IgA Targeting Human Immunodeficiency Virus-1 Envelope gp41 Triggers Antibody-Dependent Cellular Cytotoxicity Cross-Clade and Cooperates with gp41-Specific IgG to Increase Cell Lysis

Abstract

The protective efficacy of human immunodeficiency virus-1 (HIV-1) antibodies (Abs) remains mostly correlated with their in vitro neutralizing activity engaging their Fab region. However, anti-HIV-1 Abs also mediate a broad array of Fc-mediated effector functions including Ab-dependent cellular cytotoxicity (ADCC), which depend primarily on the Ab isotype. While ADCC is commonly associated with HIV-1 gp120 envelope-specific IgGs, whether IgAs, especially those targeting the HIV-1 gp41 envelope, also mediate ADCC remains elusive. Therefore, to assess the capacity of IgA specific for HIV-1 to induce Fcα-mediated ADCC, we used the gp41 envelope-specific IgA transformed from the broadly neutralizing 2F5-IgG we have previously reported to induce ADCC. We demonstrate that 2F5-IgA engages FcαRI (CD89), expressed on human monocytes used as effector cells, to induce the lysis of HIV-1 Clade A- and B-infected target cells by ADCC. Furthermore, the 2F5-IgA and 2F5-IgG cooperate to enhance target cells lysis by ADCC. Cooperation in ADCC is also observed between 2F5-IgA and the broadly neutralizing 10E8-IgG. These results provide a new perspective for IgA in protection against HIV-1 acquisition or reservoir eradication and suggest that inducing IgA by vaccination, in particular when targeting gp41, in combination with IgG could strengthen protection by complementary and cooperative activities with IgG.

Keywords: IgA; antibody-dependent cellular cytotoxicity; human immunodeficiency virus envelope protein gp41; human immunodeficiency virus-1; mucosal immune system.

Figure 1

2F5-IgA and 2F5-IgG bind to P1 Clades A and B in a dose-dependent manner. The specificity of (A) 2F5-IgA (solid line) and (B) 2F5-IgG (dotted line) for P1 Clade B (square), Clade A (circle), and Clade C (triangle) was evaluated by ELISA. Mouse anti-human kappa light chain was used for direct comparison of both 2F5 isotypes. Specific binding (OD, 450 nm) is plotted as a function of 2F5 antibody (Ab) concentration (ng/ml). No binding was detected for the irrelevant IgA or IgG used as negative controls (OD 450 nm: <0.1). Values represent mean ± SEM, derived from three independent experiments performed in duplicate.

Figure 2

Human immunodeficiency virus-1 (HIV-1)-specific antibody-dependent cellular cytotoxicity (ADCC) triggered by 2F5-IgA compared to 2F5-IgG. Target CD4⁺ T CEM-NK^r cells infected with (A) HIV-1 Clade B (JR-CSF) or (B) green fluorescent protein (GFP)-reporter CD4^high CCR5^high T-cell lines infected with HIV-1 Clade A (92UG031) were stained with PKH26 and then incubated with the indicated concentrations of 2F5-IgA or 2F5-IgG for 30 min at room temperature (RT), prior to addition of effector monocytes (effector:target ratio, 10:1). After 4 h, the percent of infected living (Gag⁺ or GFP⁺) target cells was measured by flow cytometry. The percent of ADCC was calculated using the formula: 100 × (% of infected/PKH26⁺ target cells without antibody − % of infected/PKH26⁺ target cells with antibody)/(% of infected/PKH26⁺ target cells without antibody). Values represent means of HIV-1-infected target cell-specific lysis ± SEM, from six independent experiments performed in duplicate or triplicate for each clade, *p < 0.05, **p < 0.02, and ***p < 0.005, unpaired Student’s t-test of 2F5-IgA versus 2F5-IgG-mediated ADCC.

Figure 3

2F5-IgA and 2F5-IgG induce the lysis of P1-A- and P1-B-coated cells in a dose-dependent manner. The efficacy of 2F5-IgA (solid line, square) and 2F5-IgG (dotted line, circle) to induce the lysis of P1-B (A), and P1-A coated cells (B) was evaluated by flow cytometry. Target P1 Clade A and B cells were double stained with PKH26 and carboxyfluorescein diacetate succinimidyl ester and then incubated with the indicated concentrations of 2F5-IgA or 2F5-IgG for 30 min at room temperature (RT), prior to addition of effector monocytes (effector/target ratio: 10:1). Specific lysis determined as we reported (9) is plotted as a function of 2F5 antibody (Ab) concentration (ng/ml). Values represent mean ± SEM, showing the percentage of specific Ab-dependent cellular cytotoxicity (ADCC) killing derived from five independent experiments performed in duplicate. *p < 0.05, **p < 0.02, and ***p < 0.005, unpaired Student’s t-test of 2F5-IgA versus 2F5-IgG mediated ADCC.

Figure 4

Antibody-dependent cellular cytotoxicity (ADCC) depends on FcαRI and FcγRI. Monocytes were preincubated or not with either anti-CD89- or anti-CD64-blocking antibody for 1 h at 37°C before incubation with double-stained CD4⁺ T CEM-NK^r cells coated with P1. Target cell lysis was evaluated as shown in Figure 3. Values represent the percent of ADCC-induced cell lysis reduction in the presence of monocytes preincubated with anti-CD89 or anti-CD64 antibody. Values represent mean inhibition ± SEM. *p < 0.005, unpaired Student’s t-test compared to ADCC in the absence of antibodies.

Figure 5

2F5-IgA cooperates with 2F5-IgG to induce lysis of P1-A target cells by antibody-dependent cellular cytotoxicity (ADCC) as a result from increased binding to target cells. (A,C) CD4⁺ T CEM-NK^r cells coated with P1-A and P1-B and double stained with PKH26/carboxyfluorescein diacetate succinimidyl ester were incubated with either 2F5-IgA (black bars), 2F5-IgG or 10E8-IgG (gray bars), or with a combination of both, at indicated concentrations, for 30 min at room temperature (RT). Then, effector cells were added at an effector/target ratio of 10:1. Hatched bars represent the experimental values ± SEM of ADCC induced by the combination of 2F5-IgA and 2F5-IgG or 10E8-IgG at indicated concentrations. n = 3 independent experiments performed in duplicate. *p < 0.05, Student’s t-test between the arithmetical sum of cell lysis induced by each isotype separately and cell lysis induced by both isotype together. (B,D) CD4⁺ T CEM-NK^r cells coated with P1-A and P1-B were incubated with 2F5-IgA (black bars), 2F5-IgG or 10E8-IgG (gray bars), or with a combination of both at indicated concentrations for 1 h at 4°C. Specific binding was detected using either mouse fluorescein isothiocyanate (FITC)-conjugated anti-human IgA or allophycocyanin (APC)-conjugated anti-human IgG, or the combination of both, and analyzed by flow cytometry, as indicated in the Section “Materials and Methods.” Values represent MFI of specific binding. n = 5 independent experiments. ***p < 0.0001 and p < 0.0002, **p < 0.001, Student’s t-test between MFI values of the binding of IgA or IgG alone with that of IgA or IgG in the combination mix, respectively.