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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丙型肝炎病毒结构蛋白高效组装成丙型肝炎病毒样颗粒的系统。这些非传染性的丙型肝炎病毒样颗粒与从丙型肝炎病毒感染者中分离的假定的病毒粒子具有相似的形态、血清学和生物物理特性。与重组亚单位疫苗相比，类丙型肝炎病毒颗粒的病毒蛋白可能以天然的类病毒粒子构象存在，因此在诱导保护性体液和细胞免疫反应方面可能更具优势。丙型肝炎病毒低密度脂蛋白的体液和细胞免疫原性已在小鼠和狒狒模型中得到证实。此外，我们还研究了丙型肝炎病毒蛋白在黑猩猩体内的免疫原性和保护性免疫诱导作用。用丙型肝炎病毒Lp或丙型肝炎病毒Lp佐剂ASO1B免疫黑猩猩，每组两只。在8个月的4次免疫后，所有动物都产生了强烈的丙型肝炎病毒特异性细胞免疫反应，包括针对CORE、E1和E2的干扰素-伽马和IL-2、CD4和CD8 T细胞以及增殖淋巴细胞反应。两组黑猩猩都接种了100CID50的丙型肝炎病毒CG1B疫苗。用丙型肝炎病毒攻击后，一只黑猩猩在攻击后第三周和第四周出现一过性病毒血症，病毒滴度较低(10E3-4拷贝)。另外三只黑猩猩感染了更高水平的病毒血症(10E4-5拷贝毫升)，但它们的病毒水平在10周后变得无法量化(&lt；1000拷贝毫升)。在丙型肝炎病毒攻击后，所有四只黑猩猩的外周干扰素-γT细胞和增殖反应均显著增加，肝内T细胞对丙型肝炎病毒结构蛋白的反应也显著增加。这些T细胞反应与丙型肝炎病毒RNA水平的下降相一致。相比之下，4只幼年黑猩猩感染相同的丙型肝炎疫苗，3只持续感染10E5-6拷贝的病毒血症。我们的研究表明，在黑猩猩模型中，丙型肝炎病毒-LP免疫诱导了强烈的丙型肝炎病毒特异性细胞免疫反应，并对丙型肝炎病毒攻击具有部分保护作用。为了提高和扩大丙型肝炎病毒蛋白的免疫原性，我们将非结构基因中的T细胞表位改造到丙型肝炎病毒蛋白中。我们的初步数据显示，嵌合的丙型肝炎病毒-LP携带的非结构表位的T细胞反应在小鼠中被诱导。此外，我们正在将丙型肝炎病毒-LP方法与其他免疫方式结合起来，例如以PRIME-Boost方案进行的质粒DNA免疫。了解丙型肝炎病毒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来从感染源中产生足够数量的病毒粒子，以进行深入的三维(3D)结构分析。通过负染和冷冻电子显微镜观察，我们发现从细胞培养中分离到的丙型肝炎病毒粒子大小比较均匀，直径为50 nm，重组表达的丙型肝炎病毒样颗粒具有相似的形态、生物物理和抗原特征。在存在和不存在与E1糖蛋白结合的单抗的情况下，从与丙型肝炎病毒粒子大小相同的丙型肝炎病毒脂蛋白中获得三维重建。HCV-LP的3D重建显示了一个多层结构，外层密度光滑，呈鱼骨状排列。对与抗E1抗体形成的复合体中的颗粒的重建显示，E1表位的位置暴露并围绕着5-、3-和2-折叠轴。抗E1抗体的结合模式和登革病毒E糖蛋白的结构与我们的3D重建结果相吻合，进一步表明丙型肝炎病毒-LPE1和E2蛋白形成了一个在形态和功能上与黄病毒E同源二聚体相对应的四聚体(或二聚体)。首次对丙型肝炎病毒颗粒的3D结构分析为了解丙型肝炎病毒组装和成熟的难以捉摸的机制提供了重要的见解。