The infection of CD4؉ host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.

Epitopes located in and around the coreceptor binding site of HIV-1 envelope glycoprotein (gp120) exhibit enhanced exposure after attachment to the CD4 receptor and comprise some of the most conserved and functionally important residues on the viral envelope. Therefore, antibody responses to these epitopes [designated as CD4-induced (CD4i)] should be highly cross-reactive and potentially useful for HIV vaccine development. To address this question, rhesus macaques were vaccinated with subunit immunogens designed to raise humoral responses against CD4i epitopes and challenged rectally with SHIV 162P3, which encodes a heterologous envelope versus the immunogen. We found that animals vaccinated with a rhesus full-length single-chain (rhFLSC) complex exhibited significantly accelerated clearance of plasma viremia and an absence of long-term tissue viremia compared with unvaccinated control animals. Such control of infection correlated with stronger responses to CD4i epitopes in the rhFLSC-vaccinated animals, compared with macaques immunized with gp120, crosslinked gp120 -CD4 complexes, or soluble CD4 alone. These responses were strongly boosted in the rhFLSC-vaccinated animals by SHIV 162P3 infection. The control of infection was not associated with anti-CD4 responses, overall anti-gp120-binding titers, or neutralizing activity measured in conventional assays. Vaccine-naive animals also developed anti-CD4i epitope responses after simian/ human immunodeficiency virus (SHIV) challenge, which appeared later than the overall anti-gp120 responses and in concert with the decline of viremia to a low set point. Collectively, these data suggest that antibodies to CD4i epitopes may play a role in controlling SHIV infection and provide insights for HIV vaccine development.vaccine ͉ infection ͉ immunity

The identification of HIV envelope structures that generate broadly cross-reactive neutralizing antibodies is a major goal for HIVvaccine development. In this study, we evaluated one such structure, expressed as either a gp120 -CD4 or a gp140 -CD4 complex, for its ability to elicit a neutralizing antibody response. In rhesus macaques, covalently crosslinked complexes of soluble human CD4 (shCD4) and HIV-1 IIIB envelope glycoproteins (gp120 or gp140) generated antibodies that neutralized a wide range of primary HIV-1 isolates regardless of the coreceptor usage or genetic subtype. Ig with cross-reactive neutralizing activity was recovered by affinity chromatography with a chimeric single-chain polypeptide containing sequences for HIV BaL gp120 and a mimetic peptide that induces a CD4-triggered envelope structure. These results suggest that covalently crosslinked complexes of the HIV-1 surface envelope glycoprotein and CD4 elicit broadly neutralizing humoral responses that, in part, may be directed against a novel epitope(s) found on the HIV-1 envelope.gp120 ͉ gp140 ͉ gp120 -CD4 complex ͉ vaccine A safe and effective vaccine against HIV should elicit humoral responses that are effective against a broad spectrum of primary HIV strains (1). Unfortunately, an immunogen capable of generating such antibodies in humans remains elusive. The structural and antigenic characteristics of the free HIV envelope (Env) have been studied intensively in hopes of identifying a configuration that might serve as a subunit immunogen. However, these efforts have produced Env-based immunogens that typically fail to elicit antibodies capable of neutralizing more than a minor fraction of primary isolates (2-9).The gp120-CD4 complex, which forms during virus attachment to cell surface receptor CD4 (10), has been considered as another potential target for broadly neutralizing antibodies. This complex is antigenically distinct from free gp120 and is essential for viral entry via 7-transmembrane domain (7-TM) chemokine receptors (10). Only a few studies have attempted to characterize immune sera raised by gp120-CD4 complexes for broadly cross-reactive neutralizing antibodies (11)(12)(13)(14). These studies showed that broadly neutralizing antibody responses were generated in mice by immunization with mixtures of gp120 and CD4. However, these responses were directed, in part, against the interactive domains of CD4 and gp120 and were not qualitatively different from what are elicited by the uncomplexed antigens (11, 13). In contrast, a gp120-CD4 complex, stabilized by covalent crosslinking, elicited antibody responses in goats that neutralized primary HIV isolates (12). Such an antigen can be useful for characterizing the immunogenic characteristics of HIV Env-CD4 complexes. Accordingly, in this study we evaluated the immunogenicity of crosslinked gp120-CD4 complexes in rhesus macaques, a nonhuman primate commonly used as an animal model for developing strategies to elicit protective anti-HIV immune responses. Expression and Production of HIV En...