We report striking differences in the ability of sCD4 and different CD4bs ligands to neutralize macrophage-tropic and non-macrophage-tropic R5 Env+
pseudovirions. While sCD4 mainly neutralized macrophage-tropic Envs, VRC01 conferred efficient neutralization of all Env+
pseudovirions regardless of tropism. The CD4bs MAb b12 conferred an intermediate pattern where all macrophage-tropic Envs that carried the b12 epitope were neutralized, while some but not all of the non-macrophage-tropic Envs were sensitive. The new and potent CD4bs MAbs, e.g., VRC01, have been described as “highly active agonistic antibodies against the CD4 binding site (HAADs) that mimic binding of the host receptor CD4” (41
). The binding sites for VRC01, b12, and sCD4 overlap on the outer domain of gp120 (26
). However, these reagents have different requirements for binding the bridging sheet and vary in their capacities to induce conformational changes in the trimer (43
). CD4 recruits determinants on the stem of the V1V2 loops and at the junction of the β20-β21 strands to assemble the bridging sheet present on the gp120/sCD4 structure (26
). However, the V1V2 stem determinant that binds CD4 is not critical for binding either of these MAbs (28
). Nevertheless, b12 does induce conformational changes in the trimer, although less extensively than CD4 (32
). In contrast, VRC01, like other recently described potent CD4bs MAbs (PGV04, 3BNC117, and NIH45-46) (42
), does not induce conformational changes on the trimer (43
). Finally, while mutation of bridging-sheet determinants was reported to confer profound effects on sCD4 and, to a lesser extent, b12 neutralization, they barely affect VRC01 (43
). These distinct specificities of CD4, b12, and VRC01 for the bridging sheet along with their different abilities to neutralize via macrophage-tropic and non-macrophage-tropic Envs have led us to propose that R5 macrophage tropism is determined by the ability of CD4 to recruit contact residues on the V1V2 stem and β20-21 junction to assemble the bridging sheet ().
Fig 6 Model showing how Env substitutions affect macrophage tropism of R5 Envs. Substitutions in gp120 residues that increase macrophage tropism occur in residues directly contacting CD4 or that affect their exposure. These substitutions confer an increased (more ...)
The ability of CD4 to recruit the bridging sheet may be influenced by changes within or proximal to CD4 contact residues, which directly affect the affinity of the Env for CD4. An increased Env-CD4 affinity potentially leads to a slower “off rate” (as reported for gp120 carrying N283 [7
]), thus increasing the time frame for the V1V2 stem to be bound by CD4. Alternatively, an increased affinity for CD4 may enhance the induction of conformational changes that move V1V2 at the apex of the trimer, again increasing the likelihood that the V1V2 stem will be exposed and bound by CD4. An additional mechanism involves the ease with which conformational changes are elicited by CD4 binding, i.e., movement of the V1V2 loops away from V3 at the trimer apex (31
). Our experiments with 447-52D, PG9, and PG16 have suggested that (at least for some Envs) the V3 loop may be more exposed or that the arrangement of V1V2 and V3 loops is altered at the trimer apex. For C98-15, a conserved V1 residue controls macrophage tropism as well as sCD4 and 447-52D sensitivity (39
), unequivocally associating a single V1 residue at the trimer apex with V3 loop exposure, CD4 binding, and macrophage tropism. The data presented here incorporating VRC01 sensitivity now implicate accessibility of CD4 contact residues on the bridging-sheet segments as the major factor in determining low CD4 use and macrophage tropism.
Our data show that the Env determinants that confer HIV-1 R5 macrophage tropism are focused on the Env's interaction with CD4 and the immediate ensuing conformational changes and do not reflect major changes in envelope structure. This is evident from the lack of correlations between macrophage tropism and sensitivity to other neutralizing MAbs or inhibitors that target gp120-coreceptor interactions or gp41 conformational changes as well as by the consistent insensitivity of all Envs to the CD4i MAb 17b (data not shown) The absence of significant variation in sensitivities to these reagents further emphasize the marked difference in sensitivity to trimer apex reagents 447-52D, PG9, and PG16 discussed above. Perhaps most surprisingly, we were unable to detect differences in sensitivity to heterologous neutralizing antibodies in HIV-1+
human sera. Since most of the macrophage-tropic Envs used here were derived from the immune-privileged environment of brain tissue (50
), we had expected them to have evolved a more open structure that would expose antibody epitopes and confer increased sensitivity to neutralizing antibodies. However, while Envs may evolve enhanced exposure of CD4 contact residues and subtle exposure of V3 residues at the trimer apex, they have not incurred more extensive alterations that would confer a more global neutralization sensitivity.
Previously, we had hypothesized that a more open structure of macrophage-tropic R5 Envs evolving in the brain would efficiently expose the CD4bs along with conserved neutralization epitopes and have relevance for vaccine development. Our data do not support a wide-open structure for such Envs. However, the identification of residues proximal to CD4 contact sites on the outer domain of gp120 that modulate macrophage tropism (37
) and evidence for alterations in the glycan shield (as indicated by the increased resistance of macrophage-tropic Envs to 2G12) may indicate increased exposure of sites on the gp120 outer domain that are the initial contacts for CD4. Such Envs may more readily elicit antibodies targeting CD4 contact sites on the outer domain and have application for vaccine development.
In summary, our data point to a model where R5 macrophage tropism depends on the ability of CD4 to recruit bridging-sheet determinants on the V1V2 stem and the β20-21 junction. Different mechanisms can be envisaged to achieve this, which involve changes in (i) CD4 contact residues that directly affect affinity for CD4, (ii) proximal or distal residues that affect exposure of CD4 contact residues, including the loss or change in the orientation of glycans that protect CD4 contact residues, and (iii) residues at the trimer apex that facilitate conformational changes induced by CD4. HIV-1 Envs in the brain may evolve to be highly macrophage-tropic via all three mechanisms. Thus, their increased capacity to bind and respond to CD4 will enable the efficient formation and exposure of the bridging sheet and coreceptor binding site in the presence of low levels of CD4 on the surfaces of macrophages.