After washing with ultra pure water and blocking with 2 mM EDTA and 0.05% Tween-20 in PBS for 30 min at room temperature, mutant proteins N6-Cyclohexyladenosine were captured at 20 g/ml for 2 h. antibody binding, form an immunodominant triad at the outer domain/inner domain junction of gp120. The mutation of these residues to alanine impairs viral fusion and fitness. Thus, the core epitope, a frequent target of antiCHIV-neutralizing antibodies, including the broadly neutralizing antibody HJ16, is conserved and indispensible for viral infectivity. We conclude that the core epitope should be considered as a target for vaccine design. A fraction of patients infected with HIV-1 develop broadly neutralizing antibodies against the virus (McMichael et al., 2010). In vitro studies indicate that these antibodies can reduce infectivity by interfering with virusCtargetCcell interactions or by blocking viral fusion (Dimmock, 1993; Robbins et al., 1995; Shibata et al., 1999; Zolla-Pazner, 2004). In addition, passive administration of mABs with broadly neutralizing activity to macaques or humans can provide sterilizing immunity or delay HIV-1 rebound (Emini et al., 1992; Gauduin et al., 1995; Mascola et al., 2000; Trkola et al., 2005). Therefore, it is generally believed that reproducing this type of serologic activity by immunization would be important for the development of an effective HIV vaccine (Stamatatos et al., 2009). Although several different broadly neutralizing mABs that target HIV-1 envelope epitopes have been described (Zolla-Pazner, 2004; Burton et al., 2005), there have been few comprehensive efforts to clone and characterize the antibodies from patients with broadly neutralizing serologic responses. In an effort to understand the human antibody response PSK-J3 to HIV-1, we cloned 502 anti-HIV-1 gp140 antibodies from the memory B cell N6-Cyclohexyladenosine compartment N6-Cyclohexyladenosine of six individuals with variable viral loads and high titers of broadly neutralizing antibodies (Scheid et al., 2009). We found that the memory B cell response to gp140 is composed of high affinity antibodies binding to the gp120 variable loops (VLs), the CD4 binding site (CD4bs), the induced coreceptor-binding site (CD4is), several different epitopes on gp41 (Pietzsch et al., 2010), and a group of potentially heterogeneous antibodies to one or more epitopes near the CD4bs, termed core (Scheid et al., 2009). The core antigen was not characterized molecularly; however, antibodies to this region accounted for 18% of all anti-gp140 antibodies and 32% of all antibodies with N6-Cyclohexyladenosine neutralizing activity (Table S1; Scheid et al., 2009). Anti-core was the largest single group of neutralizers in the six patients studied. In addition, antibodies with characteristics similar to anti-core antibodies were also reported in a collection of mABs obtained from EBV-transformed B cells (partial CD4 binding site antibodies; Corti et al., 2010). Anti-core antibodies bind to gp120, gp120core (a mutant that lacks V1-V3; Kwong et al., 1998), gp120D368R (which interferes with binding by CD4 and anti-CD4bs antibodies; Olshevsky et al., 1990; Thali et al., 1991; Pantophlet et al., 2003; Li et al., 2007), and gp120I420R (a mutant that interferes with the binding of anti-CD4Cinduced site [CD4is] antibodies; Thali et al., 1993). Anti-core antibodies do not bind to a stabilized gp120core protein that retains CD4 and b12 binding sites, but is mutated to reduce the flexibility of gp120 to improve presentation of conserved but discontinuous epitopes (Zhou et al., 2007; Scheid et al., 2009). Furthermore, anti-CD4bs and some anti-CD4is antibodies inhibit the binding of anti-core antibodies, suggesting that anti-core antibodies recognize an N6-Cyclohexyladenosine epitope that is closer to the CD4bs than to the CD4is (Scheid et al., 2009). Here, we report on the characteristics of this new epitope. The data show that anti-core antibodies target a conformational epitope on gp120 found within the 5-helix of the molecule, which is highly conserved across different HIV-1 clades. This high degree of conservation correlates to viral fitness, as mutating the epitope results in loss of infectivity. RESULTS Fine mapping of anti-core antibodies cloned by single cell sorting To map the epitope or epitopes recognized by anti-core antibodies, we assayed all anti-core antibodies for binding to 72 different alanine mutants of HIV-1 gp120 by ELISA..
After washing with ultra pure water and blocking with 2 mM EDTA and 0