括IFN-GAMMA和IL-2，CD4和CD8 T细胞，以及针对CORE，E1和E2的增殖性淋巴细胞反应。两组中的黑猩猩都被HCV CG1B接种物的100 CID50挑战。在对HCV挑战时，一位黑猩猩在挑战后的第三周和第四周就患有低HCV RNA滴度（10E3-4 Copiesml）的短暂性病毒血症。其他三只黑猩猩感染了较高水平的病毒血症（10E4-5拷贝），但挑战后10周，它们的病毒水平变得无法量化（<1000份）。在HCV挑战之后，所有四个黑猩猩均表现出外围IFN-GAMMA T细胞和增殖反应的显着增加，以及针对HCV结构蛋白的肝内T细胞反应的存在。这些T细胞反应与HCV RNA水平下降相吻合。相比之下，用相同的HCV接种物感染了四个中殿黑猩猩，三种在10E5-6 CopiesML范围内发出了持续感染的病毒血症。我们的研究表明，HCV-LP免疫可诱导强大的HCV特异性细胞免疫反应，并在黑猩猩模型中赋予针对HCV挑战的部分保护。为了改善和扩大HCV-LP的免疫原性，我们将T细胞表位从非结构基因设计为HCV-LP。我们的初步数据表明，在小鼠中诱导了嵌合HCV-LP携带的非结构表位的T细胞反应。此外，我们将HCV-LP方法与其他免疫方法（例如质粒DNA）相结合，在素促进方案中。了解HCV的结构特征对于设计诱导保护性免疫的免疫原至关重要。由于缺乏强大的组织培养系统，无法从传染源产生足够量的病毒源来进行深入的三维（3D）结构分析，因此丙型肝炎病毒（HCV）的结构细节是难以捉摸的。使用阴性染色和冷冻电子显微镜（冷冻），我们表明从细胞培养中分离出的HCV病毒体的直径相当均匀，并且重组表达的HCV样颗粒（HCV-LPS）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糖蛋白的单克隆抗体的情况下，从具有与HCV病毒相同的HCV-LPS获得3D重建。 HCV-LP的3D重建揭示了一个多层结构，具有光滑的外层密度，以鱼骨构型排列。与抗E1抗体复合物中颗粒的重建表明，E1表位的位点被暴露并包围5倍，3倍和2倍轴。抗E1抗体的结合模式以及登革热病毒E糖蛋白的结构进一步表明，HCV-LP E1和E2蛋白形成四聚体（或异源二聚体的二聚体）的HCV-LP E1和E2蛋白在形态学上和与Flavivivirus e Homidodimersy symodimologology and应用。 HCV颗粒的第一个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。