DNA & SOLUBLE MULTIMERIC IMMUNOGEN FOR HUMMORAL IMMUNITY

描述：(改编自申请者摘要)：多部早期作品 疾病模型的研究表明，含有重复序列的免疫原 表位的免疫原性可能比仅包含 序列的单一拷贝。申请者的研究表明 小鼠抗重组明矾免疫原的抗血清 MN分离物V3PND序列的拷贝能够中和主要的ADA HIV-I的M嗜性分离株。然而，尽管有希望产生免疫原 能够诱导能够中和初级分离株的抗体， HIV V3环的多变量性质显然需要 在任何原型中整合多个毒株特异性V3环序列 艾滋病疫苗。与人们对……作用的不断加深的认识不谋而合 然而，决定细胞趋向性的V3环有了新的见解 V3环序列如何与细胞内的辅助受体相互作用 促进艾滋病毒感染。 CCR5是一种G蛋白偶联受体，被认为是进入细胞的完整受体 嗜M的HIV病毒分离进入巨噬细胞。由于M嗜性病毒分离株 尤其是早期与艾滋病毒感染有牵连的人 32个碱基缺失的纯合导致非功能性-CCR5 受体被保护不受艾滋病毒感染，但似乎具有正常的 表型，CCR5胞外受体序列具有吸引力 针对HIV-I的体液免疫靶标。我们已经分析了CCR5 胞外受体序列结构域，并已确定可能 代表潜在抗体靶点的细胞外序列。这个 目前研究的主要目标是：a)发展串联重复 表示来自V3环的序列的可溶性和DNA构建体 原代NSI M嗜性分离株；b)分析抗血清对这些菌株的能力 在BALB/c小鼠中提出的中和同源和异源的结构 初级NSI嗜M HIV分离株；c)发展串联重复序列可溶性和DNA 代表假定的胞外结构域序列的构建 CCR5；d)分析针对这些构建物的抗血清的能力 BALB/c小鼠中和同源和异源原发NSI嗜中性 HIV分离和；e)利用靶向V3和CCR5的结果 序列，构建包含来自两者的序列的杂交免疫原 主要NSI嗜M病毒的V3环，以及来自 CCR5共受体。这些免疫原的构建是由我们的 多决定簇串联克隆新载体BMX7的研制 Repeat免疫原和BMX7EX，一种保留了 BMX7载体的基本克隆特征。理想情况下，这些研究将 最终发展出能够诱导抗体的免疫原 能够中和M-热带原发分离株的传染性并做出贡献 对我们理解多肽成分的免疫学有重要意义 DNA和可溶性免疫原。

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.