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Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

ABSTRACT: Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins.HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.

SUBMITTER: Veillette M

PROVIDER: S-EPMC3958102 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

Veillette Maxime M Désormeaux Anik A Medjahed Halima H Gharsallah Nour-Elhouda NE Coutu Mathieu M Baalwa Joshua J Guan Yongjun Y Lewis George G Ferrari Guido G Hahn Beatrice H BH Haynes Barton F BF Robinson James E JE Kaufmann Daniel E DE Bonsignori Mattia M Sodroski Joseph J Finzi Andrés A

Journal of virology 20131218 5

<h4>Unlabelled</h4>Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed t ...[more]

PMID: 24352444

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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: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.

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.

Project description:Antibody-dependent cellular cytotoxicity (ADCC) has been correlated with reduced risk of human immunodeficiency virus type 1 (HIV-1) infection in several preclinical vaccine trials and in the RV144 clinical trial, indicating that this is a relevant antibody function to study. Given the diversity of HIV-1, the breadth of vaccine-induced antibody responses is a critical parameter to understand if a universal vaccine is to be realized. Moreover, the breadth of ADCC responses can be influenced by different vaccine strategies and regimens, including adjuvants. Therefore, to accurately evaluate ADCC and to compare vaccine regimens, it is important to understand the range of HIV Envelope (Env) susceptibility to these responses. These evaluations have been limited because of the complexity of the assay and the lack of a comprehensive panel of viruses for the assessment of these humoral responses. Here, we used 29 HIV-1 infectious molecular clones (IMCs) representing different Envelope subtypes and circulating recombinant forms to characterize susceptibility to ADCC from antibodies in plasma from infected individuals, including 13 viremic individuals, 10 controllers, and six with broadly neutralizing antibody responses. We found in our panel that ADCC susceptibility of the IMCs in our panel did not cluster by subtype, infectivity, level of CD4 downregulation, level of shedding, or neutralization sensitivity. Using partitioning around medoids (PAM) clustering to distinguish smaller groups of IMCs with similar ADCC susceptibility, we identified nested panels of four to eight IMCs that broadly represent the ADCC susceptibility of the entire 29-IMC panel. These panels, together with reagents developed to specifically accommodate circulating viruses at the geographical sites of vaccine trials, will provide a powerful tool to harmonize ADCC data generated across different studies and to detect common themes of ADCC responses elicited by various vaccines. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) responses were found to correlate with reduced risk of infection in the RV144 trial of the only human HIV-1 vaccine to show any efficacy to date. However, reagents to understand the breadth and magnitude of these responses across preclinical and clinical vaccine trials remain underdeveloped. In this study, we characterize HIV-1 infectious molecular clones encoding 29 distinct Envelope strains (Env-IMCs) to understand factors that impact virus susceptibility to ADCC and use statistical methods to identify smaller nested panels of four to eight Env-IMCs that accurately represent the full set. These reagents can be used as standardized reagents across studies to fully understand how ADCC may affect efficacy of future vaccine studies and how studies differ in the breadth of responses developed.

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Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

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Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

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