CD4-GP120 COMPLEX IMMUNOGENS

HIV-1的外膜糖蛋白gp120与其细胞表面受体CD4结合后，暴露出gp120上新的“依赖于复合体”的构象表位，可引发广泛的中和抗体反应。Gp120-CD4复合体的晶体结构表明，其中一些表位占据了gp120的结构域，该结构域与病毒进入所需的7-跨膜区辅助受体结合。我们以前的研究表明，化学交联的gp120-CD4复合体可以诱导广泛的中和体液反应，对抗HIV的主要分离株。最近，含有可被细胞表面受体结合改变构象的gp120的“融合能力”免疫原也被证明对来自不同分支的各种HIV分离株产生广泛的中和体液反应。综上所述，这些结果值得进一步探索疫苗策略，使用结构性表达复杂依赖表位的免疫原来诱导广泛的中和抗体。为了克服化学交联型gp120-CD_4复合体的大规模合成所面临的制造难题，我们利用重组DNA技术制备了新型的HIV-1BA-L分离株的单链gp_(120)-CD_4嵌合体(SCGP/120BA-L-CD_4)，该嵌合体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，这种scgp120BA-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复合体所不可能的。我们的scgp120BA-L CD4嵌合体能够与7-TM辅受体结合；在转人CD_4和CCR5的小鼠(huCDR5-鼠)和转人CD_4的兔(huCD_4-兔)中都能产生抗体。我们这些临床前研究的实验设计还将评估scgp120BA-L-CD_4信息是否将被用于决定是否在项目3的目标2中对scgp120BA-L-CD_4嵌合体进行I期评估。我们的假设将在两个特定目标中得到验证：1)产生并鉴定结构性地暴露R5病毒的7-TM结合域的scgp120BA-L-CD_4嵌合体；以及2)在转人CD_4和CCR5的小鼠和转人CD_4的转基因兔中评价scgp120BA-L-CD_4嵌合体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.