Identification of a HIV Gp41-Specific Human Monoclonal Antibody With Potent Antibody-Dependent Cellular Cytotoxicity

Abstract

Antibody-Dependent Cellular Cytotoxicity (ADCC) is a major mechanism of protection against viral infections in vivo. Identification of HIV-1-specific monoclonal antibodies (mAbs) with potent ADCC activity may help develop an effective HIV-1 vaccine. In present study, we isolated such human mAb, designated E10, from an HIV-1-infected patient sample by single B cell sorting and single cell PCR. E10 bound to gp140 trimer and linear peptides derived from gp41 membrane proximal external region (MPER). E10 epitope (QEKNEQELLEL) overlapped with mAb 2F5 epitope. However, E10 differentiated from 2F5 in neutralization breadth and potency, as well as ADCC activity. E10 showed low neutralization activity and narrow spectrum of neutralization compared to 2F5, but it mediated higher ADCC activity than 2F5 at low antibody concentration. Fine mapping of E10 epitope may potentiate MPER-based subunit vaccine development.

Keywords: ADCC; HIV; MPER; antibody; epitope; gp41.

Figure 1

Single cell sorting of gp140 trimer specific memory B cell from PBMC of HIV infected patient. (A) gate on lymphocyte; (B) gate on IgM (PerCP-cy5.5) negative, 7AAD (PerCP-cy5.5) negative, CD3(PE-CY7) negative and CD14 (PE-CY7) negative population; the purpose is to remove IgM positive cells and T cells. (C) Gate on CD19 (FITC) positive and IgG (Pacific blue) positive population; the purpose is to gate the IgG positive B cells. (D) Gate on SF162gp140 trimer binder (PE), the purpose is to fish out antigen specific IgG positive B cells.

Figure 2

Affinity detection of Mab E10 against gp120 (bal) and gp41-Fc (bal) by ELISA. (A) IgG E10 size and purity detection by denatured and none-denatured SDS-PAGE; binding (B) of IgG E10 against SF162gp140 trimer, RSC3 and delta RSC3; (C) binding of IgG E10 against bal gp120; (D) binding of IgG E10 against gp41-Fc fusion protein.

Figure 3

Neutralization and ADCC activity of IgG E10 and its binding to MPER peptide F7. (A) Infection inhibition percentage of IgG b12 and E10 against 6 pseudo type HIV strains at different concentrations were shown, calculated IC50 in μg/ml were labeled after the strain and clade names. (B) IgG1 E10, 2F5, and mz76 mediated cellular cytotoxicity against HIV envelope expressing TF228 at different concentrations (μg/ml) were shown by percentage. Experiments were repeated three times. (C) Affinity detection of IgG E10, 2F5, and b12 to MPER peptide fusion protein F7-Fc by ELISA; OD450 at different antibody concentration were shown. (D) FlowJo generated figure showing binding of E10 SCFV expression yeast clone with bal gp120-Fc and F7-Fc fusion proteins, APC positive population indicate positive binding. Statistical analysis, ^**P ≤ 0.01.

Figure 4

Epitope amino acid sequence comparison between mAb E10, 2F5 and 4E10 (A) and epitope mapping of mAb E10 by gp41 peptide microarray ELISA (B). CDR sequences of antibodies in bright red (A), peptides bind by antibodies were labeled with number and sequence. E10 core epitope in dark red and 2F5 core epitope in bright blue (B).

Figure 5

Reactivity of hMAbs with autoantigens. The ELISA for antibody binding to CL was performed as described in section Materials and Methods. Statistical analysis, ^**P ≤ 0.01.