The CH1α domain of mucosal gp41 IgA contributes to antibody specificity and antiviral functions in HIV-1 highly exposed Sero-Negative individuals

Abstract

The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine.

Fig 1

FabA and G of clones 43 and 177 binding to HIV envelope gp41 clades A, B and C in a dose dependent manner. A and B: The specificity of FabA (solid line) and FabG (dotted line) for clade A gp140 (red), clade B gp41 (blue) clade C gp41 (green) was evaluated by ELISA. For direct comparison of FabA and G isotypes, detection was performed using an anti-kappa light chain. Specific binding (OD450 nm) is plotted as a function of Fab concentration (μg/ml). Values represent mean ± SEM, derived from 3 independent experiments performed in duplicate. (A) Fab 43, (B) Fab 177. C: The specificity of FabA 43 (solid line) and FabG 43 (dotted line) for the peptide P1 cross clade, namely clade A (red), clade B (blue) and clade C (green) was evaluated by ELISA. One representative out of three independent experiments performed in duplicate is shown. D and E: Binding of FabA and G of clones 43 and 177 to HIV-1 infected CD4⁺T-cells. FabA (solid bars) and FabG (hatched bars) from clones 43 (D) and 177 (E) were incubated with Clade A (red) or clade B (blue) and clade C (green) HIV-infected cells or uninfected cells as negative control overnight at 4°C. Irrelevant IgA and IgG were used as negative controls. Specific binding was detected using FITC conjugated anti-human kappa light chain to allow for direct comparison of both FabA and G isotypes, and analyzed by flow cytometry, as indicated in Materials and methods section. Values represent the % of Fab+± SEM among HIV-1-infected (Gag-p24+) CD4+T-cells. Binding of all Fabs to non-infected cells was negligible. Binding of irrelevant IgA and IgG to infected cells it was fixed at 1% and subtracted from the specific binding values; n = 3 independent experiments. Student’s t–test, * = p<0.05.

Fig 2

Fab 43 and 177-A and -G antiviral efficacy against clades A, B and C HIV-1. (A and B) Dose-dependent inhibition of HIV-1 infection mediated by Fab 43 (A) and Fab 177 (B) assessed in a single cycle infectivity assay, using p24 staining on CD4⁺T-cells. For each Fab clone, FabA (solid line), and FabG (dotted line), were incubated for 1 h at 37°C with either HIV-1 clade A (red), clade B (blue) or with clade C (green) before the addition of CD4⁺T cells for 36 h. Irrelevant Fab-IgA (solid grey line) and Fab-IgG (dotted grey line) at similar concentrations were used as negative controls. The percentage of neutralization, analyzed by flow cytometry as indicated in the Materials and methods section, was defined as the reduction of Gag-p24⁺ HIV-1-infected cells compared with HIV-1-infected cells in the absence of Fab. Values represent mean ± SEM, derived from at least 4 independent experiments performed in triplicate. Student’s t–test, * = p<0.01; ** = p<0.001; *** = p<0.00001. (C and D) Inhibition of HIV-1 transfer from LCs to autologous CD4⁺T cells evaluated by measuring the p24 released by LCs/T cell co-cultures at day 5. HIV-1 of either clade A (red), clade B (blue) or clade C (green) was pre-incubated with LCs for 2 h before addition of the Fabs (FabA solid line, FabG dotted line) and CD4⁺T-cells. Irrelevant Fab-IgA (solid grey line) and Fab-IgG (dotted grey line) at similar concentrations were used as negative controls. The LC–T cell cocultures were incubated at 37°C for 7d. LC–T cocultures without Fab were considered 100% HIV-1 transfer from LC to autologous CD4⁺T-cells. Virus transfer was evaluated by measuring p24-Ag by ELISA as described in the Material and Methods section. Results correspond to the % inhibition of transfer in the presence of Fabs. Values represent mean ± SEM of at least five independent experiments performed in triplicate. Student’s t–test, * = p<0.01**; = p<0.01. (E) Correlation between HIV-1 neutralizing activity and transfer inhibition. Neutralization and transfer inhibition measured for clone 43 and 177 FabA and G at 100ng/ml, were correlated. Spearman rank correlation r and corresponding values are indicated. (F) Correlation between performance in each assay of each clone 43 and 177. Ratio of values obtained in each indicated assay for paired FabA and G of each clone, were obtained and plotted as a heatmap with using the complete linkage clustering method and ranking according to Spearman statistics. Binding represents 1/HIV envelope ELISA OD values; Affinity corresponds to 1/log KD values.

Fig 3. Characterization of epitopes of the FabA clones 43 and 177 on the 6-Helix bundle conformation of HIV-1 envelope gp41.

Each set of antibody specific mimotopes obtained by biopanning for both isotype FabA clones 43 (A) and 177 (B) were docked on the crystal structure of gp41 under its 6-Helix bundle conformation. Each gp41 monomer in the trimer is highlighted by a different hue of blue. Epitopes are visualized in a color scale according to the scores returned by the PEPsurf algorithm at each position of the sequence (mean of the 3 chains, over 15 conformations extracted each 10 ns from the molecular dynamic simulations) for each set of mimotope. A common color scale varying from never (yellow) to the most frequent on all clades and structures (red) is used, allowing to directly compare epitopes on the three gp41 clades. For each score, a set of random sequences at each position of the sequence had been subtracted to remove background noise.

Fig 4. Characterization of epitopes of the FabA clones 43 and 177 on the pre-fusion conformation of HIV-1 envelope gp41.

Each set of antibody specific mimotopes obtained by biopanning for both isotype FabA clones 43 (A) and 177 (B) were docked on the crystal structure of gp41 under its trimeric pre-fusion conformation. Each gp41 monomer in the trimer is highlighted by a different hue of blue. Epitopes are visualized as described in Fig 3 using the same scale to facilitate direct comparison.

Fig 5. FabA 43 conformational epitope P7 designed in silico, blocks FabA 43 binding to gp41 and reduces FabA 43 neutralization activity.

(A) Preincubation of FabA 43 with the conformational epitope P7 (5 μM, hatch bars) but not with control HA (5μM, plain bars) interferes significantly with FabA 43 binding to clade A (red), clade B (blue) and clade C (green) P1 of clades A and C gp140 and clade B gp41, as evaluated by competition ELISA as described in Material and Method section. FabA 43 preincubated with P7 or control HA were added to gp41 clade B, gp140 (clades A, C) or P1 (clades A, B, C) coated on the ELISA and their binding was detected by using HRP-coupled anti-human IgA. The percentage of binding was calculated relative to the binding of Fab in the absence of the P7 or control HA. (B) The P7-induced reduction of FabA 43 binding to each antigen is dose dependent. Competition ELISA was done in the same conditions described in A. Binding inhibition to FabA 43 binding to each antigen in the presence of P7 is calculated relative to the binding inhibition in the presence of the irrelevant HA peptide serving as negative control. In A and B, values represent mean ± SEM, derived from 3 independent experiments performed in triplicate. (C) Localization of the amino acid paths corresponding to P7 peptide on the 6-Helix bundle conformation of clades A, B and C gp41 frame15. Each gp41 monomer of the trimer is depicted using a different level of gray. The amino acid paths corresponding to the FaA 43-specific peptide P7 are highlighted in orange. Note residues 532–535 belong to the N-Helix of one monomer while residues 669–672 belong to the C-Helix of another monomer of the trimer. Red: the P7-predicted conformation, superimposed on the structure of gp41. RMSD values over the paired residues of gp41 and P7 are of 2.45, 2.97 and 1.97 Å for clade A, B and C, respectively. Note that in this region of the gp41, only residue 535 differs between clade B in one hand and clade A and C in the other hand. (D) Conformational variability of the predicted conformations of P7 using PEP-FOLD3. Left: conformation best fitting gp41. Right: top 10 predicted conformations superimposed. (E) Interference of FabA 43 neutralization by a 400-fold molar excess of P7 peptide. FabA 43 preincubated with P7 or control HA were incubated with the HIV-1 of one of each three clades (A, B, C) before adding the activated CD4⁺T cells, and cultures were incubated at 37°C for 2 days. Cultures without Fab were served as positive control of infection. The percentage of neutralization, analyzed by flow cytometry as indicated in Materials and Methods section, is shown relative to FabA 43 neutralization using primary CD4⁺T cells in the presence of the irrelevant HA peptide, tested in parallel. Values represent mean ± SEM, derived from 3 independent experiments performed in triplicate.