HCV REPLICATION AND IN VITRO NEUTRALIZATION ASSAY

HCV 复制和体外中和测定

基本信息

批准号：
2098665
负责人：
Robert E LANFORD
金额：
$ 22.18万
依托单位：
TEXAS BIOMEDICAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-01 至 1995-08-31
项目状态：
已结题

项目摘要

Hepatitis C virus (HCV) infections represent a worldwide health problem, with approximately 0.5 to 1.5% of most populations testing positive for antibodies to HCV. An estimated 50% of infected individuals will become chronic carriers of HCV and will be at increased risk for the development of cirrhosis and hepatocellular carcinoma and will also act as a source of infection for others. Transmission of HCV occurs most readily through contact with contaminated blood as in blood transfusions and IV drug abuse; however, the true epidemiology of this agent is only now emerging through the use of diagnostic assays. Other routes of transmission include sexual contact, intrafamilial contact, and transmission from mother to child at birth. The development of a vaccine for this virus must be considered a high priority. Currently, the only available method of testing vaccine candidates is the immunization and challenge of chimpanzees, a very costly and time consuming approach. The development of an in vitro assay for evaluating the presence of neutralizing antibodies would greatly facilitate vaccine development. This proposal will employ a recently developed primary hepatocyte tissue culture system for the in vitro propagation of HCV to address these needs. The specific aims of this proposal are: 1) to optimize methods for detecting HCV polypeptides using a human hepatoma cell line transfected with recombinant expression vectors encoding various domains of the HCV polyprotein; 2) to produce antibodies to the capsid and envelope proteins of HCV expressed in the insect cell/baculovirus system; 3) to define the authentic HCV proteins expressed in HCV-infected primary hepatocytes using immunoprecipitation and immunoblot methodology; 4) to develop a quantitative assay for measuring in vitro infections of primary hepatocytes with HCV and to use this assay to develop an in vitro neutralization assay for HCV; 5) to examine panels of human and chimpanzee sera for the presence of neutralizing antibodies and to evaluate antisera produced against the envelope proteins of HCV for neutralizing activity; and 6) to create immortalized human and chimpanzee hepatocyte cell lines permissive for HCV infections.

丙型肝炎病毒（HCV）感染是一个全球性的健康问题，大多数人群中约 0.5% 至 1.5% 的检测结果呈阳性 HCV 抗体。预计 50% 的感染者将成为慢性丙型肝炎病毒携带者，罹患丙型肝炎病毒的风险会增加肝硬化和肝细胞癌，也将作为一个来源以免感染他人。 HCV 的传播最容易通过接触受污染的血液，如输血和静脉注射药物虐待;然而，这种病原体的真正流行病学现在才刚刚浮出水面通过使用诊断分析。其他传播途径包括性接触、家庭内接触和传播婴儿出生时的母亲。针对这种病毒的疫苗的开发必须被视为高度优先事项。目前唯一可用的方法测试候选疫苗的关键是免疫和挑战黑猩猩，这是一种非常昂贵且耗时的方法。发展历程评估中和作用存在的体外测定抗体将极大地促进疫苗的开发。这个提议将采用最近开发的原代肝细胞组织培养系统 HCV 的体外繁殖可以满足这些需求。具体的该提案的目的是： 1) 优化 HCV 检测方法多肽使用转染的人肝癌细胞系编码HCV不同结构域的重组表达载体多聚蛋白； 2) 产生针对衣壳和包膜蛋白的抗体在昆虫细胞/杆状病毒系统中表达的HCV； 3）定义在 HCV 感染的原代肝细胞中表达的真实 HCV 蛋白使用免疫沉淀和免疫印迹方法； 4）开发一个用于测量原发性细菌体外感染的定量测定肝细胞与 HCV 并使用该测定法开发体外 HCV 中和测定； 5）检查人类小组和黑猩猩血清中是否存在中和抗体评估针对 HCV 包膜蛋白产生的抗血清中和活性； 6）创造永生的人类和黑猩猩允许HCV感染的肝细胞系。