A New Family of Small-Molecule CD4-Mimetic Compounds Contacts Highly Conserved Aspartic Acid 368 of HIV-1 gp120 and Mediates Antibody-Dependent Cellular Cytotoxicity.

The HIV-1 envelope glycoprotein (Env) trimer mediates virus entry into cells. The "closed" conformation of Env is resistant to nonneutralizing antibodies (nnAbs). These antibodies mostly recognize occluded epitopes that can be exposed upon binding of CD4 or small-molecule CD4 mimetics (CD4mc). Here, we describe a new family of small molecules that expose Env to nnAbs and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC). These compounds have a limited capacity to inhibit virus infection directly but are able to sensitize viral particles to neutralization by otherwise nonneutralizing antibodies. Structural analysis shows that some analogs of this family of CD4mc engage the gp120 Phe43 cavity by contacting the highly conserved D368 residue, making them attractive scaffolds for drug development.IMPORTANCE HIV-1 has evolved multiple strategies to avoid humoral responses. One efficient mechanism is to keep its envelope glycoprotein (Env) in its "closed" conformation. Here, we report on a new family of small molecules that are able to "open up" Env, thus exposing vulnerable epitopes. This new family of molecules binds in the Phe43 cavity and contacts the highly conserved D368 residue. The structural and biological attributes of molecules of this family make them good candidates for drug development.

Two Families of Env Antibodies Efficiently Engage Fc-Gamma Receptors and Eliminate HIV-1-Infected Cells.

HIV-1 conceals epitopes of its envelope glycoproteins (Env) recognized by antibody (Ab)-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These Abs, including anti-coreceptor binding site (CoRBS) and anti-cluster A antibodies, preferentially recognize Env in its "open" conformation. The binding of anti-CoRBS Abs has been shown to induce conformational changes that further open Env, allowing interaction of anti-cluster A antibodies. We explored the possibility that CoRBS Abs synergize with anti-cluster A Abs to engage Fc-gamma receptors to mediate ADCC. We found that binding of anti-CoRBS and anti-cluster A Abs to the same gp120 is required for interaction with soluble dimeric FcγRIIIa in enzyme-linked immunosorbent assays (ELISAs). We also found that Fc regions of both Abs are required to optimally engage FcγRIIIa and mediate robust ADCC. Taken together, our results indicate that these two families of Abs act together in a sequential and synergistic fashion to promote FcγRIIIa engagement and ADCC.IMPORTANCE The "open" CD4-bound conformation of HIV-1 envelope glycoproteins is the primary target of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies present in HIV-positive (HIV+) sera, such as anti-coreceptor binding site and anti-cluster A antibodies. Here we report that the binding of these two families of antibodies is required to engage FcγRIIIa and mediate ADCC.

Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors.

Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations.IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the transitions from the unliganded state to the CD4-bound state.

Soluble Envelope Glycoprotein Trimers from a CD4-Independent HIV-1 Elicit Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Guinea Pigs.

CD4-independent HIV-1 variants can infect coreceptor-expressing cells lacking CD4. The envelope (Env) glycoproteins on these HIV-1 variants expose a coreceptor binding site that overlaps some CD4-induced (CD4i) epitopes. Reports have demonstrated that CD4i antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). Here we investigated the immunogenicity of soluble Env trimers (sgp140) from a CD4-independent HIV-1 in guinea pigs and found that the sgp140 elicited ADCC-mediating antibodies. Therefore, these sgp140 might be useful in vaccine regimens aimed at eliciting ADCC responses.

Optimization of Small Molecules That Sensitize HIV-1 Infected Cells to Antibody-Dependent Cellular Cytotoxicity.

With approximately 37 million people living with HIV worldwide and an estimated 2 million new infections reported each year, the need to derive novel strategies aimed at eradicating HIV-1 infection remains a critical worldwide challenge. One potential strategy would involve eliminating infected cells via antibody-dependent cellular cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to conceal epitopes located in its envelope glycoprotein (Env) that are recognized by ADCC-mediating antibodies present in sera from HIV-1 infected individuals. Our aim is to circumvent this evasion via the development of small molecules that expose relevant anti-Env epitopes and sensitize HIV-1 infected cells to ADCC. Rapid elaboration of an initial screening hit using parallel synthesis and structure-based optimization has led to the development of potent small molecules that elicit this humoral response. Efforts to increase the ADCC activity of this class of small molecules with the aim of increasing their therapeutic potential was based on our recent cocrystal structures with gp120 core.

Paring Down HIV Env: Design and Crystal Structure of a Stabilized Inner Domain of HIV-1 gp120 Displaying a Major ADCC Target of the A32 Region.

Evidence supports a role of antibody-dependent cellular cytotoxicity (ADCC) toward transitional epitopes in the first and second constant (C1-C2) regions of gp120 (A32-like epitopes) in preventing HIV-1 infection and in vaccine-induced protection. Here, we describe the first successful attempt at isolating the inner domain (ID) of gp120 as an independent molecule that encapsulates the A32-like region within a minimal structural unit of the HIV-1 Env. Through structure-based design, we developed ID2, which consists of the ID expressed independently of the outer domain and stabilized in the CD4-bound conformation by an inter-layer disulfide bond. ID2 expresses C1-C2 epitopes in the context of CD4-triggered full-length gp120 but without any known neutralizing epitope present. Thus, ID2 represents a novel probe for the analysis and/or selective induction of antibody responses to the A32 epitope region. We also present the crystal structure of ID2 complexed with mAb A32, which defines its epitope.

Co-receptor Binding Site Antibodies Enable CD4-Mimetics to Expose Conserved Anti-cluster A ADCC Epitopes on HIV-1 Envelope Glycoproteins.

Human immunodeficiency virus type 1 (HIV-1) has evolved a sophisticated strategy to conceal conserved epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies, which are present in the sera of most HIV-1-infected individuals, preferentially recognize Env in its CD4-bound conformation. Accordingly, recent studies showed that small CD4-mimetics (CD4mc) able to "push" Env into this conformation sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. Here we test whether CD4mc also expose epitopes recognized by anti-cluster A monoclonal antibodies such as A32, thought to be responsible for the majority of ADCC activity present in HIV+ sera and linked to decreased HIV-1 transmission in the RV144 trial. We made the surprising observation that CD4mc are unable to enhance recognition of HIV-1-infected cells by this family of antibodies in the absence of antibodies such as 17b, which binds a highly conserved CD4-induced epitope overlapping the co-receptor binding site (CoRBS). Our results indicate that CD4mc initially open the trimeric Env enough to allow the binding of CoRBS antibodies but not anti-cluster A antibodies. CoRBS antibody binding further opens the trimeric Env, allowing anti-cluster A antibody interaction and sensitization of infected cells to ADCC. Therefore, ADCC responses mediated by cluster A antibodies in HIV-positive sera involve a sequential opening of the Env trimer on the surface of HIV-1-infected cells. The understanding of the conformational changes required to expose these vulnerable Env epitopes might be important in the design of new strategies aimed at fighting HIV-1.

Flow cytometry-based assay to study HIV-1 gp120 specific antibody-dependent cellular cytotoxicity responses.

Increased attention on the role of Fc-mediated effector functions against HIV-1 has led to renewed interest into the role that antibody-dependent cellular cytotoxicity (ADCC) could play in controlling viral transmission and/or the rate of disease progression. While (51)Chromium release assays have traditionally been used to study ADCC responses against HIV-1, a number of alternative flow-cytometry-based assays were recently developed. In this study, an alternative flow-cytometry-based assay was established to allow non-radioactive measurement of ADCC-mediated elimination of HIV-1 gp120 envelope glycoprotein (Env)-coated target cells. This assay relies on staining target and effector cells with different dyes, which allows precise gating and permits the calculation of the number of surviving target cells by normalization to flow-cytometry particles. By using small concentrations of recombinant gp120 Env, suitable targets cells that recapitulate the ADCC response mediated against HIV-1-infected cells were generated. Finally, this method was applied successfully to screen human sera for ADCC activity directed against HIV-1 gp120 Env.