Broad HIV-1 neutralization mediated by CD4-binding site antibodies

Abstract

We have identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from selected gp120 variants, the neutralizing specificities of the two most broadly reactive sera were mapped to the primary receptor CD4-binding region of HIV-1 gp120. Novel antibodies to the CD4-binding site are elicited in some HIV-1-infected individuals, and new approaches to present this conserved region of gp120 to the immune system may result in improved vaccine immunogens.

Figure 1

Adsorption of sera with gp120WT and CD4-binding site mutant proteins. (a) Left, molecular surface of the gp120 core. Off-white, CD4-binding site; red, surface-exposed aspartic acid residue Asp368; yellow, glutamic acid residue Glu370. Center and right, flow cytometric analysis of gp120WT (center) and gp120-D368R (right) proteins covalently linked to paramagnetic beads. Protein-linked beads were reacted with the conformational mAbs 2G12 (glycan-dependent), b12 (CD4 binding site) or 447D (V3-directed) at 10 μg/ml, or polyclonal immune globulin from HIV-infected individuals (HIVIG) at 4.0 mg/ml. A PE-conjugated goat antibody to human IgG was used to detect binding. Number of binding events as a percentage of the maximum was plotted against fluorescence intensity. (b) End-point ELISA data for sera 1 and 45. Each serum was adsorbed with blank beads or with beads covalently linked to BSA, BaL gp120WT, YU2 gp120WT, BaL gp120-D368R or YU2 gp120-D368A/E370A. The latter two mutant proteins are unable to bind soluble CD4 or most known CD4-binding site antibodies. The protein used to coat ELISA plates is indicated underneath each set of bars. Of note, adsorption with the gp120-D368R or gp120-D368A/E370A mutant proteins left behind a fraction of serum antibodies, presumably to the CD4-binding site, that were detected in the gp120WT protein ELISA. (c) Neutralization curves for sera 1 and 45 after adsorption with gp120WT and CD4-binding site–mutant proteins. Control is adsorption with BSA. For serum 1 (two left columns), the CD4-binding site–defective mutants adsorbed little or no neutralizing activity, suggesting that CD4-binding site antibodies were the predominant fraction of neutralizing antibodies in this serum. For serum 45 (two right columns), the CD4-binding site–mutant proteins removed a portion of neutralizing activity against some viruses, suggesting that besides CD4-binding site antibodies, atypical CD4-binding site antibodies or antibodies that bind outside the CD4-binding site were also mediating virus neutralization.

Figure 2

Analysis of antibodies eluted from Env protein beads and after additional adsorption to enrich for CD4-binding site antibodies. In each panel, serum 1 data are shown on the left and serum 45 data are on the right. (a) Concentration of IgG after elution from the indicated proteins. (b) Neutralization by IgG eluted from the indicated protein. Top, eluate neutralization of the clade A virus RW20. Bottom, neutralization of the clade B virus PVO. For each virus, left chart shows neutralization by mAb b12 with serum 1 IgG fractions. The BSA eluate contained little IgG; amounts shown here are based on the same physical dilutions as the other IgG fractions. (c) Competition ELISA using Fab F105 to block antibody binding to the CD4-binding site of the BaL gp120 used to coat the plate. Monoclonal antibody b12 and 2G12 controls show that increasing concentrations of the Fab F105 have no effect on 2G12 binding but completely block b12 binding to gp120. Open symbols represent the antibody fractions eluted from gp120WT or the core protein and then adsorbed with the gp120-D368R mutant; for example, ‘gp120 eluate/368ft’ refers to the final flow-though after IgG was eluted from gp120WT and adsorbed with the gp120-D368R protein. (d) HIV-1 neutralization of designated isolates by the same IgG fractions described in c.