Short Communication: Small-Molecule CD4 Mimetics Sensitize HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity by Antibodies Elicited by Multiple Envelope Glycoprotein Immunogens in Nonhuman Primates

ABSTRACT To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here, we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of antibody-dependent cellular cytotoxicity (ADCC). Using flow cytometry and confocal microscopy, we show that broadly neutralizing antibodies (bNAbs) targeting the “closed” conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibilities of infected cells to ADCC. Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the “closed” conformation of Env expressed on HIV-1-infected cells. IMPORTANCE HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus, a better understanding of this mechanism might help develop antibodies with improved capacities to mediate ADCC.

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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

Journal of Virology ◽

10.1128/jvi.01325-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 3

Author(s):

Shilei Ding ◽

Melissa C. Grenier ◽

William D. Tolbert ◽

Dani Vézina ◽

Rebekah Sherburn ◽

...

Keyword(s):

Drug Development ◽

Small Molecules ◽

Small Molecule ◽

Envelope Glycoprotein ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Closed Conformation ◽

New Family ◽

Humoral Responses ◽

Hiv 1

ABSTRACT 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.

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Cross-Reactive Antibodies With the Capacity to Mediate HIV-1 Envelope Glycoprotein–Targeted Antibody-Dependent Cellular Cytotoxicity Identified in HIV-2–Infected Individuals

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz001 ◽

2019 ◽

Vol 219 (11) ◽

pp. 1749-1754 ◽

Cited By ~ 3

Author(s):

Ingrid Karlsson ◽

Jeanette Linnea Tingstedt ◽

Gülşen Özkaya Şahin ◽

Mikkel Hansen ◽

Zsofia Szojka ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Hiv 1

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Differences in the Binding Affinity of an HIV-1 V2 Apex-Specific Antibody for the SIVsmm/macEnvelope Glycoprotein Uncouple Antibody-Dependent Cellular Cytotoxicity from Neutralization

mBio ◽

10.1128/mbio.01255-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 6

Author(s):

Benjamin von Bredow ◽

Raiees Andrabi ◽

Michael Grunst ◽

Andres G. Grandea ◽

Khoa Le ◽

...

Keyword(s):

Binding Affinity ◽

Specific Antibody ◽

Neutralizing Antibody ◽

Glycosylation Site ◽

Antibody Binding ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Broadly Neutralizing Antibody ◽

Infected Cells ◽

Hiv 1

ABSTRACTAs a consequence of their independent evolutionary origins in apes and Old World monkeys, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses of the SIVsmm/maclineage express phylogenetically and antigenically distinct envelope glycoproteins. Thus, HIV-1 Env-specific antibodies do not typically cross-react with the Env proteins of SIVsmm/macisolates. Here we show that PGT145, a broadly neutralizing antibody to a quaternary epitope at the V2 apex of HIV-1 Env, directs the lysis of SIVsmm/mac-infected cells by antibody-dependent cellular cytotoxicity (ADCC) but does not neutralize SIVsmm/macinfectivity. Amino acid substitutions in the V2 loop of SIVmac239 corresponding to the epitope for PGT145 in HIV-1 Env modulate sensitivity to this antibody. Whereas a substitution in a conserved N-linked glycosylation site (N171Q) eliminates sensitivity to ADCC, a lysine-to-serine substitution in this region (K180S) increases ADCC and renders the virus susceptible to neutralization. These differences in function correlate with an increase in the affinity of PGT145 binding to Env on the surface of virus-infected cells and to soluble Env trimers. To our knowledge, this represents the first instance of an HIV-1 Env-specific antibody that cross-reacts with SIVsmm/macEnv and illustrates how differences in antibody binding affinity for Env can differentiate sensitivity to ADCC from neutralization.IMPORTANCEHere we show that PGT145, a potent broadly neutralizing antibody to HIV-1, directs the lysis of SIV-infected cells by antibody-dependent cellular cytotoxicity but does not neutralize SIV infectivity. This represents the first instance of cross-reactivity of an HIV-1 Env-specific antibody with SIVsmm/macEnv and reveals that antibody binding affinity can differentiate sensitivity to ADCC from neutralization.

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Elicitation of Cluster A and Co-Receptor Binding Site Antibodies Are Required to Eliminate HIV-1 Infected Cells

Microorganisms ◽

10.3390/microorganisms8050710 ◽

2020 ◽

Vol 8 (5) ◽

pp. 710 ◽

Cited By ~ 2

Author(s):

Guillaume Beaudoin-Bussières ◽

Jérémie Prévost ◽

Gabrielle Gendron-Lepage ◽

Bruno Melillo ◽

Junhua Chen ◽

...

Keyword(s):

Binding Site ◽

Envelope Glycoprotein ◽

Antibody Dependent Cellular Cytotoxicity ◽

Receptor Binding Site ◽

Coreceptor Binding ◽

Infected Cells ◽

Cluster A ◽

Hiv 1 ◽

Coreceptor Binding Site

HIV-1-infected individuals raise a polyclonal antibody response targeting multiple envelope glycoprotein (Env) epitopes. Interestingly, two classes of non-neutralizing CD4-induced (CD4i) antibodies, present in the majority of HIV-1-infected individuals have been described to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of small CD4 mimetic compounds (CD4mc). These antibodies recognize the coreceptor binding site (CoRBS) and the constant region one and two (C1C2 or inner domain cluster A) of the gp120. In combination with CD4mc they have been shown to stabilize an antibody-vulnerable Env conformation, known as State 2A. Here we evaluated the importance of these two families of Abs in ADCC responses by immunizing guinea pigs with gp120 immunogens that have been modified to elicit or not these types of antibodies. Underlying the importance of anti-CoRBS and anti-cluster A Abs in stabilizing State 2A, ADCC responses were only observed in the presence of these two types of CD4i antibodies. Altogether, our results suggest that these two families of CD4i antibodies must be taken into account when considering future strategies relying on the use of CD4mc to eliminate HIV-1-infected cells in vivo.

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Uninfected Bystander Cells Impact the Measurement of HIV-Specific Antibody-Dependent Cellular Cytotoxicity Responses

mBio ◽

10.1128/mbio.00358-18 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 35

Author(s):

Jonathan Richard ◽

Jérémie Prévost ◽

Amy E. Baxter ◽

Benjamin von Bredow ◽

Shilei Ding ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Ex Vivo ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Antibody Recognition ◽

Vaccine Trials ◽

Infected Cells ◽

Bystander Cells ◽

Hiv 1

ABSTRACTThe conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a “closed” conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affectin vitromeasurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells inin vitrocultures orex vivosamples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands.IMPORTANCEEmerging evidence supports a role for antibody-dependent cellular cytotoxicity (ADCC) in protection against HIV-1 transmission and disease progression. However, there are conflicting reports regarding the ability of nonneutralizing antibodies targeting CD4-inducible (CD4i) Env epitopes to mediate ADCC. Here, we performed a side-by-side comparison of different methods currently being used in the field to measure ADCC responses to HIV-1. We found that assays which are unable to differentiate virus-infected from uninfected cells greatly overestimate ADCC responses mediated by antibodies to CD4i epitopes and underestimate responses mediated by broadly neutralizing antibodies (bNAbs). Our results strongly argue for the use of assays that measure ADCC against HIV-1-infected cells expressing physiologically relevant conformations of Env to evaluate correlates of protection in vaccine trials.

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CD4- and Time-Dependent Susceptibility of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

Journal of Virology ◽

10.1128/jvi.01901-18 ◽

2019 ◽

Vol 93 (10) ◽

Cited By ~ 6

Author(s):

Wen Shi Lee ◽

Jérémie Prévost ◽

Jonathan Richard ◽

Reneé M. van der Sluis ◽

Sharon R. Lewin ◽

...

Keyword(s):

Late Stage ◽

De Novo ◽

Nk Cell ◽

Early Stage ◽

Time Dependent ◽

Target Cells ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Infected Cells ◽

Hiv 1

ABSTRACTHIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) antibodies within HIV-1-positive (HIV-1+) individuals predominantly target CD4-induced (CD4i) epitopes on HIV-1 envelope glycoprotein (Env). These CD4i epitopes are usually concealed on the surface of infected cells due to CD4 downregulation by the HIV-1 accessory proteins Nef and Vpu. We hypothesized that early-stage infected cells in the process of downregulating CD4 could be more susceptible to ADCC than late-stage infected cells that have fully downregulated CD4. There was significantly higher binding of antibodies within plasma from HIV-1-infected individuals to early-stage infected cells expressing intermediate levels of CD4 (CD4-intermediate cells) than in late-stage infected cells expressing low levels of CD4 (CD4-low cells). However, we noted that HIV-1-uninfected bystander cells and HIV-1-infected cells, at various stages of downregulating CD4, were all susceptible to NK cell-mediated ADCC. Importantly, we observed that the cytolysis of bystander cells and early infected cells in this culture system was driven by sensitization of target cells by inoculum-derived HIV-1 Env or virions. This phenomenon provided Env to target cells prior tode novoEnv expression, resulting in artifactual ADCC measurements. Future studies should take into consideration the inherent caveats ofin vitroinfection systems and develop improved models to address the potential role for ADCC against cells with nascent HIV-1 infection.IMPORTANCEAn increasing body of evidence suggests that ADCC contributes to protection against HIV-1 acquisition and slower HIV-1 disease progression. Targeting cells early during the infection cycle would be most effective in limiting virus production and spread. We hypothesized that there could be a time-dependent susceptibility of HIV-1-infected cells to ADCC in regard to CD4 expression. We observed NK cell-mediated ADCC of HIV-1-infected cells at multiple stages of CD4 downregulation. Importantly, ADCC of early infected cells appeared to be driven by a previously unappreciated problem of soluble Env and virions from the viral inoculum sensitizing uninfected cells to ADCC prior tode novoEnv expression. These results have implications for studies examining ADCC against cells with nascent HIV-1 infection.

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Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

Journal of Virology ◽

10.1128/jvi.02717-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 2021-2030 ◽

Cited By ~ 26

Author(s):

Wen Shi Lee ◽

Jonathan Richard ◽

Marit Lichtfuss ◽

Amos B. Smith ◽

Jongwoo Park ◽

...

Keyword(s):

Immune Responses ◽

Autologous Serum ◽

Cellular Cytotoxicity ◽

Primary Cells ◽

Antibody Dependent Cellular Cytotoxicity ◽

Serum Antibodies ◽

Latently Infected ◽

Infected Cells ◽

Antibody Epitopes ◽

Hiv 1

ABSTRACTLifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+T cells from HIV-1+subjects were used as targets for ADCC. Theseex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted.IMPORTANCEAn HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune responses within HIV-1+individuals can efficiently eliminate the reactivated cells. HIV-1-specific antibodies can potentially eliminate cells reactivated from latency via Fc effector functions by recruiting innate immune cells. Our study highlights the potential role that antibody-dependent cellular cytotoxicity might play in antilatency cure approaches.

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Faculty Opinions recommendation of Differences in the Binding Affinity of an HIV-1 V2 Apex-Specific Antibody for the SIVsmm/mac Envelope Glycoprotein Uncouple Antibody-Dependent Cellular Cytotoxicity from Neutralization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736126194.793562589 ◽

2019 ◽

Author(s):

Noah Sather

Keyword(s):

Binding Affinity ◽

Specific Antibody ◽

Envelope Glycoprotein ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Hiv 1

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Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses

Journal of Virology ◽

10.1128/jvi.00484-18 ◽

2018 ◽

Vol 92 (13) ◽

pp. e00484-18 ◽

Cited By ~ 20

Author(s):

Jérémie Prévost ◽

Jonathan Richard ◽

Halima Medjahed ◽

Audrey Alexander ◽

Jennifer Jones ◽

...

Keyword(s):

Cell Surface ◽

Reporter Gene ◽

Large Scale ◽

Surface Expression ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Content Type ◽

Infected Cells ◽

Infectious Molecular Clones ◽

Hiv 1

ABSTRACTHIV-1-infected cells expressing envelope glycoproteins (Env) in the CD4-bound conformation on their surfaces are targeted by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) antibodies and sera from HIV-1-infected individuals (HIV+sera). By downregulating the surface expression of CD4, Nef prevents Env-CD4 interaction, thus protecting HIV-1-infected cells from ADCC. HIV-1 infectious molecular clones (IMCs) are widely used to measure ADCC. In order to facilitate the identification of infected cells and high-throughput ADCC analysis, reporter genes (e.g., theRenillaluciferase [LucR] gene) are often introduced into IMC constructs. We evaluated the susceptibility of HIV-1-infected CD4+T lymphocytes to ADCC using a panel of parental IMCs and derivatives that expressed the LucR reporter gene, utilizing different molecular strategies, including one specifically designed to retain Nef expression. We found that in some of these constructs, Nef expression in CD4+T cells was suboptimal, and consequently, CD4 downregulation was incomplete. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Strikingly, protection from ADCC was observed when cells were infected with the parental IMC, which exhibited strong CD4 downregulation. This discrepancy between the parental and Nef-impaired viruses was independent of the strains of Env expressed, but rather, it was correlated with the levels of CD4 surface expression. Overall, our results indicate that caution should be taken when selecting IMCs for ADCC measurements and that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.IMPORTANCEIn-depth understanding of the susceptibility of HIV-1-infected cells to ADCC might help establish correlates of vaccine protection and guide the development of HIV-1 vaccine strategies. Different ADCC assays have been developed, including those using infectious molecular clones (IMCs) carrying a LucR reporter gene that greatly facilitates large-scale quantitative analysis. We previously reported different molecular strategies for introducing LucR while maintaining Nef expression and function and, consequently, CD4 surface downregulation. Here, we demonstrate that utilizing IMCs that exhibit impaired Nef expression can have undesirable consequences due to incomplete CD4 downregulation. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Overall, our results indicate that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

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