For the majority of HIV+
patients, AIDS and death result from replication by HIV-1 R5 viruses in the absence of detectable CXCR4-using variants. The mechanisms of CD4+
T-cell loss and immune destruction conferred by R5 viruses are unclear. Whether R5 variants with increased virulence emerge in late disease and contribute to CD4+
T-cell loss remains an open question. Several groups have reported the presence of R5 variants in late disease that are highly macrophage-tropic [31
]. The capacity of highly macrophage-tropic R5 viruses to infect cells with low levels of CD4 and/or CCR5 may confer a broader tropism for CD4+
T-cells and exacerbate their depletion late in disease. Our previous studies have highlighted the variation of R5 viruses at different tissue sites [26
], showing that highly macrophage-tropic R5 envelopes predominated in brain tissue but were less prevalent in immune tissue (lymph node), blood and semen.
In this study we have examined the sensitivity of envelopes amplified from these different sites to a range of inhibitors and antibodies that target CD4, CCR5, or various sites on the HIV envelope and block different stages in the entry process. We focused entirely on R5 envelopes and did not include R5X4 or X4 envs. We evaluated whether the variation in macrophage-tropism estimated for all R5 envelopes correlated with sensitivity to each of these reagents using a two-tailed, non-parametric Spearman test with 95% confidence limits. Care must be taken in interpreting these analyses since the panel of envelopes evaluated included several sets that originated from individual subjects i.e. thirty-six envelopes from nine subjects. Thus, it is possible that envelopes with a particular phenotype may be predominant in an individual due to a founder effect or other extenuating circumstances and shift the statistical significance in its favor. Nonetheless, envelope sensitivity to reagents that block CD4: gp120 interactions (sCD4, IgG-CD4 and Q4120) correlated with R5 macrophage-tropism with very high significance. Thus, our data strongly indicates that R5 macrophage tropism predominantly correlates with sensitivity to reagents that interfere with envelope binding to CD4. Macrophage-tropic R5 viruses were more sensitive to sCD4 and tetravalent IgG-CD4 (PRO 542), but more resistant to inhibition by the CD4 mab, Q4120. These data are consistent with an increased envelope affinity for CD4, although there are other potential mechanisms e.g. gp120 shedding, that could explain different sensitivities to sCD4 and PRO 542. An increased envelope affinity for CD4 could result from gp120 substitutions that that result in tighter binding to CD4, in better exposure of the CD4 binding site, or both. Certainly brain-derived envelopes are more likely to carry the N283 in the C2 CD4 binding site as reported by Dunfee et al. [35
] and confirmed by our group [27
]. N283 appears to confer a higher affinity for CD4 by facilitating the formation of a hydrogen bond between N283 on envelope gp120 and Q40 on CD4 [35
]. We also tested envelope sensitivity to BMS-378806, a reagent reported to inhibit gp120:CD4 interactions [39
] and gp120 conformational changes [42
]. Since BMS-378806 is a small molecule, binding to gp120 will not be restricted by variable loops or glycan residues. Intriguingly, decreasing sensitivity to BMS-378806 correlated with increasing R5 macrophage-tropism. There was only minimal variation in the amino acids implicated in BMS-378806 binding which did not associate with sensitivity (not shown) [43
]. The variation in BMS-378806 sensitivity must therefore be due to other mechanisms but could be explained by changes in envelope: CD4 affinities.
Protection of the CD4 binding site may be conferred by V1V2 shielding or by glycan groups [53
]. Recently, Dunfee et al. reported that a glycosylation site at N386 may protect the proximal CD4 binding loop from neutralizing antibodies while also compromising env:CD4 interactions [36
]. Curiously, N386 is a contact residue for b12 in the reported structure for b12 complexed with the HXBc2 envelope [58
]. We have recently confirmed a role of N386 in protecting some envelopes from b12 (Duenas-Decamp et al. Personal communication). However, N386 contributed only modestly to the lack of macrophage infection conferred by a non-macrophage-tropic R5 envelope. Rather, we showed that residues on the N-terminal flank of the CD4 binding loop had a more significant effect on R5 macrophage-tropism and may influence the extent to which this loop is exposed (Duenas-Decamp et al. Personal communication).
Enhanced macrophage-tropism of HIV in brain tissue may result from an adaptation for infection of macrophage-lineage cells, while HIV-1 replicating in immune tissue may have adapted for replication in CD4+
T-cells. However, it is unclear to what extent neutralizing antibodies in immune tissue act to modulate these different tropisms by selecting for envelopes that protect the critical envelope sites e.g. the CD4 binding site. The brain is protected by the blood brain barrier, which usually limits penetration by antibodies [37
], although the barrier may become compromised in late disease [60
]. We failed to show an overall significant correlation between R5 macrophage-tropism and sensitivity to any of the neutralizing mabs tested except for 2G12. The increased resistance of brain macrophage-tropic envelopes to 2G12 is not likely to be due to the presence of 2G12-like antibodies in brain tissue. Rather, 2G12 resistance may be a side effect of the evolution of variants that are less protected by glycosylation and thus lack N-linked glycosylation sites that are critical for 2G12 sensitivity. Such variants therefore may have evolved in response to the absence of neutralizing antibodies in the brain. The carbohydrate epitope for 2G12 is comprised of a cluster of α1–2 mannose residues on the outer face of gp120, which are associated with potential glycosylation sites at N295, N332, N339, N386, N392 and N448. Of these, N295 and N332 are the most important sites [46
]. Overall, three of nine brain envelopes were resistant to 2G12, while only one of nine LN envelope were resistant. For patient NA420, all five envelopes, including those from brain and LN, lack the critical 2G12 glycosylation site at N339 [46
] (Table ), yet one of these envelopes (LN40) retains sensitivity to 2G12. All other envelopes retain the critical N295 and N332 residues indicating that the determinants of 2G12 sensitivity and resistance are unclear but must include other determinants in addition to these glycosylation sites.
For the CD4bs mab, b12, the lack of a correlation with macrophage-tropism is intriguing. A trend of increased b12 sensitivity for brain envelopes was observed, with all but one of the brain envelopes sensitive, while most LN-derived envelopes were resistant. The sensitivity of envelopes to b12 may also depend on whether their host patient carried antibodies that bound epitopes close to or overlapping the b12 binding site and that acted as a selective force. For example, since three non-macrophage-tropic envelopes from subject NA118 were sensitive to b12, it may be that this person did not develop such antibodies. In contrast, for patients NA20 and NA420, brain-derived envelopes were substantially more sensitive to b12 compared to LN-derived envelopes. Together, these results are consistent with selection by neutralizing antibodies that target the CD4bs or proximal epitopes in immune lymphoid tissue but not in brain. So, it seems probable that neutralizing antibodies present in immune tissue play an important role in selecting for envelopes that protect the CD4bs via variable loops, glycosylation or other mechanisms. Such envelopes may evade neutralization by antibodies, but appear to be compromised in their interactions with CD4 and limited to infection of cells that carry high amounts of CD4 e.g. CD4+ T-cells. Curiously increased R5 macrophage-tropism may have resulted in increased resistance to 2G12 but increased sensitivity to b12. Thus a vaccine designed to induce both b12-like and 2G12-like neutralizing antibodies may protect against the entire range of macrophage-tropic and non-macrophage-tropic R5 viruses.
An earlier study using HIV-1 viral isolates from brain tissue suggested that their envelopes conferred a higher affinity for both CD4 and CCR5 [28
]. However, our data do not support this conclusion. R5 envelopes from the brain tended to be more sensitive to CCR5 inhibitors compared to non-macrophage-tropic R5 envelopes from other sites (Figure ), although this was not statistically significant. Thus, an increase in affinity for CD4 may reduce the requirement for a high affinity for CCR5, as suggested by Platt et al. [62
]. The modulation in sensitivity to CCR5 inhibitors is clearly observed in dose dependent inhibition curves that show the majority of brain-derived envelopes from patients NA20 and NA420 are more sensitive to both TAK779 and SCH350581 (Figure ), and the anti-CCR5 mab, 2D7 (data not shown), compared to LN-derived envelopes. NA20 LN14 is an exception that was more sensitive to CCR5 antagonists than other brain and LN-derived envelopes tested. However, LN14 carries the N283 motif present in the C2 CD4 binding site that has been associated with enhanced macrophage-tropism in the brain and increased gp120:CD4 affinity, even though this envelope was non-macrophage-tropic.
Although this study has concentrated on brain and LN envelopes, we have also included envelopes that were amplified from blood and semen. Previously, we reported that most of these additional R5 envelopes were non-macrophage tropic, although several macrophage-tropic envelopes were detected. These included C98-15 and C98-18 from the same pediatric plasma sample and the semen-derived envelopes SQ43 380.1 and 380.4. These macrophage-tropic R5 envelopes conferred increased resistance to Q4120 and enhanced sensitivity to sCD4 and PRO 542 indicating that the association of macrophage-tropism with sensitivity to reagents that interfere with envelope:CD4 interactions, holds true, regardless of envelope tissue origin.
In summary, we have studied how variation in HIV-1 R5 macrophage-tropism relates to sensitivity to neutralizing antibodies that target conserved envelope epitopes and to reagents that inhibit virus entry. We have investigated HIV-1 envelopes amplified directly from patient material without culture. Such envelopes are expected to represent those in vivo and have a distinct advantage over primary isolates that will have been altered by culture. Our data demonstrate considerable phenotypic variation conferred by R5 envelopes that impacts on macrophage-tropism and sensitivity to entry inhibitors including the CD4 binding site mab, b12. It is currently unclear whether this variation affects the capacity of R5 viruses to transmit. Regardless, our results strongly indicate that macrophage-tropism is modulated by changes in gp120 that predominantly impact on the CD4 binding site consistent with an increased gp120:CD4 affinity. Our results have relevance for therapies that target HIV entry and for the design of vaccines that aim to induce neutralizing antibodies.