CD4-GP120 COMPLEX IMMUNOGENS

HIV-1的外包膜糖蛋白gp120与其细胞表面受体CD4结合，暴露出gp120上新的“复合物依赖”构象表位，可以引发广泛中和的抗体反应。gp120-cd4复合物的晶体结构表明，其中一些表位占据gp120的一个结构域，该结构域与病毒进入所需的7-跨膜结构域共受体结合。我们之前的研究表明，化学交联的gp120-CD4复合物可引起广泛中和的体液反应，以对抗原发HIV分离物。最近，含有gp120的“融合能力”免疫原也被证明可以通过细胞表面受体结合改变构象，从而引发针对来自不同分支的多种HIV分离株的广泛中和性体液反应。综上所述，这些结果值得进一步探索疫苗策略，使用组成性表达复杂依赖表位的免疫原来引发广泛中和抗体。为了克服大规模合成化学交联gp120-CD4复合物所面临的制造问题，我们利用重组DNA技术制备了HIV-1Ba-L分离物（scgp/120Ba-L-CD4）的新型单链gp120-CD4嵌合体，该嵌合体易于在细胞系中表达，提供可溶性亚基蛋白免疫原。此外，这种scgp120Ba-L- CD4嵌合体可以作为DNA疫苗递送（项目1），这是通过化学交联制备的gp120-CD4复合物无法实现的。我们的scgp120Ba-L CD4嵌合体能够组成性地结合7-TM辅助受体；在转染人CD4和CCR5的小鼠（hucdr5 -小鼠）和转染人CD4的家兔（hucd4 -家兔）中均存在抗体。我们对这些临床前研究的实验设计还将评估scgp120Ba-L-CD4信息是否将用于决定是否在项目3的Aim 2中进行scgp120Ba-L-CD4嵌合体的人体I期评估。我们的假设将在两个特定目标中得到验证：1)生产和表征scgp120Ba-L-CD4嵌合体，该嵌合体可组成性地暴露R5病毒的7-TM结合结构域；2)评价scgp120Ba-L-CD4嵌合体在人CD4和CCR5转基因小鼠和人CD4转基因家兔中的安全性和免疫原性。

Binding of the outer envelope glycoprotein, gp120, of HIV-1 to its cell surface receptor, CD4, exposes novel "complex-dependent" conformational epitopes on gp120 that can elicit broadly neutralizing antibody responses. The crystal structure of gp120-cd4 complexes shows that some of these epitopes occupy a domain of gp120 that binds to the 7- transmembrane domain co-receptors that are required for virus entry. Our previous studies showed that chemically crosslinked gp120-CD4 complexes elicit broadly neutralizing humoral responses against primary HIV isolates. More recently, "fusion competent" immunogens containing gp120 that is conformationally altered by cell surface receptor binding were also shown to elicit broadly neutralizing humoral responses against a wide variety of HIV isolates from different clades. Taken together, these results warrant further exploration of vaccine strategies that use immunogens which constitutively express complex dependent epitopes to elicit broadly neutralizing antibodies. To overcome the manufacturing problems facing the large scale synthesis of chemically cross-linked gp120-CD4 complexes, we have used recombinant DNA techniques to make a novel single chain gp120-CD4 chimera of the HIV-1Ba-L isolate (scgp/120Ba-L-CD4) that is expressed readily in cell lines to provide soluble subunit protein immunogens. Furthermore, this scgp120Ba-L- CD4 chimera can be delivered as a DNA vaccine (Project 1), which is not possible using gp120-CD4 complexes that are prepared by chemical crosslinking. Our scgp120Ba-L CD4 chimera is able to constitutively bind to 7-TM coreceptors ; antibodies both in mice transgenic for human CD4 and CCR5 (huCDR5-mice) and rabbits transgenic for human CD4 (huCD4-rabbits). Our experimental design for these preclinical studies will also evaluate whether the scgp120Ba-L-CD4 information will be used to decide whether to go forward with a Phase I evaluation of the scgp120Ba-L-CD4 chimera in humans in Aim 2 of Project 3. Our hypothesis will be tested in two specific aims: 1) to produce and characterize a scgp120Ba-L-CD4 chimera that constitutively exposes the 7-TM binding domain for R5 viruses; and 2) to evaluate the safety and immunogenicity of the scgp120Ba-L-CD4 chimera in mice transgenic for human CD4 and CCR5 and in rabbits transgenic for human CD4.