Isotype modulates epitope specificity, affinity, and antiviral activities of anti-HIV-1 human broadly neutralizing 2F5 antibody

Abstract

The constant heavy chain (CH1) domain affects antibody affinity and fine specificity, challenging the paradigm that only variable regions contribute to antigen binding. To investigate the role of the CH1 domain, we constructed IgA2 from the broadly neutralizing anti-HIV-1 2F5 IgG1, and compared 2F5 IgA2 and IgG binding affinity and functional activities. We found that 2F5 IgA2 bound to the gp41 membrane proximal external region with higher affinity than IgG1. Functionally, compared with IgG1, 2F5 IgA2 more efficiently blocked HIV-1 transcytosis across epithelial cells and CD4(+) cell infection by R5 HIV-1. The 2F5 IgG1 and IgA2 acted synergistically to fully block HIV-1 transfer from Langerhans to autologous CD4(+) T cells and to inhibit CD4(+) T-cell infection. Epitope mapping performed by screening a random peptide library and in silico docking modeling suggested that along with the 2F5 IgG canonical ELDKWA epitope on gp41, the IgG1 recognized an additional 3D-conformational epitope on the gp41 C-helix. In contrast, the IgA2 epitope included a unique conformational motif on the gp41 N-helix. Overall, the CH1 region of 2F5 contributes to shape its epitope specificity, antibody affinity, and functional activities. In the context of sexually transmitted infections such as HIV-1/AIDS, raising a mucosal IgA-based vaccine response should complement an IgG-based vaccine response in blocking HIV-1 transmission.

Fig. 1.

The 2F5 IgA2 binds to gp41 MPER more robustly than 2F5 IgG1. 2F5 IgG1 and 2F5 IgA2 bind to P1, and ELDKWA in a dose-dependent manner. The specificity of 2F5 IgG1 (light gray) and of 2F5 IgA2 (black) for P1 (solid diamond, solid line) and for ELDKWA (solid diamond, broken line) was evaluated by ELISA. W₆₆₆A-mutated P1 (solid diamond, dotted line) served as a negative control. (A and B) Goat anti-human IgG or IgA (A) or mouse anti-human kappa light chain (B) were used as detection reagent to allow for direct comparison of both 2F5 isotypes. Specific binding (OD 450 nm) is plotted as a function of 2F5 Ab concentration (nM). (C and D) Competitive binding of 2F5 IgA2 to P1 and ELDKWA (C) or gp41 (D) in the presence of an excess of 2F5 IgG1 (solid diamond, solid line), or irrelevant IgG (solid diamond, dotted line) used as a negative control, was evaluated in competitive ELISA; 2F5 IgA2-specific binding (OD 450 nm) is plotted as function of the competitor IgG concentration (nM). Values in (A–D) represent mean ± SD of three independent experiments performed in duplicate.

Fig. 2.

The 2F5 IgA2 has greater affinity for gp41 MPER compared with 2F5 IgG1. P1 was immobilized on a CM-5 chip for surface plasmon resonance evaluation of an antibody affinity constant for P1. (A and B) 2F5 IgG1 (at 0, 0.1, 0.2, 0.3, and 0.4 nM) (A) and IgA2 (at 0, 0.5, 1, 1.5, and 2 nM) (B) were the analytes. The K_d and corresponding Pearson's χ² test (Chi2) values shown were estimated by global curve fitting of the specific binding responses. Fitted curves are in black. Injections were carried out in duplicate and gave essentially the same results. Only one of the duplicates is shown. (C) 2F5 IgA2- and IgG1-specific binding to HIV-1 JR-CSF virions. 2F5 IgG1 (gray bars) or IgA2 (black bars) bound to goat anti-human IgG- or IgA-coated ELISA plates were incubated with HIV-1 JR-CSF for 1 h at 37 °C. HIV-1 binding was evaluated by measuring the p24 content after removal of unbound virus. Results are presented as HIV-1 p24 captured by 2F5 IgA2 or IgG1 after subtraction of the nonspecific binding (HIV-1 p24 captured by irrelevant IgA2 or IgG). Values represent mean ± SD of two independent experiments performed in triplicate.

Fig. 3.

The 2F5 IgA2 and IgG1 have different anti-viral efficiencies. (A) Transcytosis of HIV-1, induced by HIV-1 R5-infected PBMCs, across epithelial cells for 2 h was measured after preincubation of HIV⁺ PBMCs with 2F5 IgG1 (gray bars) or IgA2 (black bars). Results are presented as percentage of transcytosis blockade in the absence of Abs. (B) Inhibition of HIV-1 transfer from LCs to autologous CD4⁺ T cells evaluated by measuring the p24 released by LCs/T cells cocultures at day 5. HIV-1 JR-CSF was preincubated with LCs for 2 h before addition of the antibodies, either alone (2F5 IgG1, gray bars; 2F5 IgA2, black bars) or in combination (hatched bars), and T cells. Results are presented as percentage of transfer inhibition in the absence of Abs. (C and D) Dose-dependent inhibition of HIV-1 neutralization mediated by 2F5 Abs assessed in a single cycle infectivity assay, using p24 staining on primary CD4⁺ T cells (C) and CEM-CCR5⁺ cell line (D); 2F5 IgG1 (gray bars), IgA2 (black bars), or their combination (hatched bars), were incubated for 1 h at 37 °C with HIV-1JR-CSF before the addition of CD4⁺ cells for 36 h. The percentage of neutralization was defined as the reduction of p24⁺ cells compared with control-infected cells in the absence of Abs. Values represent mean ± SD of at least three independent experiments performed in duplicate for A and B and in triplicate for C and D.

Fig. 4.

Multiple alignment of sequences specific for gp 41 and 2F5 IgG1 or IgA2. Sequences of epitopes retrieved after panning the random 12-mer library on 2F5 IgA2 (A) or IgG1 (B) used for in silico analyses. The multiple alignment on selected sequences together with the gp41 C-helix sequence was performed with the T-Coffee server (32). Frequency refers to the retrieval occurrence obtained for each sequence. Specific sequences retrieved with a frequency of ≥3 are framed. Each residue is colored following the T-Coffee conservation color scheme (CORE), ranging from blue (low conservation) to red (high conservation). The last line in each panel (consensus sequence) highlights amino acids feature conservation, along the reference gp41 sequence, following the same color code.

Fig. 5.

Both 2F5 IgA2 and IgG1 recognize different nonlinear epitopes on gp41 when organized as a six-helix bundle. 2F5IgG1-predicted (A and B) and IgA2-predicted (C and D) epitope amino acid frequencies are mapped using a red gradient (the more red, the higher the frequency) onto the gp41 structure (36), represented as a ribbon diagram (A and C) or molecular surface (B and D).