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

ABSTRACT: HIV-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 to de novo Env expression, resulting in artifactual ADCC measurements. Future studies should take into consideration the inherent caveats of in vitro infection systems and develop improved models to address the potential role for ADCC against cells with nascent HIV-1 infection.IMPORTANCE An 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 to de novo Env expression. These results have implications for studies examining ADCC against cells with nascent HIV-1 infection.

SUBMITTER: Lee WS

PROVIDER: S-EPMC6498039 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Lee Wen Shi WS Prévost Jérémie J Richard Jonathan J van der Sluis Reneé M RM Lewin Sharon R SR Pazgier Marzena M Finzi Andrés A Parsons Matthew S MS Kent Stephen J SJ

Journal of virology 20190501 10

HIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) antibodies within HIV-1-positive (HIV-1<sup>+</sup>) 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 infe ...[more]

PMID: 30842324

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Project description:Lifelong 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. These ex 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.An 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.

Project description:BackgroundAntibody-dependent cellular cytotoxicity (ADCC) has been associated with improved infant outcome in mother-to-child transmission (MTCT) of HIV-1. Epitopes of these ADCC-mediating antibodies remain unidentified. CD4-inducible (CD4i) epitopes on gp120 are common ADCC targets in natural infection and vaccination. We tested whether CD4i epitope-specific ADCC mediated by maternal antibodies or passively-acquired antibodies in infants is associated with reduced MTCT and improved infant survival.MethodsWe used variants of CD4i cluster A-specific antibodies, A32 and C11, and a cluster C-specific antibody, 17b, with mutations abolishing Fc-Fc receptor interactions as inhibitors in a competition rapid and fluorometric ADCC assay using gp120-coated CEM-nkr target cells with plasma from 51 non-transmitting and 21 transmitting breastfeeding mother-infant pairs.FindingsCluster A-specific ADCC was common. Individually, neither A32-like nor C11-like ADCC was statistically significantly associated with risk of MTCT or infected infant survival. In combination, total maternal cluster A-specific ADCC was statistically significantly associated with decreased infected infant survival in a log-rank test (p = 0·017). There was a non-significant association for infant passively-acquired total cluster A-specific ADCC and decreased infected infant survival (p = 0·14). Surprisingly, plasma ADCC was enhanced in the presence of the defective Fc 17b competitor. Defective Fc 17b competitor-mediated maternal ADCC enhancement was statistically significantly associated with reduced infected infant survival (p = 0·011). A non-significant association was observed for passively-acquired infant ADCC enhancement and decreased survival (p = 0·19).InterpretationsThese data suggest that ADCC targeting CD4i epitopes is not associated with protection against breast milk HIV transmission but is associated with decreased survival of infected infants. FUND: This study was funded by NIH grant R01AI076105 and NIH fellowship F30AI136636.

Project description:Measuring Envelope (Env)-specific antibody (Ab)-dependent cellular cytotoxicity (ADCC)-competent Abs in HIV+ plasma is challenging because Env displays distinctive epitopes when present in a native closed trimeric conformation on infected cells or in a CD4-bound conformation on uninfected bystander cells. We developed an ADCC model which distinguishes Env-specific ADCC-competent Abs based on their capacity to eliminate infected, bystander, or Env rgp120-coated cells as a surrogate for shed gp120 on bystander cells. A panel of monoclonal Abs (MAbs), used to opsonize these target cells, showed that infected cells were preferentially recognized/eliminated by MAbs to CD4 binding site, V3 loop, and viral spike epitopes whereas bystander/coated cells were preferentially recognized/eliminated by Abs to CD4-induced (CD4i) epitopes. In HIV-positive (HIV+) plasma, Env-specific Abs recognized and supported ADCC of infected cells, though a majority were directed toward CD4i epitopes on bystander cells. For ADCC activity to be effective in HIV control, ADCC-competent Abs need to target genuinely infected cells.IMPORTANCE HIV Env-specific nonneutralizing Abs (NnAbs) able to mediate ADCC have been implicated in protection from HIV infection. However, Env-specific NnAbs have the capacity to support ADCC of both HIV-infected and HIV-uninfected bystander cells, potentially leading to misinterpretations when the assay used to measure ADCC does not distinguish between the two target cell types present in HIV cultures. Using a novel ADCC assay, which simultaneously quantifies the killing activity of Env-specific Abs on both infected and uninfected bystander cells, we observed that only a minority of Env-specific Abs in HIV+ plasma mediated ADCC of genuinely HIV-infected cells displaying Env in its native closed conformation. This assay can be used for the development of vaccine strategies aimed at eliciting Env-specific Ab responses capable of controlling HIV infection.

Project description:HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specificmemory B cells encoding Abs that mediate ADCC froma subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activitywhen compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to Fc?Rs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise fromunique B-cell lineages in the same individual. Further, thesemAbsmediate potent plasma IgG-specificADCC breadth and potency and contribute to ADCC activity in other HIV-infected individuals.

Project description:HIV-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., the Renilla luciferase [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.IMPORTANCE In-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.

Dataset Information

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

Publications

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

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