Effector function activities of a panel of mutants of a broadly neutralizing antibody against human immunodeficiency virus type 1

Abstract

The human antibody immunoglobulin G1 (IgG1) b12 neutralizes a broad range of human immunodeficiency virus-type 1 (HIV-1) isolates in vitro and is able to protect against viral challenge in animal models. Neutralization of free virus, which is an antiviral activity of antibody that generally does not require the antibody Fc fragment, likely plays an important role in the protection observed. The role of Fc-mediated effector functions, which may reduce infection by inducing phagocytosis and lysis of virions and infected cells, however, is less clear. To investigate this role, we constructed a panel of IgG1 b12 mutants with point mutations in the second domain of the antibody heavy chain constant region (CH2). These mutations, as expected, did not affect gp120 binding or HIV-1 neutralization. IgG1 b12 mediated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of HIV-1-infected cells, but these activities were reduced or abrogated for the antibody mutants. Two mutants were of particular interest. K322A showed a twofold reduction in FcgammaR binding affinity and ADCC, while C1q binding and CDC were abolished. A double mutant (L234A, L235A) did not bind either FcgammaR or C1q, and both ADCC and CDC functions were abolished. In this study, we confirmed that K322 forms part of the C1q binding site in human IgG1 and plays an important role in the molecular interactions leading to complement activation. Less expectedly, we demonstrate that the lower hinge region in human IgG1 has a strong modulating effect on C1q binding and CDC. The b12 mutants K322A and L234A, L235A are useful tools for dissecting the in vivo roles of ADCC and CDC in the anti-HIV-1 activity of neutralizing antibodies.

FIG. 1

Binding of wild-type and mutant IgG1 b12 to recombinant HIV-1_JR-FL gp120 in ELISA (a) and neutralization of HIV-1_JR-FL (b).

FIG. 2

ADCC of CEM-Nkr cells infected with HIV-1_MN by PBMC and purified human monocytes. Uninfected and HIV-1_MN-infected CEM-NKr cells were labeled with ⁵¹Cr for 2 h at 37°C. The labeled target cells were incubated with wild-type or mutant IgG1 b12 before addition of cultured PBMCs (a) or purified human monocytes (b) as effector cells. Wild-type and mutant IgG1 b12 were used at 12.5 μg/ml. Serum from an HIV-1-seropositive patient (FDA-2 [44]) was used at a 1/4,000 dilution as a positive control. Uninfected CEM-NKr cells incubated with wild-type IgG1 b12 were included as a negative control. The assays were performed twice with similar results.

FIG. 3

Inhibition of ADCC by anti-FcγR antibodies. ADCC of HIV-1_MN-infected CEM-NKr cells by PBMC (a) or purified human monocytes (b) in the presence and absence of F(ab′)₂ fragments of anti-FcγRIII MAb 3G8 (20 μg/ml), Fab fragments of anti-FcγRII MAb IV.3 (5 μg/ml), and Fab fragments of anti-FcγRI MAb 10.1 (20 μg/ml). IgG1 b12 was used at a concentration of 12.5 μg/ml. Serum from an HIV-1-seropositive patient (FDA-2 [44]) was used at a 1/4,000 dilution as a positive control. The assays were performed twice with similar results.

FIG. 4

Binding of wild-type and mutant IgG1 b12 to C1q (a) and complement-mediated lysis of HIV-1_MN-infected CEM-Nkr-MN cells by IgG1 b12 and IgG1 b12 mutants (b). Isotype variants of anti-CD3 MAb OKT3 (IgG1 and IgG4) were used as controls.