DNA & SOLUBLE MULTIMERIC IMMUNOGEN FOR HUMMORAL IMMUNITY

脱氧核糖核酸

• 批准号: 2887880
• 负责人: JON OSCHERWITZ
• 金额: $ 18.9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-29 至 2002-06-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887880
• 关键词: AIDS vaccines; G protein; chimeric proteins; gene targeting; human immunodeficiency virus; humoral immunity; laboratory mouse; neutralizing antibody; nucleic acid repetitive sequence; protein sequence; receptor coupling; vaccine development; vector vaccine; virus antigen

DESCRIPTION: (Adapted from applicant's abstract): Earlier work in a number of disease models has shown that immunogens containing tandemly repeated epitopes can be more immunogenic than a respective immunogen containing only a single copy of the sequence. The applicant's studies have revealed that antisera raised in mice to a recombinant immunogen in alum containing eight copies of an MN isolate V3PND sequence is able to neutralize ADA, a primary M-tropic isolate of HIV-I. However, despite hopes of generating immunogens able to induce antibodies capable of primary isolate neutralization, the hypervariable nature of the V3 loop of HIV would apparently necessitate incorporation of multiple strain specific V3 loop sequences in any prototype AIDS vaccine. Coincident with the increased understanding of the role of the V3 loop in determining cellular tropism, however, has come new insights into how the V3loop sequences interact with cellular coreceptors in the facilitation of HIV infection. CCR5 is a G-protein coupled receptor identified as integral for entry by M-tropic HIV isolates into macrophages. Since M-tropic viral isolates are especially implicated early in HIV infection and individuals who are homozygous for the 32 bp deletion which results in a non-functional-CCR5 receptor are protected from infection from HIV, yet appear to have a normal phenotype, the CCR5 extracellular receptor sequences represent attractive targets for humoral immunity to HIV-I. We have analyzed the CCR5 extracellular receptor sequence domains and have identified putative extracellular sequences which represent potential antibody targets. The major objectives of the current study are to: a) Develop tandem repeat soluble and DNA constructs representing sequences from the V3 loop of primary NSI M-tropic isolates; b) Analyze the ability of antisera to these constructs raised in BALB/c mice to neutralize homologous and heterologous primary NSI M-tropic HIV isolates; c) Develop tandem repeat soluble and DNA constructs representing sequences from the putative extracellular domains of CCR5; d) Analyze the ability of antisera to these constructs raised in BALB/c mice to neutralize homologous and heterologous primary NSI M-tropic HIV isolates and; e)Utilizing the results from targeting the V3 and CCR5 sequences, construct hybrid immunogens which contain sequences from both the V3 loop of primary NSI M-tropic viruses, and immunogenic sequences from the CCR5 coreceptor. The construction of these immunogens is enabled by our development of BMX7, a novel vector for cloning multi-determinant tandem repeat immunogens, and BMX7EX, a DNA expression vector which retains the essential cloning features of the BMX7 vector. These studies will ideally culminate in the development of immunogens capable of inducing antibodies able to neutralize M-tropic primary isolate infectivity and contribute significantly to our understanding of the immunology of peptide component DNA and soluble immunogens.

描述：（改编自申请人的摘要）：早期的一些工作 疾病模型的研究表明，含有串联重复序列的免疫原 表位可以比仅含有的相应免疫原更具免疫原性 序列的单个副本。 申请人的研究表明 小鼠体内产生的针对明矾中重组免疫原的抗血清含有八种 MN 分离株 V3PND 序列的副本能够中和 ADA，ADA 是一种主要的 HIV-I的M向性分离株。 然而，尽管希望产生免疫原 能够诱导能够初级分离物中和的抗体， HIV V3 环的高度可变性质显然需要 在任何原型中合并多个菌株特定的 V3 环序列 艾滋病疫苗。 与对角色的进一步了解相一致 然而，V3 环在确定细胞趋向性方面有了新的见解 研究 V3loop 序列如何与细胞辅助受体相互作用 促进艾滋病毒感染。 CCR5 是一种 G 蛋白偶联受体，被确定为进入的组成部分 M-tropic HIV 分离进入巨噬细胞。 由于 M 向性病毒分离株是 特别是与艾滋病毒感染早期和患有艾滋病毒的人有关 32 bp 缺失纯合，导致非功能性 CCR5 受体受到保护，免受艾滋病毒感染，但似乎具有正常的 表型中，CCR5胞外受体序列代表有吸引力 HIV-I 体液免疫的目标。 我们已经分析了CCR5 细胞外受体序列域并已鉴定出假定的 代表潜在抗体靶标的细胞外序列。 这 当前研究的主要目标是： a) 开发串联重复序列 可溶性和DNA构建体代表来自V3环的序列 原发性 NSI M-tropic 分离株； b) 分析抗血清对这些的能力 在 BALB/c 小鼠中产生的构建体用于中和同源和异源 原发性 NSI M 向性 HIV 分离株； c) 开发串联重复可溶性DNA 构建代表来自假定的细胞外结构域的序列 CCR5； d) 分析抗血清对这些构建体的能力 BALB/c 小鼠中和同源和异源原发性 NSI M 向性 HIV 分离株； e) 利用针对 V3 和 CCR5 的结果 序列，构建杂合免疫原，其中包含来自两个序列的序列 原代 NSI M 向性病毒的 V3 环，以及来自 CCR5 辅助受体。 这些免疫原的构建是由我们的 BMX7的开发，一种用于克隆多决定簇串联的新型载体 重复免疫原和 BMX7EX，一种 DNA 表达载体，保留了 BMX7 载体的基本克隆特征。 这些研究将理想地 最终发展出能够诱导抗体的免疫原 能够中和 M-tropic 初级分离株的传染性并有助于 对我们对肽成分免疫学的理解具有重要意义 DNA 和可溶性免疫原。