CD4-GP120 COMPLEX IMMUNOGENS

HIV-1的外包膜糖蛋白gp 120与其细胞表面受体CD 4的结合暴露了gp 120上新的“复合物依赖性”构象表位，其可以引起广泛的中和抗体应答。gp 120-cd 4复合物的晶体结构表明，这些表位中的一些占据了gp 120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。我们以前的研究表明，化学交联的gp 120-CD 4复合物引起广泛中和的体液反应，对主要的艾滋病毒分离株。最近，含有gp 120的“融合感受态”免疫原也显示出对来自不同进化枝的多种HIV分离株的广泛中和体液应答，所述gp 120通过细胞表面受体结合而构象改变。综上所述，这些结果保证了疫苗策略的进一步探索，所述疫苗策略使用组成型表达复合物依赖性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的gp 120-CD 4复合物所面临的生产问题，我们使用重组DNA技术制备了HIV-1Ba-L分离物的新型单链gp 120-CD 4嵌合体（scgp/120 Ba-L-CD 4），其易于在细胞系中表达以提供可溶性亚单位蛋白免疫原。此外，该scgp 120 Ba-L-CD 4嵌合体可以作为DNA疫苗递送（项目1），这是使用通过化学交联制备的gp 120-CD 4复合物不可能实现的。我们的scgp 120 Ba-L CD 4嵌合体能够组成型结合7-TM辅助受体;人CD 4和CCR 5转基因小鼠（huCDR 5-小鼠）和人CD 4转基因兔（huCD 4-兔）中的抗体。我们这些临床前研究的实验设计还将评估scgp 120 Ba-L-CD 4信息是否将用于决定是否在项目3的目标2中继续进行人体scgp 120 Ba-L-CD 4嵌合体的I期评价。我们的假设将在两个特定目标中进行检验：1）产生和表征组成性暴露R5病毒7-TM结合结构域的scgp 120 Ba-L-CD 4嵌合体; 2）评价scgp 120 Ba-L-CD 4嵌合体在人CD 4和CCR 5转基因小鼠和人CD 4转基因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.