ANIMAL LENTIVIRUSES--STRATEGIES FOR HIV VACCINATION

人类免疫缺陷病毒(Hiv)是慢病毒的一种。 亚科，非致癌逆转录病毒的分类群， 持续感染宿主免疫系统的细胞。这些是 具有高度糖基化包膜糖蛋白的包膜病毒 (Env GP)，在受感染的宿主中刺激强烈的免疫反应。 然而，在自然感染过程中激发的免疫反应未能 控制病毒复制，导致持续感染 最终导致慢性多系统疾病和死亡。许多. 调节细胞亲和性、细胞致病性和 抗体对病毒的中和作用与病毒的包膜糖蛋白 爱滋病毒。然而，作为基础的慢病毒包膜的属性 无效的抗慢病毒免疫反应知之甚少 很难用艾滋病毒本身来评估。对这些领域的洞察是 对于制定有效的基于免疫的对抗艾滋病毒的战略至关重要。 山羊关节炎脑炎病毒(CAEV)和山羊Visna病毒 绵羊是两种密切相关的、特征良好的慢病毒 在许多生物学和结构特征上反映了艾滋病毒。 CAEV和Visna的感染性分子克隆 完全测序可供研究，并提供了一个系统 以解决慢病毒的免疫原性和致病机制问题 在自然宿主中。该提案侧重于测绘和 绵羊/山羊慢病毒功能结构域的鉴定 环境GP。将构建一组截短和突变的env gp基因。 利用合成的寡核苷酸进行聚合酶链式反应 并将其亚克隆到杆状病毒表达载体中。 表达不同形式包膜糖蛋白的重组杆状病毒将 用于感染昆虫细胞的旋转瓶以获得毫克 纯化的包膜糖蛋白的量。各种纯化的糖蛋白 将对构造进行比较，看它们是否能够阻塞各种 功能分析。在4-5年内，这些测绘研究将被延长 利用聚合酶链式反应构建被膜的感染性分子克隆 CAEV和Visna的嵌合体。在环境之间切换特定域 这两种病毒的基因将映射细胞嗜性、抗体的决定因素 绵羊/山羊的中和、免疫原性和致病性 慢病毒在它们的自然宿主中。这些生物学特性是 至少部分由env gp调节，并且在井之间有明显的差异 表征了亲本病毒、CAEV和Visna病毒。 应聘者在应用分子技术方面的经验有限。 生物学对疾病发病机制的研究。主管是一名 慢病毒分子生物学领域公认的专家，具有 大型活动实验室。这位候选人已被任命为独立候选人 约翰·霍普金斯大学湾景研究园区的实验室空间 大学、医学院以及来自 比较医学部和大学。这种组合 一系列因素应为完成 建议学习并极大地促进候选人作为一名 独立研究人员。

The human imunodeficiency virus (HIV) is a member of the lentivirus subfamily, a taxonomic group of nononcogenic retroviruses that persistently infect cells of the host immune system. These are enveloped viruses that have a highly glycosylated envelope glycoprotein (env gp) that stimulates strong immune responses in the infected host. However, immune responses elicited during natural infection fail to control virus replication resulting in a persistent infection that eventually leads to chronic multisystemic disease and death. Many of the functional domains that mediate cell tropism, cytopathicity, and neutralization of virus by antibody have been mapped to the env gp of HIV. However, the properties of the lentivirus envelope that underlie ineffective anti-lentivirus immune responses are poorly understood and difficult to evaluate with HIV itself. Insight into these areas are essential to developing effective immune based strategies against HIV. Caprine arthritis encephalitis virus (CAEV) of goats and visna virus of sheep are two closely related, well-characterized lentiviruses that mirror HIV in many of their biological and structural characteristics. Infectious molecular clones of both CAEV and visna that have been completely sequenced are available for study and provide a system in which to address questions of lentivirus immunogenicity and pathogenesis in the natural host. This proposal focuses on mapping and characterizing the functional domains of the ovine/caprine lentivirus env gp. A set of truncated and mutated env gp genes will be constructed using the polymerase chain reaction with synthetic oligonucleotides as primers and the subcloned into a baculovirus expression vector. Recombinant baculovirus expressing the various forms of the env gp will be used to infect spinner flasks of insect cells to obtain milligram quantities of purified env gp. The various purified glycoprotein constructs will be compared for their ability to block in various functional assays. In years 4-5 these mapping studies will be extended by using PCR to construct infectious molecular clones that are envelope chimeras of CAEV and visna. Switching specific domains between the env genes of the two viruses will map determinants of cell tropism, antibody neutralization, immunogenicity and pathogenicity for the ovine/caprine lentiviruses in their natural host. These biological properties are mediated at least in part by env gp and differ markedly between the well characterized parental viruses, CAEV and visna virus. The candidate has limited experience in applying techniques of molecular biology to the study of disease pathogenesis. The supervisor is a recognized expert in the field of lentivirus molecular biology with a large active lab. The candidate has been provided with independent laboratory space at the Bayview Research Campus of the Johns Hopkins University, School of Medicine along with equipment and support from the Division of Comparative Medicine and the university. This combination of factors should provide an excellent environment for completing the proposed studies and greatly enhance the candidate's development as an independent researcher.