In this study, we characterized PGV04, a newly identified broad and potent CD4bs-directed HIV-1 neutralizing MAb. PGV04 neutralization was determined on two different pseudovirus panels. For the 162-pseudovirus panel, PGV04 and PG9 displayed comparable potency while PGV04 exhibited greater breadth. For the 97-pseudovirus panel, PG9 exhibited similar breadth but increased potency compared to that of PGV04. PG9 neutralized the viruses in the latter panel with greater potency than the viruses in the 162-virus panel, while PGV04 displayed similar neutralization potency on both panels. This may be due to the isolates chosen on both panels, since 7.4% of the viral isolates on the 162-virus panel were from acute/early infection while 44.3% of the viral isolates on the 97-virus panel were from acute/early infection (see Tables S1 and S2 in the supplemental material). Also, the target cell lines used were different: TZM-bl cells were used for the 97-pseudovirus panel and U87 cells for the 162-virus panel.
Overall, PG9 and -16, VRC01, and PGV04 neutralized more than 70% of circulating viruses with a mean IC50 demonstrating approximately 10-fold greater potency than the earlier established HIV-1 bnMAbs b12, 2G12, 2F5, and 4E10. Interestingly, the virus isolated from the PGV04 donor was subtyped as clade A1/D recombinant, which differs from the donors associated with the other CD4bs bnMAbs, 3BNC117 (clade B virus), VRC01 (clade B virus), HJ16 (clade C virus), and b12 (presumed clade B virus). Therefore, it seems that elicitation of broadly neutralizing CD4bs-directed MAbs is not dependent on the clade of the infecting isolate. Of note, the PGV04 donor virus does not appear to be of any known circulating recombinant form (CRF) listed in the Los Alamos HIV databases.
Our results show significant differences between CD4bs bnMAbs in their mode of recognition of the CD4bs region on monomeric gp120. CD4-IgG, VRC01, and NIH45-46 enhance exposure of the coreceptor binding site as assessed by 17b binding, while PGV04, VRC03, and b12 do not. Furthermore, the entropy change associated with PGV04 binding to gp120 is much less than that associated with VRC01 binding, consistent with a smaller conformational change in gp120 on PGV04 binding. Nevertheless, PGV04 binding to gp120 is associated with relatively large entropic and compensating enthalpic changes that are suggestive of protein conformational changes or extensive solvent rearrangements. Further insight is provided by the structures of CD4 (11
), VRC01 (33
), NIH45-46 (4
), VRC03 (28
), and PGV04 (28
) complexed with gp120 core. These structures show that the conformational changes induced by these CD4bs ligands in gp120 core are similar but have critical differences. In particular, while VRC01 and NIH45-46 induce a conformation of the bridging sheet that is similar to that induced by CD4 and therefore compatible with 17b and X5 recognition, PGV04 and VRC03 induce a different conformation between residues 428 and 431 in strands β20/21. Additionally, a critical 17b-interacting residue, Met434, on β21 in both PGV04- and VRC03-bound forms assumes a position that clashes with CDRH2 of 17b, while that in the VRC01-, NIH45-46-, and CD4-bound forms adopts a nonclashing conformation.
In contrast to the enhancement of binding on monomeric gp120, only CD4 induced the coreceptor site as assessed by 17b binding on JR-FL functional trimers; the MAbs VRC01, VRC03, b12, and PGV04 were ineffective. These observations suggest that the CD4bs is presented differently in the context of recombinant gp120 and the functional trimer; that is, the conformation and/or flexibility of gp120 around the CD4bs is different, allowing a change in the monomer but not the trimer. Furthermore, the finding that none of the CD4bs bnMAbs induced CD4i MAb binding to surface-expressed trimers contrasts with the results found earlier (22
). The difference in the two findings may stem, in part, from the use in the previous study (22
) of surface-expressed YU2 and BaL Envs, which are known to be a mixture of cleaved and uncleaved Envs (15
). Using YU2 and BaL Envs, one cannot differentiate between the expression and induction of the CD4i site on cleaved and uncleaved trimers, the latter of which are not used in viral entry. In contrast, JR-FL surface-expressed trimers are fully cleaved (15
) and competent for viral entry. The induction of the CD4i site in this system therefore represents changes occurring on functional Env spikes. A further difference between the two studies is that here we used the CD4i MAb 17b, whereas the earlier study used MAb 3-67, and the two MAbs may bind somewhat different residues and sense somewhat different conformational changes.
We found that several alanine substitutions affected CD4bs MAb neutralization and binding differently, again illustrating that PGV04, VRC01, b12, and CD4-IgG recognize the CD4bs in somewhat different ways. For example, alanine substitutions at positions D279, I420, and I423 greatly decreased neutralization by PGV04 but varied in their effects on VRC01, CD4-IgG, and b12. The D279A substitution decreased neutralization by VRC01 and CD4-IgG but did not substantially affect neutralization by b12. The I420A and I423A substitutions decreased VRC01 neutralization but increased both CD4-IgG and b12 neutralization. In addition, certain substitutions in the V3 loop decreased neutralization by PGV04 and VRC01 but increased neutralization by CD4 and b12. The highly conserved nature of the residues important for PGV04 recognition probably explains how PGV04 is able to achieve broad neutralization. Additionally, mapping the residues found to be important in neutralization and binding onto the gp120 core in its PGV04-bound state allowed us to gain insight into the contribution of each residue in binding to gp120 in the context of the trimer (A and B).
In summary, the recent isolation of several new CD4bs MAbs, notably, VRC01 (27
) and 3BNC117 (22
), has led to the idea that the success of a CD4bs MAb in broad and potent neutralization may depend on its ability to precisely induce changes in gp120 that mimic those induced by CD4. Indeed, certain bnMAbs, such as VRC01, do enhance binding of the CD4i MAb 17b to monomeric gp120. However, we show here that the equally broad and potent bnMAb PGV04 does not enhance such binding. Furthermore and crucially, in contrast to CD4, none of the CD4bs MAbs tested induced the 17b site on trimeric cleaved Env. Thus, a certain degree of mimicry of CD4 by anti-CD4bs bnMAbs may simply be a consequence of binding to the CD4 epitope on monomeric gp120 rather than a neutralization mechanism. As argued previously (10
), the binding of an IgG molecule to an envelope spike may be sufficient to lead to neutralization irrespective of the precise epitope. Finally, the description of differences in recognition of the CD4bs by different bnMAbs is encouraging for immunogen design in relaxing somewhat the stringency of the types of Abs that should be elicited.