The CD4bs of gp120 is an attractive target for HIV-1 vaccine design because it is functionally conserved and contains epitopes of potently neutralizing antibodies. The isolation of VRC01 and similar CD4bs MAbs demonstrates that the immune system is capable of generating antibodies to this region of gp120. To characterize the circulating Env variants that coexist with VRC01 and to understand how the HIV-1 env
gene evolves under the selection pressure of a broadly reactive antibody, we isolated Env variants from the VRC01 donor and studied their neutralization sensitivities. While numerous published studies have shown that autologous plasma virus is often resistant to concurrent serum neutralization (4
), there had not previously been a detailed assessment of the Env quasispecies in a donor with a broadly reactive serum neutralizing antibody such as VRC01.
Using SGA followed by DNA Sanger sequencing, we found that plasma-derived Env variants from donor 45 from three time points, spanning from 2001 to 2009, displayed almost uniform resistance to VRC01 neutralization, indicating a strong selection pressure on the viral Env quasispecies by VRC01 and related autologous CD4bs antibodies. While the Env SGA and Sanger sequencing approach has an inherent limitation in the overall depth of the sequences identified, our data show that the vast majority of the viruses circulating in plasma were VRC01 resistant. Donor 45 was first found to be HIV-1 infected in 1990; thus, the time points studied here were in excess of 10 years after HIV-1 infection. The VRC01 MAb was isolated from IgG+ memory B cells from a 2008 PBMC sample (), but since memory B cells may circulate for a long time, it is not clear when VRC01 first developed. Our finding that all nine plasma Env variants from 2001 were VRC01 resistant suggests that VRC01 arose prior to 2001. It was only through the isolation of HIV-1 env sequences from proviral DNA that we were able to identify archival Env clones that are highly sensitive to VRC01, with IC50s of <1 μg/ml. Thus, it seems unlikely that donor 45 was infected with a VRC01-resistant virus but rather likely that the Env variants evolved to escape from this potently neutralizing antibody.
The neutralization sensitivity of some of the VRC01 escape variants to MAbs VRC03, VRC06, and VRC06b suggests the possibility of continuous evolution of CD4bs neutralizing antibodies in response to viral escape. VRC03 was isolated from the same PBMC samples as VRC01, and its liganded structure has been solved (75
). While the VRC03 heavy chain derives from the same IGHV1-2*02 allele as VRC01, it has a different light chain, indicating that it arose from a different B-cell clone. The crystal structure of VRC03 in complex with a gp120 core revealed that the antibody forms contacts with the bridging sheet between the inner and outer domains of gp120, which is part of the coreceptor binding site (77
). Therefore, we speculate that the donor immune system responded to VRC01 escape variants by generating antibody specificities extending from the VRC01 epitope toward the coreceptor binding region and resulting in efficient neutralization of some VRC01 escape variants. MAbs VRC06 and VRC06b are clonal relatives of VRC03 and will be described in detail in a separate report (Yuxing Li et al., unpublished data). The sensitivity of some 2001 viral Env clones (which are all resistant to VRC01) to VRC03, VRC06, and VRC06b suggests that these MAbs might have evolved later than VRC01. Indeed, suboptimal 454 pyrosequencing of the IgG+
memory B-cell heavy-chain transcripts from the 2008 PBMC sample identified a population of antibody sequences with >90% identity to VRC03 but not VRC01-related sequences (77
), suggesting the presence of greater numbers of memory B cells for VRC03 than VRC01 in 2008. We are currently optimizing PCR and sequencing conditions to analyze donor 45 longitudinal samples to determine the dynamics of these neutralizing antibody clones.
Although MAbs VRC03, VRC06, and VRC06b were able to neutralize VRC01 escape Env variants in donor 45, the neutralization breadth of these MAbs against heterologous viruses (<60%) was less than that of VRC01 (90%) (75
). Therefore, the development or maintenance of these MAbs after VRC01 in donor 45 was specific to the relevant autologous viruses but not necessarily associated with better neutralization breadth of “irrelevant” heterologous viruses circulating globally. This highlights the complexity of underlying factors that influence the broadening of antibody neutralization (16
). Although most of the donor 45 Env variants from 2001 plasma was neutralized by VRC03, VRC06, and VRC06b, Env variants from 2006 plasma were resistant, suggesting that the virus was able to escape these newly developed antibody variants. Note that the 2006 Env variants remained highly sensitive to the 2009 serum IgG, indicating the presence of unidentified antibody specificities, including the possibility of those outside the CD4bs, in this serum that likely arose in response to the escaped variants.
We also analyzed Env variants from five additional subtype B-infected individuals who developed strong broadly neutralizing antibody responses. We previously reported that the serum samples of donors 1 and B7B5 contained a major fraction of CD4bs-directed neutralizing antibodies (43
); thus, we hypothesized that Env sequences from these two donors would contain some VRC01-resistant clones. However, we were surprised to find a majority of Env clones in donor N26 also resistant to VRC01, as our mapping did not definitively indicate CD4bs neutralizing antibodies in this donor. This was in contrast to donor N90, whose Env isolates were fully sensitive to VRC01 neutralization, suggesting a lack of VRC01-like antibody pressure in this donor. Overall, the rather high level of VRC01-resistant clones in four of five donors (other than donor 45) suggests that the CD4bs-directed neutralizing antibodies similar to VRC01 may not be uncommon in donors whose serum samples are broadly neutralizing.
Lastly, we observed that viral variants fully resistant to VRC01 retained their sensitivity to CD4-Ig and required CD4 for entry. These results suggest that VRC01 escape mutations do not substantially impair the gp120 interaction with CD4, at least not enough to have a major impact on viral entry. Our prior studies of VRC01-resistant viruses demonstrated that mutations in the D-loop and V5 and regions of gp120 were often responsible for VRC01 resistance and that some of these mutations were not contact sites for CD4 (44
). It remains possible that initial escape from VRC01 is associated with some loss of binding to CD4 and that compensatory mutations restore viral entry and replication. More systematic studies of viral fitness and the affinity of interaction with CD4 are in progress. Donor 45 is a slow progressor, as are several other donors from whom broadly neutralizing CD4bs MAbs have been isolated (63
). More systematic and unbiased sampling is required to know if CD4bs neutralizing MAbs are more commonly found in donors with slow progression.
In summary, our data show that neutralizing antibodies to the conserved CD4bs exert selection pressure on HIV-1 Env and that the viruses evolve to escape from such neutralization. The B-cell response to the CD4bs also evolves by generating antibodies that neutralize viral escape mutants. Hence, even for this functionally conserved region of gp120, there is ongoing viral evolution matched by antibody evolution. The facts that escape from VRC01-like CD4bs antibodies appears to occur commonly within a donor but that the large majority of heterologous viral strains are sensitive to VRC01 suggest some constraint on resistance to such CD4bs antibodies, although whether there is any measurable fitness cost to VRC01 escape remains to be determined. Finally, the study of Env species in donors with broadly neutralizing antibodies may reveal clues about the viral antigenic stimulus that led to the development of this antibody. Such conclusions are difficult in the case of donor 45, who had been infected for more than 10 years prior to the time when the samples we studied were taken. Similar studies of known seroconverter donors with longitudinal sampling could reveal the relationship between the early circulating Env sequences and the early B-cell clones that ultimately develop into mature broadly neutralizing antibodies.