The ultimate means for stopping the HIV epidemic is a prophylactic vaccine that blocks virus transmission in the general population. It is now accepted that such a vaccine will have to elicit anti-HIV antibody responses as well as cellular immunity to provide protection. To meet this goal, it is necessary to identify an immunogen that will elicit broadly neutralizing antibodies capable of preventing (neutralizing) infection by primary HIV strains found worldwide. Our approach focuses on the "constrained" transition state structure of gp120 induced by CD4 binding. In previous studies, we showed that crosslinked gp120-soluble CD4 complexes elicited antibodies in macaques that neutralized a wide variety of primary isolates regardless of Clade. We also showed that these "broadly neutralizing" antibodies were isolated from immune sera by affinity chromatography with a constrained single chain complex (called SCBaL/M9) containing gp120 linked to a CD4 mimetic miniprotein (CD4M9). Thus, single chain gp120-CD4 mimetic complexes warrant exploration as vaccine subunit immunogens to elicit broadly neutralizing antibodies in humans. However, in preliminary experiments SCBaL/M9 elicited broadly neutralizing antibodies less efficiently than a single chain gp120-CD4 complex (FLSC). This could mean that a significant portion of SCBaL/M9 fails to maintain an intrachain interaction under steady state conditions and consequently does not present the key constrained determinants on gp120 that are needed to elicit broadly neutralizing antibodies. Such instability is consistent with the known properties of CD4M9, which has a binding affinity for gp120 that is only about 1% that of CD4. In agreement, our preliminary studies show that the intramolecular interactions in SCBaL/M9 are less stable than in FLSC. Accordingly, our central hypothesis, which we will evaluate in this project, is that we will improve the immunogenicity of SCBaL/M9 by sequence modifications that produce highly stabilized intramolecular complexes. In order to explore this hypothesis, Aim 1 of this project will be to design and evaluate modified versions of SCBaL/M9 for improved intrachain binding stability. Aim 2 will be to compare the immunogenicity of modified complexes versus SCBaL/M9 in rabbits. Binding and functional assays will be performed to verify that none of the modified immunogens elicits antibodies that crossreactive with human or rabbit CD4. We expect these efforts to yield new candidate immunogens that can be feasibly used to elicit broadly neutralizing antibodies in a variety of vaccine contexts.