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.
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