The inability to induce antibodies capable of neutralizing diverse HIV-1 isolates has been one of the most important obstacles to the development of an effective AIDS vaccine1,2
. Whereas current HIV-1 envelope protein (Env)–based vaccines do not elicit potent neutralizing antibody responses1,3,4
, the sera of a subset of HIV-1–infected subjects contain both potent and broadly reactive neutralizing antibodies1,2
. Little is known about the antibody specificities that mediate this breadth of reactivity5–7
, 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 coreceptor–binding site, both on HIV-1 gp120, and regions of HIV-1 gp41 involved in 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 against the gp41 membrane-proximal external region (MPER), which consists of 20–30 highly conserved residues that are important in virus fusion. Only two broadly neutralizing mAb are directed against the gp120 region of 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 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 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 site–defective mutant Env proteins (, left) by mutation of residues previously described as crucial for recognition by both CD4 and most CD4-binding site antibodies, including mAb b12 (refs. 9–12
). 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-1–specific antibodies (, center and right). 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 ELISA (). The gp120WT proteins adsorbed a considerable fraction of serum neutralizing activity, whereas the CD4-binding site mutants had either no effect or a much reduced effect ( and Supplementary Table 1 online
). These results suggest that both sera contain cross-clade neutralizing antibodies directed against the gp120 CD4-binding site.
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, (more ...)
To further characterize antibody specificities, gp120-specific IgG was eluted from each protein. We incorporated the YU2 gp120 core protein into the analysis because it contains the CD4-binding site but does not bind major variable-loop antibodies or those directed against the coreceptor-binding site13
. IgG was eluted from the gp120WT, gp120-D368R and core proteins (). As the quantity of IgG eluted from each protein was limited, we chose to focus on the neutralization of a non–clade B virus (RW20, clade A) and a clade B virus (PVO) that is highly resistant to mAb b12 ( and Supplementary Methods
). For both sera, the IgG eluted from gp120WT and core protein could neutralize both viruses tested ( and Supplementary Table 2 online
). For serum 1, the Env-specific IgG eluted from gp120-D368R, which presumably contained no or very few CD4-binding site antibodies, showed only minimal neutralizing activity. This suggests that the predominant neutralizing activity in serum 1 is directed against the CD4-binding site of gp120. For serum 45, the IgG eluted from gp120-D368R was able to modestly neutralize both viruses, suggesting the existence of some neutralizing antibodies directed to a region other than the central region of the CD4-binding site.
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 (more ...)
To further isolate and characterize the fraction of neutralizing antibodies specific to the CD4-binding site in both sera, we readsorbed the IgG eluted from gp120WT and core protein with the gp120-D368R protein to remove non–CD4-binding site antibodies. The resulting flow-through fractions would presumably be enriched with CD4-binding site antibodies and were termed gp120WTeluate/368ft and core eluate/368ft, respectively (). Of note, the D368R protein adsorbed only about 40%–50% of the total IgG that had been eluted from gp120WT or core protein (Supplementary Table 2
), suggesting that a large proportion of the initial eluted antibodies were directed against the CD4-binding site. To confirm this, we analyzed these IgG fractions in a competition ELISA. Both the CD4-binding site Fab F105 () and soluble CD4 (data not shown) completely blocked binding of the core eluate/368ft IgG to gp120 and blocked most binding of the gp120WT eluate/368ft IgG, indicating that these antibody fractions were highly enriched with CD4-binding site antibodies. We then showed that these antibody fractions neutralize several strains of HIV-1, including the mAb b12–resistant virus PVO ( and Supplementary Table 2
In summary, we tested sera from a well-characterized cohort of HIV-1–infected patients to find those capable of neutralizing diverse strains of HIV-1. Selective antibody adsorption and elution analysis of the two most broadly reactive sera revealed a fraction of neutralizing antibodies directed against the functionally conserved CD4-binding site of gp120. This novel fraction of CD4-binding site antibodies was able to neutralize viruses that were partially or fully resistant to IgG1 b12, the one mAb to the CD4-binding site region that is known to be capable of neutralizing primary patient isolates. Vaccine immunogens capable of generating this type of antibody response are likely to be protective, as mAb b12 can protect monkeys against a viral challenge14
. These data show that it is possible for B cells in the normal human repertoire to effectively target epitopes located in the gp120 CD4-binding site. Further studies will be needed to determine the prevalence of broadly neutralizing CD4-binding site–directed antibodies elicited in HIV-1–infected individuals. In conjunction with the recent atomic resolution of the mAb b12–binding surface on gp120 (ref. 15
), our results suggest that the CD4-binding site of gp120 should be a major target of HIV-1 vaccines designed to better elicit broadly neutralizing antibodies.