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. 2024 Nov 13;15(11):e0182724.
doi: 10.1128/mbio.01827-24. Epub 2024 Oct 7.

CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies

Jonathan Richard #  1   2 , Gérémy Sannier #  1   2 , Li Zhu #  3 , Jérémie Prévost  1   2 , Lorie Marchitto  1   2 , Mehdi Benlarbi  1   2 , Guillaume Beaudoin-Bussières  1   2 , Hongil Kim  3 , Yaping Sun  3 , Debashree Chatterjee  1 , Halima Medjahed  1 , Catherine Bourassa  1 , Gloria-Gabrielle Delgado  1 , Mathieu Dubé  1 , Frank Kirchhoff  4 , Beatrice H Hahn  5   6 , Priti Kumar  3 , Daniel E Kaufmann  7   8   9 , Andrés Finzi  1   2
Affiliations

CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies

Jonathan Richard et al. mBio. .

Abstract

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy.

Importance: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

Keywords: A32; ADCC; Env; HIV-1; RNA-flow fish; bNAbs; hu-mice; nnAbs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Recognition of HIV-1-infected primary CD4+ T cells by bnAbs and nnAbs. Primary CD4+ T cells, mock infected or infected with the transmitted-founder virus CH077, either wild type (WT) or defective for Nef expression (nef-), were stained with a panel of bnAbs and nnAbs, followed with appropriate secondary Abs. Cells were then stained for cell-surface CD4 prior to detection of intracellular HIV-1 p24. (A, E) Example of flow cytometry gating strategy based on cell-surface CD4 and intracellular p24 detection. (B, F) Histograms depicting representative staining with bnAbs (green) and nnAbs (black). (C, G) Graphs shown represent the median fluorescence intensities (MFI) obtained for at least six independent staining with the different mAbs. Error bars indicate means ± standard errors of the means. (D, H) Graphs shown represent the mean MFI obtained with each mAb. Statistical significance was tested using Mann–Whitney U test (**P < 0.01; ns, non-significant).
Fig 2
Fig 2
Nef is expressed in HIV-1-infected cells undergoing CD4 downregulation and expressing high levels of p24. Primary CD4+ T cells, mock infected or infected with the transmitted-founder virus CH077, either WT or defective for Nef expression (nef-) were stained for cell-surface CD4 prior to detection of intracellular HIV-1 p24 and Nef expression. (A) Histograms depicting representative intracellular Nef staining when gating on CD4highp24−, CD4highp24low, or CD4lowp24high cells. In the context of cells infected with CH077TF nef-, in the absence of Nef-mediated CD4 downmodulation, the p24high remained CD4high (CD4highp24high). (B) Quantification of the median fluorescence intensities (MFI) obtained for six independent experiments. Error bars indicate means ± standard errors of the means. Statistical significance was tested using multiple Mann–Whitney tests with a Holm–Sidak post-test (**P < 0.01; ns, non-significant).
Fig 3
Fig 3
HIV-1 late transcripts are mostly detected among cells that downregulated CD4. Purified primary CD4+ T cells, mock infected or infected with the transmitted-founder virus CH077 WT, were stained for cell-surface CD4 prior to detection of intracellular HIV-1 p24 and env mRNA and nef mRNA by RNA flow-FISH. (A) Representative example of flow cytometry gating strategy based on cell-surface CD4 and intracellular p24 detection and representative example of RNA flow-FISH detection of env and nef mRNA among the different cell populations. (B) Quantification of the percentage of env mRNA+ nef mRNA+ cells detected among the different cell populations in three different donors. (C) Alternatively, productively infected cells were first identified based on env and nef mRNA detection (D) Quantification of the percentage of CD4highp24−, CD4highp24low, CD4lowp24low, and CD4lowp24high cells among the env and nef mRNA+ cells with three different donors. Statistical significance was tested using one-way analysis of variance (ANOVA) with a Holm–Sidak post-test (****P < 0.0001; ns, non-significant).
Fig 4
Fig 4
Productively infected cells are resistant to recognition by A32. Purified primary CD4+ T cells, mock infected or infected with the transmitted-founder virus CH077 WT, were stained with A32 or PGT126, followed with appropriate secondary Abs. Cells were then stained for cell-surface CD4 prior to detection of intracellular HIV-1 p24 and env mRNA and nef mRNA by RNAflow FISH. (A–C) In a first analysis, HIV-infected cells were identified, then A32 and PGT126 binding was evaluated. (A) Example of RNAflow FISH gating strategy based on env and nef mRNA detection. (B) Example of antibody binding among the env/nef mRNA− and env/nef mRNA+ cell population. (C) Quantification of the percentage of cells recognized by either A32 or PGT126 among the env/nef mRNA− and env/nef mRNA+ cell population with three different donors. (D–F) In a second alternative analysis, Ab-binding cells were first identified, and the HIV-infection status was then evaluated. (D) Example of flow cytometry gating strategy based on A32 or PGT126 binding. (E) Example of env/nef mRNA detection among the cells recognized (Ab+) or not (Ab−) by indicated mAbs. (F) Quantification of the percentage of env/nef mRNA+ cells among the cells recognized (Ab+) or not (Ab−) by indicated mAbs with three donors. Statistical significance was tested using a two-way ANOVA with a Holm–Sidak post-test (*P < 0.05,**P < 0.01, ****P < 0.0001; ns, non-significant).
Fig 5
Fig 5
Ex vivo expanded CD4+ T cells isolated from PLWH are preferentially targeted by bNAbs. Ex vivo expanded CD4+ T cells from six PLWH were stained with bnAbs and nnAbs, followed by appropriate secondary Abs. Cells were then stained for surface CD4 prior to detection of intracellular HIV-1 p24. (A) Percentage of p24+ upon activation overtime. (B) Example of flow cytometry gating based on CD4 and p24 detection. (C) Histograms depicting representative staining with bnAbs (Green) and nnAbs (Black). (D) Median fluorescence intensities (MFI) obtained with primary CD4+ T cells from six PLWH. (E) Graphs shown represent the mean MFI obtained with each mAb. Each symbol represents a different HIV +donor. Statistical significance was tested using Mann–Whitney U test (*P < 0.05, ****P < 0.0001).
Fig 6
Fig 6
Ex vivo expanded CD4+ T cells isolated from PLWH are resistant to ADCC mediated by nnAbs. Ex vivo expanded CD4 T cells from PLWH were used as target cells, while autologous PBMCs were used as effector cells in our FACS-based ADCC assay. (A) Graph shown represents the percentage of ADCC against the CD4lowp24high cells with the single mAbs and (B) nnAbs vs bnAbs. (A) Each symbol represents results obtained with cells from a different PLWH. Statistical significance was tested using unpaired t-test (****P < 0.0001).
Fig 7
Fig 7
A32 nnAb does not impact viral replication or the size of the reservoir in vivo. (A) Experimental outline. NSG-15-Hu-PBL mice were infected with HIV-1 JRCSF intraperitoneally. At days 6 and 9 post infection, mice were administered 1.5 mg of A32 or 3BNC117 (WT or GRLR) mAb subcutaneously (S.C.). (B) Mice were bled routinely for plasma viral load (PVL) and flow cytometry analysis. PVL levels were measured by quantitative real-time PCR (limit of detection = 300 copies/mL, dotted line). (C) Percentage of CD4+ T cells in peripheral blood was evaluated by flow cytometry. At least six mice were used for each treatment. (D, E) Tissues of JRCSF-infected NSG-15 hu-PBL mice, treated or not with A32 or 3BNC117 (WT or GRLR), were harvested at day 11. (D) Percentage of CD4+ T cells was evaluated by flow cytometry. (E) CD4+ T cells were isolated for real-time PCR analysis of HIV DNA. Each dot represents the mean values ± SEM. S.C., subcutaneous; I.P., intraperitoneal; BM, bone marrow; mock treated, no antibody. Statistical significance was tested using one-way ANOVA with a Holm–Sidak post-test or a Kruskal–Wallis test with a Dunn’s post-test (*P < 0.05,**P < 0.01,***P < 0.001).
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