REPLICATION AND CAPSID ANTIGENS OF HEPATITIS A VIRUS

甲型肝炎病毒（HAV）是一种常见的 人类感染，仍然是一个世界性的公共卫生问题。 病毒在培养细胞中繁殖的能力，以及 最近构建的cDNA克隆和确定的 病毒基因组的核苷酸序列， 许多新的方法来理解生物学， HAV感染的发病机制，并提供了深入了解 开发潜在的亚单位疫苗。 在本申请中，我们 提出旨在鉴定特定病毒序列的实验 导致复制周期缓慢而漫长的原因 这种病毒的一个特征，使它区别于所有其他 其属（肠道病毒）和科（小核糖核酸病毒科）的成员。 将构建重组病毒，其由以下组成： HAV 5 ′端调控序列和脊髓灰质炎病毒编码序列， 或HAV聚合酶编码序列替换同源的 脊髓灰质炎序列。 插入这些潜在的努力 下调HAV序列，使其成为快速生长的 脊髓灰质炎病毒将通过检查空斑大小进行评估， 形态学、温度敏感性、病毒RNA和蛋白质 合成和病毒产量。 另一个未知的HAV基因， 将分析2A蛋白酶编码区的功能 处理宿主细胞相互作用和病毒形态发生， 表达含有2A的基因工程蛋白 序列，在体内和体外。 最后，我们制作了 HAV衣壳蛋白（VP 1）抗原在原核（E.大肠杆菌） 和真核（杆状病毒感染的SF 9）细胞。 我们计划 系统评价不同抗原蛋白的能力， 从多种不同的载体表达并通过 不同的程序，以诱导中和抗体和/或 让免疫系统产生中和性回忆反应 我们希望这些研究能鉴定出一种重组蛋白 其将有效作为亚单位的候选免疫原 疫苗

Hepatitis A virus (HAV) causes one of the most prevalent infections of man, and remains a worldwide public health problem. The ability to propagate the virus in cultured cells, as well as the recent construction of cDNA clones and the determination of the nucleotide sequence of the viral genome, have made possible numerous new approaches to understanding the biology and pathogenesis of HAV infection and provided insight towards the development of potential subunit vaccines. In this application, we propose experiments designed to identify specific viral sequences that are responsible for the slow and protracted replication cycle of this virus, a characteristic that distinguishes it from all other members of its genus (enterovirus) and family (Picornaviridae). Recombinant viruses will be constructed that are composed of HAV 5' end regulatory sequences and poliovirus coding sequences, or HAV polymerase coding sequences replacing the homologous polio sequences. The efforts of the insertion of these potentially down-regulating HAV sequences into a rapidly-growing, lytic poliovirus will be evaluated by examining plaque size and morphology, temperature sensitivity, viral RNA and protein synthesis, and virus yields. Another uncharacterized HAV gene, the 2A protease coding region, will be analyzed for functions dealing with host cell interaction and virus morphogenesis by expressing genetically engineered proteins containing 2A sequences, both in vivo and in vitro. Finally, we have produced HAV capsid protein (VP1) antigens in both prokaryotic (E. coli) and eukaryotic (baculovirus-infected SF9) cells. We plan to systematically evaluate the ability of different antigenic proteins, expressed from a variety of different vectors and purified by different procedures, to induce neutralizing antibodies and/or to prime the immune system for a neutralizing anamnestic response. We hope that these studies will identify a recombinant protein that will be effective as a candidate immunogen for a subunit vaccine.