Timothy Fouts

Expression and Characterization of a Single-Chain Polypeptide Analogue of the Human Immunodeficiency Virus Type 1 gp120-CD4 Receptor Complex

ABSTRACT 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.

Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques

The identification of HIV envelope structures that generate broadly cross-reactive neutralizing antibodies is a major goal for HIV-vaccine 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-1IIIB 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 HIVBaL 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.

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.

Antibodies to CD4-induced sites in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit immunogens

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 SHIV162P3, 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, cross-linked gp120–CD4 complexes, or soluble CD4 alone. These responses were strongly boosted in the rhFLSC-vaccinated animals by SHIV162P3 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.

Cholera Toxin and Heat-Labile Enterotoxin Activate Human Monocyte-Derived Dendritic Cells and Dominantly Inhibit Cytokine Production through a Cyclic AMP-Dependent Pathway

ABSTRACT Cholera toxin (CT) and heat-labile enterotoxin (LT) are powerful mucosal adjuvants whose cellular targets and mechanism of action are unknown. There is emerging evidence that dendritic cells (DC) are one of the principal cell types that mediate the adjuvant effects of these toxins in vivo. Here we investigate the effects of CT and LT on the maturation of human monocyte-derived DC (MDDC) in vitro. We found that an enzymatically active A domain is necessary for both CT and LT to induce the maturation of MDDC and that this activation is strictly cyclic AMP (cAMP) dependent. ADP-ribosylation-defective derivatives of these toxins failed to induce maturation of MDDC, whereas dibutyryl-cyclic-3′,5′-AMP and Forskolin mimic the maturation of MDDC induced by CT and LT. In addition, an inhibitor of cAMP-dependent kinases, Rp-8-Br-cAMPs, blocked the ability of CT, LT, and Forskolin to activate MDDC. CT, LT, dibutyryl-cyclic-3′,5′-AMP, and Forskolin also dominantly inhibit interleukin 12 and tumor necrosis factor alpha production by MDDC in the presence of saturating concentrations of lipopolysaccharide. Taken together, these results show that the effects of CT and LT on MDDC are mediated by cAMP.

Correlation between Humoral Responses to Human Immunodeficiency Virus Type 1 Envelope and Disease Progression in Early-Stage Infection

Human immunodeficiency virus (HIV)-1-infected rapid and slow progressors showed differential humoral responses against HIV envelope peptides and proteins early in infection. Sera from slow progressors reacted more strongly with short envelope peptides modeling gp160NL4-3, predominantly in gp41. Reactivity to six peptides (in constant regions C3, C4, and C5 of gp120 and in gp41) correlated with slower progression. In a novel association, reactivity to three peptides (in constant regions C1 and C3 and variable region V3 of gp120) correlated with faster progression. Envelope peptide reactivity correlated with subsequent course of disease progression as strongly as did reactivity to gag p24. Patients heterozygous for 32-bp deletions in the CCR5 coreceptor reacted more frequently to an epitope in gp41. Rapid progressors had greater gp120 native-to-denatured binding ratios than did slow progressors. While antibody-dependent cellular cytotoxicity against gp120 did not strongly differentiate the groups, slow progressors showed a broader neutralization pattern against 5 primary virus isolates.

Balance of cellular and humoral immunity determines the level of protection by HIV vaccines in rhesus macaque models of HIV infection

Significance Our candidate HIV vaccine, a single-chain gp120-CD4 chimera, elicits protection against acquisition of simian-human immunodeficiency virus (SHIV)/simian immunodeficiency virus (SIV) in rhesus macaques. Antibody-dependent cellular cytotoxicity was an inverse correlate of infection risk. However, it is attenuated when antigen-specific T-cell responses exceed a threshold, presumably due to the generation of CD4+ CCR5+ T cells, the preferred cellular targets of SHIV/SIV. Multiple studies strongly suggest that HIV/SIV-specific T-cell responses are a double-edged sword. On one hand, they are required for T-cell help in the protective antibody response. On the other hand, they appear to mitigate protection by creating new targets for viral replication. Determining the balance between protective antibody responses and attenuating T-cell responses is a key challenge confronting HIV vaccine development. A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.

Discordant memory B cell and circulating anti-Env antibody responses in HIV-1 infection.

Long-lived memory B cells (BMem) provide an archive of historic Ab responses. By contrast, circulating Abs typically decline once the immunogen is cleared. Consequently, circulating Abs can underestimate the nature of cognate humoral immunity. On the other hand, the BMem pool should provide a comprehensive picture of Ab specificities that arise over the entire course of infection. To test this hypothesis, we compared circulating Ab and BMem from natural virus suppressors who control HIV-1 without therapy and maintain a relatively intact immune system. We found high frequencies of BMem specific for the conserved neutralizing CD4 induced or CD4 binding site epitopes of gp120, whereas low Ab titers to these determinants were detected in contemporaneous plasma. These data suggest that plasma Ab repertoires can underestimate the breadth of humoral immunity, and analyses of BMem should be included in studies correlating Ab specificity with protective immunity to HIV-1.

Induction of Neutralizing Antibodies against Human Immunodeficiency Virus Type 1 Primary Isolates by Gag-Env Pseudovirion Immunization

ABSTRACT A major challenge for the development of an effective HIV vaccine is to elicit neutralizing antibodies against a broad array of primary isolates. Monomeric gp120-based vaccine approaches have not been successful in inducing this type of response, prompting a number of approaches designed to recreate the native glycoprotein complex that exists on the viral membrane. Gag-Env pseudovirions are noninfectious viruslike particles that recreate the native envelope glycoprotein structure and have the potential to generate neutralizing antibody responses against primary isolates. In this study, an inducible cell line was created in order to generate Gag-Env pseudovirions for examination of neutralizing antibody responses in guinea pigs. Unadjuvanted pseudovirions generated relatively weak anti-gp120 responses, while the use of a block copolymer water-in-oil emulsion or aluminum hydroxide combined with CpG oligodeoxynucleotides resulted in high levels of antibodies that bind to gp120. Sera from immunized animals neutralized a panel of human immunodeficiency virus (HIV) type 1 primary isolate viruses at titers that were significantly higher than that of the corresponding monomeric gp120 protein. Interpretation of these results was complicated by the occurrence of neutralizing antibodies directed against cellular (non-envelope protein) components of the pseudovirion. However, a major component of the pseudovirion-elicited antibody response was directed specifically against the HIV envelope. These results provide support for the role of pseudovirion-based vaccines in generating neutralizing antibodies against primary isolates of HIV and highlight the potential confounding role of antibodies directed at non-envelope cell surface components.

Development of an oral prime-boost strategy to elicit broadly neutralizing antibodies against HIV-1.

Given the increasing incidence of HIV-1 infection world-wide, an affordable, effective vaccine is probably the only way that this virus will be contained. Accordingly, our group is developing an oral prime-boost strategy with the primary goal of eliciting broadly neutralizing antibodies against HIV-1 to provide sterilizing immunity for this virus. Our secondary goal is to elicit broadly cross-reactive anti-viral CD8(+) T cells by this strategy to blunt any breakthrough infections that occur after vaccination of individuals who fail to develop sterilizing immunity. This article describes our progress in the use of the live attenuated intracellular bacteria, Salmonella and Shigella, as oral delivery vehicles for DNA vaccines and the development of conformationally constrained HIV-1 Env immunogens that elicit broadly neutralizing antibodies.