Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens

Abstract

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.

FIG. 1.

Competition of free gp120_89.6 with immobilized gp140_89.6 for binding to anti-gp41 antibodies was performed as described in Materials and Methods (“Binding assays”). The binding of antibodies in the absence of free gp120 was taken as 100%. The relative binding was calculated by using the following formula: (optical density at 405 nm in the presence of free gp120/optical density at 405 nm in the absence of free gp120) × 100. Fab m14 is a CD4 binding site (CD4bs) antibody and used as a negative control.

FIG. 2.

Neutralization activity of IgG1 m44 and Fab m44 with the clade C SHIV chimera, SHIV-1157ipd3N4, in a PBMC-p27 assay. 2F5, 4E10, and b12 were used as controls and for comparison.

FIG. 3.

Binding of Fab m44, 2F5, Z13, and NC-1 to nondenatured (A) and denatured (A) gp140_89.6 in an ELISA. OD, optical density; DTT, dithiothreitol. Mouse MAb NC-1 was used as a negative control in binding to nondenatured gp140_89.6. (C) Binding of IgG1s m44, 2F5, and 4E10 and an anti-gp120 control antibody, m17, to a long peptide (QQEKNEQELLELDKWASLWNSLWNWFNITNWLWYIK) derived from the MPER from the Biacore assay. (D) Binding of Fab m44 to nonreduced N35_CCG-gp41 (lane 1), N35_CCG-N13 (Lane 2), N34_CCG (lane 3), gp41 core (lane 4), and 5HB (25.4 kDa) (lane 5) as shown by Western blotting. (E) Binding of IgG1_s m44 and b12 to 293T cells expressing uncleaved gp160 from HIV_HTLV-IIIB in fluorescence-activated cell sorter analysis.

FIG. 4.

Binding of Fab m44, 2F5, Z13, and mouse MAbs NC-1 and T3 to 5HB and 6HB. Single-chain 5HB at 1 μg/ml was used to coat microwell plates. Diluted N36/C34-formed 6HB was prepared as follows: 20 μM N36 was mixed with 20 μM C34 and incubated at 37°C for 30 min. The plates were coated with 10-fold diluted N36/C34-formed 6HB. The plates were blocked with 3% BSA in PBS, and threefold serially diluted antibodies were added to the wells. Bound Fab m44, 2F5, and Z13 were detected using HRP-conjugated anti-human IgG, F(ab′)₂, and ABTS as the substrate. Bound NC-1 and T3 were detected by using HRP-conjugated anti-mouse IgG and ABTS as the substrate. Optical densities (OD) at 405 nm were measured after color development at room temperature for 30 min.

FIG. 5.

Reactivity of hMAbs with autoantigens. The ELISA for antibody binding to CL and PS was performed as described in Materials and Methods. Shown are data for CL only. 4E10 and 2F5 bound to CL with EC₅₀ values of 23 and 340 nM, respectively. Whereas 2F5 and 4E10 did react with CL, m44 was completely negative. OD, optical density; NB, no binding.