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）：早期的工作，在一些 疾病模型的研究表明，含有串联重复的免疫原 表位的免疫原性可以比仅含有 序列的一个拷贝 申请人的研究表明， 在小鼠中产生的抗重组免疫原的抗血清， MN分离株V3 PND序列的拷贝能够中和ADA， HIV-I的M-嗜性分离株。 然而，尽管希望产生免疫原 能够诱导能够中和初级分离物的抗体， HIV的V3环的高变性质显然需要 在任何原型中掺入多个菌株特异性V3环序列 艾滋病疫苗。 随着人们对联合国作用的认识不断加深， 然而，决定细胞向性的V3环有了新的认识， V3环序列如何与细胞共受体相互作用， 促进艾滋病毒感染。 CCR 5是一种G蛋白偶联受体，被鉴定为通过免疫细胞化学（ELISA）进入细胞所必需的。 M嗜性HIV分离物进入巨噬细胞。 由于M嗜性病毒分离株是 特别是在艾滋病毒感染的早期， 纯合的32 bp缺失，导致非功能性-CCR 5 受体受到保护，免受艾滋病毒感染，但似乎有一个正常的 表型，CCR 5细胞外受体序列代表有吸引力的 对HIV-I的体液免疫的目标。 我们分析了CCR 5 细胞外受体序列结构域，并已确定推定的 代表潜在抗体靶标的细胞外序列。 的 本研究的主要目的是：a）开发串联重复序列 可溶性和DNA构建体，其代表来自V3环的 原代NSI M-嗜性分离株; B）分析抗血清针对这些分离株的能力 在BALB/c小鼠中产生的构建体，以中和同源和异源 原代NSI M-嗜性HIV分离株; c）开发串联重复可溶性和DNA 构建体代表来自推定的细胞外结构域的序列， CCR 5; d）分析抗血清针对在CCR 5中产生的这些构建体的能力。 BALB/c小鼠中和同源和异源原发性NSI M嗜性 e）利用来自靶向V3和CCR 5的结果 序列，构建含有来自两种序列的杂交免疫原。 原代NSI M嗜性病毒的V3环，以及来自NSI M嗜性病毒的免疫原性序列。 CCR 5辅助受体。 这些免疫原的构建是通过我们的 多决定簇串联克隆载体BMX 7的构建 重复免疫原，和BMX 7 EX，一种DNA表达载体，其保留了 BMX 7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嗜性原代分离株的感染性， 这对我们理解肽组分的免疫学意义重大， DNA和可溶性免疫原。