Protein-linked beads were reacted with the conformational mAbs 2G12 (glycan-dependent), b12 (CD4 binding site) or 447D (V3-directed) at 10 g/ml, or polyclonal immune globulin from HIV-infected individuals (HIVIG) at 4

Protein-linked beads were reacted with the conformational mAbs 2G12 (glycan-dependent), b12 (CD4 binding site) or 447D (V3-directed) at 10 g/ml, or polyclonal immune globulin from HIV-infected individuals (HIVIG) at 4.0 mg/ml. antibodies1,2. Little is known about the antibody specificities that mediate this breadth of reactivity5C7, and an understanding of how broad neutralization is accomplished could provide valuable insights for Env-based immunogen design. Several functionally conserved regions of the Env protein are potential targets of neutralizing antibodies, namely the binding site for the primary receptor CD4 and the chemokine coreceptorCbinding site, both on HIV-1 gp120, and regions of HIV-1 gp41 involved in VPS34-IN1 viral fusion to target cells. However, only a few broadly neutralizing monoclonal antibodies (mAb) have been isolated from infected patients over the past 20 years1. Two well-characterized mAb, 2F5 and 4E10, are directed VPS34-IN1 against the gp41 membrane-proximal external region (MPER), which consists of 20C30 highly conserved residues that are important in virus fusion. Only two broadly neutralizing mAb are directed against the gp120 region of VPS34-IN1 Env. One is mAb 2G12; it has an unusual domain-swap structure and recognizes a cluster of oligomannose residues on gp120. The second is mAb IgG1 b12; first isolated as an Fab fragment through phage-display technologies, it recognizes the functionally conserved gp120 CD4-binding site2. We tested 32 HIV-1+ sera from a well-characterized patient cohort8 to identify those that can neutralize a diverse panel of viruses. Several sera neutralized most viruses VPS34-IN1 from clades A, B and C, but two sera stood out as particularly potent and broadly reactive, sera 1 and 45 (Supplementary Fig. 1 online). These two sera were further evaluated to determine the viral epitopes targeted by their neutralizing antibodies. Because two of the most broadly reactive neutralizing mAb are directed against the MPER of gp41, assays were developed to test for neutralizing antibodies directed against this region. Neither neutralization assays based on competition with linear peptides specific for the MPER epitopes nor assays that used a chimeric HIV-2 virus engineered to contain the 25 residues of the HIV-1 MPER detected MPER-directed antibodies (data not shown). We therefore focused further mapping efforts on the gp120 surface. Serum was adsorbed with Env protein covalently linked to magnetic beads (Supplementary Methods online). Initial studies used a conformationally intact gp120 wild-type protein (gp120WT) and a denatured gp120 protein (gp120den). Adsorption with the clade B gp120WT, but not gp120den, removed neutralizing activity against clade B viruses as well as clade A and C VPS34-IN1 viruses (Supplementary Fig. 2 online). These data suggest that the neutralizing activity in both sera is directed against a conformational epitope on gp120 that is conserved across clades. To evaluate whether antibodies to the CD4-binding site are involved in neutralization, we generated two CD4-binding siteCdefective mutant Env proteins (Fig. 1a, left) by mutation of residues previously described as crucial for recognition by both CD4 and most CD4-binding site antibodies, including mAb b12 (refs. 9C12). One protein contained a mutation of an aspartic acid residue to an arginine in the CD4-binding region of HIV-1 BaL gp120 (D368R) and is denoted gp120-D368R. The other contained two mutations on the HIV-1 YU2 gp120 backbone (aspartic acid 368 to alanine, D368A, and glutamic acid 370 to alanine, E370A) and is denoted YU2 gp120-D368A/E370A. We confirmed the integrity of the wild-type and mutant proteins, both before and after bead coupling, by testing their reactivity to known panels of HIV-1Cspecific antibodies (Fig. 1a, center and right). Rabbit Polyclonal to B3GALT1 Figure 1b,c shows representative data for sera 1 and 45 adsorbed with both wild-type and mutant gp120s. Each protein adsorbed all measurable binding antibodies, as assessed by.