In this study, we assessed the interaction of the broadly neutralizing MAb VRC01 with the HIV-1 native viral spike, and where informative, to full-length monomeric gp120. We measured the effect of gp120 Ala scanning point mutations on virus neutralization and recognition of gp120 by VRC01 and the prototypic CD4bs ligands b12 and CD4-Ig. We also analyzed the contribution of amino acid variation within specific regions of Env to viral resistance to VRC01. In addition, we compared the thermodynamic properties of VRC01 and CD4-Ig (or sCD4) and assessed their impact on the conformation of gp120 and the native viral spike. Finally, we determined that there was little detectable interaction of the human MAb VRC01 with human antigens commonly recognized by self-reactive antibodies that may have escaped the normal regulatory mechanisms of B-cell self-tolerance.
There are several important findings highlighted by the results presented in our current study. We confirmed that mutations of structurally defined contact residues in loop D (N terminal to the V3 region), the CD4 binding loop, and the V5-β24-α5 region diminished VRC01-mediated binding or neutralization. With regard to natural resistance, in most cases, combinations of mutations at these sites were required to restore full VRC01 neutralization sensitivity. Interestingly, many Ala substitutions at noncontact residues increased the potency of CD4- or b12-mediated neutralization, but few of these substitutions enhanced VRC01-mediated neutralization. This suggests that VRC01 targets its epitope on the functional spike in a highly precise manner, overcoming the steric constraints that restrict the binding of many Env ligands. The observation that VRC01 targeting appears to be superior to the primary virus receptor itself is somewhat paradoxical, as one might expect that HIV-1 would maintain optimal recognition of its primary receptor, CD4. We speculate that selective pressure imposed by CD4 binding site antibodies may select for viruses that occlude optimal access to the CD4 binding region, even for CD4 itself. Obviously, the favorable energetics of the CD4-gp120 interaction allow the viral spike to engage host cell CD4, triggering the conformational changes in Env necessary for viral entry. In contrast, the relatively uncommon CD4bs ligand, VRC01, appears to target the functional spike in a manner that requires limited Env spike rearrangement to accomplish access and neutralization. The lack of VRC01-induced gp120 shedding and the lack of conformational changes of the native virus spike observed after VRC01 binding are consistent with this interpretation. Somewhat paradoxically, VRC01-induced conformational changes on the free gp120 monomer generated a signature that was very similar to that of CD4, implying that the conformation of free gp120 is considerably different than its conformation on the functional spike. Alternatively, VRC01 may indeed induce conformational changes on the functional trimer that are not detected by the assays used in this study but that serve to constrain gp120 in a (low-energy) state not compatible with coreceptor interaction or liberation of the gp41 fusion peptide required for entry.
Our studies of the mechanism of neutralization resistance to VRC01 reveal that a minority of viruses do contain alterations in key contact sites that diminish VRC01 binding and neutralization. However, we found only one example of a viral isolate that appears to resist VRC01 neutralization by restricting the antibodies' access to the CD4bs. These observations are consistent with the model in which VRC01 has achieved nearly optimal access to the CD4bs on the functional spike (70
). We further defined specific aspects of neutralization resistance among a panel of 16 VRC01-resistant Env pseudoviruses. We demonstrated that full VRC01 neutralization sensitivity could be restored to several viruses possessing natural escape variations in their respective Envs. However, restoration of VRC01 sensitivity was accomplished only after multiple changes in three gp120 hotspots, suggesting that viral resistance might require multiple, and perhaps sequential, alterations. Ongoing studies will evaluate the effect of these VRC01 resistance mutations on the efficiency of CD4-mediated cell entry and on viral replicative fitness.
In our previous study, we reported that VRC01 could enhance CD4i antibody 17b and coreceptor CCR5 binding to monomeric gp120 (70
). In the current study, we extended such analysis and observed similarly that the binding of other CD4i antibodies to gp120 was enhanced by prior VRC01 interaction, a property similar to that of CD4. However, when we compared the effects of VRC01 to sCD4 interaction in the context of the functional trimer by other means of analysis, again there were striking differences from the monomeric context. We found that sCD4 binding to the functional trimer can trigger gp120 shedding from the Env trimer and enhancement of gp41 MPER exposure, confirming previous work that CD4 can induce substantial rearrangements of the functional spike, likely necessary to permit entry. In contrast, we did not see the induction of gp120 shedding or enhancement of gp41 MPER exposure after VRC01 bound to the Env functional trimer. These data support a model in which VRC01 locks the Env functional trimer into a low-energy conformation (or trough) from which it cannot “escape” to undergo the further rearrangements that eventually coalesce in virus entry. Finally, the observation that VRC01 does not display substantial self-reactive or polyreactive properties will facilitate the use of passive protection studies of nonhuman primates and humans. This outcome is perhaps not so surprising since VRC01, as noted previously, does not possess an exceptionally long or hydrophobic HCDR3 loop (28
), both of which have correlated with some level of self-reactivity previously.
In summary, the data presented here support the concept that VRC01 interacts with the functional spike in a manner distinct from that of CD4. VRC01 achieves potent neutralization by precisely targeting a highly conserved region of the CD4bs without requiring the alterations of the Env functional spike configuration that occur upon CD4 ligation. This helps to explain how VRC01 can access the CD4bs on the large majority of virus isolates and why VRC01 resistance based on the quaternary structure of the HIV-1 Env is uncommon. These studies highlight the unique features of this broadly neutralizing CD4bs-directed antibody and point to further efforts regarding HIV immunogen design based on its gp120 epitope.