Viruses with reduced susceptibility to ibalizumab were identified in patients who experienced a rebound in the HIV-1 viral load after the addition of ibalizumab to failing antiretroviral drug regimens. The susceptibilities of these viruses and their paired baseline isolates to ibalizumab and other HIV-1 entry inhibitors were evaluated by using cell-based infectivity assays. Compared to matching baseline viruses, the infectivity of viruses isolated after viral load rebound was incompletely inhibited by ibalizumab. This phenotypic profile most likely reflects the ability of ibalizumab-resistant envelope proteins to mediate CD4-dependent HIV-1 entry in the presence of bound ibalizumab (11
). A similar inhibitory profile was reported previously for viruses that have acquired reduced susceptibility to noncompetitive CCR5 antagonists, including maraviroc (40
), vicriviroc (18
), and aplaviroc (12
). In these instances, resistant viruses regained the ability to bind CCR5 despite the presence of a bound antagonist (24
). Similarly, ibalizumab is also a noncompetitive inhibitor which does not block gp120 binding to CD4 (20
) but rather is thought to prevent downstream events required for efficient HIV-1 entry. Such events might include conformational changes in gp120 (28
) and/or CD4 (2
) that are triggered by CD4-gp120 binding, coreceptor engagement (34
), or subsequent events leading to membrane fusion. Whether ibalizumab inhibition occurs by direct or indirect interference in such events is currently unresolved.
In this study we demonstrated that viruses with reduced susceptibility to ibalizumab also exhibited reduced susceptibility to the anti-CD4 monoclonal antibody RPA-T4. This observation suggests that the competitive inhibition of CD4-gp120 binding by RPA-T4 is less efficient with ibalizumab-resistant viruses and is consistent with the previously reported behavior of viruses selected in vitro
for reduced susceptibility to the monoclonal antibody 5A8, the murine progenitor of ibalizumab (13
). In addition, our studies revealed a striking inverse relationship between the susceptibilities of viruses to ibalizumab and sCD4, which targets the CD4 binding site of gp120; that is, reductions in susceptibility to ibalizumab are accompanied by increases in susceptibility to sCD4. Interestingly, viruses with reduced ibalizumab susceptibility (week 9) displayed measurably higher levels of env
-mediated infectivity in vitro
than did the paired ibalizumab-susceptible (day 0) viruses. The observed increases in virus infectivity are consistent with increased susceptibility to sCD4 if one assumes more efficient interactions between viral gp120 and cell surface CD4. This increased susceptibility to sCD4 is not associated with a detectable increase in susceptibility to antibody neutralization, since week 9 viruses were not more susceptible than their paired day 0 viruses to the broadly neutralizing monoclonal antibodies b12, 2G12, and 4E10 or polyclonal HIV-1 plasma antibodies (data not shown). Further studies are needed to evaluate the impact of viral replication of ibalizumab-resistant viruses in vivo
To begin to experimentally delineate the genetic determinants of ibalizumab resistance, an in-depth analysis of env clones isolated from four paired baseline and on-treatment virus populations was conducted. The phenotypic properties of the 96 individual clones consistently mirrored the pooled env sequence populations sampled at day 0 and week 9 with respect to susceptibility to ibalizumab and other entry inhibitors. Phylogenetic analyses of day 0 and week 9 clones from each patient suggested that ibalizumab-resistant variants may emerge by the outgrowth of preexisting minority variants present in the baseline virus population; however, this and other potential mechanisms will require more extensive evaluation. A careful scrutiny of env sequences identified a common mutation pattern associated with reduced susceptibility to ibalizumab. Specifically, mutations that disrupt PNGSs in the V5 region of gp120 are key determinants of ibalizumab resistance, which was further substantiated by site-directed mutagenesis experiments. Among the two highly conserved PNGSs in V5 (site 1 and site 2), site 1 was most often absent in ibalizumab-resistant envelope proteins. Note that exceptions were observed, i.e., ibalizumab-resistant variants possessing two V5 PNGSs. These data suggest that additional genetic determinants can confer reductions in susceptibility to ibalizumab; however, their identification and characterization will require further study.
HIV Env contains numerous N-linked carbohydrates, some of which are important determinants of antibody neutralization (17
). The elimination of PNGS and reduced ibalizumab susceptibility are consistent with the notion that these mutations restore an essential CD4-dependent conformational change(s) in gp120 despite drug binding. The V5 loop is situated on the outer domain of gp120 and comprises a portion of the so-called “glycan shield,” which is thought to reduce the immunogenicity of this exposed facet of the gp120 protein structure (38
). An additional perspective on the mechanism of ibalizumab resistance may be gained from studies of the mechanism of sCD4 inhibition. sCD4 inhibits HIV-1 entry either by the competitive inhibition of gp120 binding or by promoting irreversible conformational changes leading to an inactivation of infectivity. Differences in the affinity of gp120 for sCD4 do not account for reductions in susceptibility to ibalizumab (3
) or the murine precursor, mu5A8 (13
). Conformational changes in gp120 triggered by sCD4 are transient and render gp120 competent, albeit briefly, for receptor-mediated virus entry (10
). These same changes are also likely involved in the host cell CD4-mediated activation of gp120, although it is unclear how this differs functionally from activation by sCD4. Thus, it is reasonable to postulate that ibalizumab escape variants can bypass this requirement by enhancing the ability to trigger CD4-induced conformational changes or, less likely, by developing an alternative mechanism of gp120 activation, since the latter is inconsistent with the observed CD4 dependency of ibalizumab-resistant isolates. Alternatively, ibalizumab may block downstream events such as coreceptor binding or other conformational changes that are required for membrane fusion.
Analysis of 1,322 nonredundant env
sequences in the Los Alamos National Laboratory HIV Database demonstrated that the V5 sequences of 97.9% and 56.3% of subtype B sequences contain one and two PNGSs, respectively. This conservation of PNGS is consistent with a determinant role in broad ibalizumab susceptibility. To further explore the role of V5 glycosylation in ibalizumab susceptibility, we modeled the interaction between gp120 and CD4 using the structure reported under Protein Data Bank (PDB) accession number 1GC1
and PNGSs using the Glycosylate software program (courtesy of Jiang Zhu). Based on this model, the V5 site 1 glycan is situated in close proximity to the CD4-gp120 interface (). This proximity elicits several potential mechanisms of ibalizumab activity and escape. In one instance, CD4-ibalizumab binding may result in a steric clash between gp120 carbohydrate moieties and CD4 that prevents CD4-gp120 binding. Such a constraint may be removed by the elimination of V5 carbohydrate side chains. However, this mechanism is inconsistent with data from previous studies demonstrating the ability of gp120 to bind CD4 in the presence of bound ibalizumab (20
). Alternatively, the loss of V5 glycosylation may increase the binding affinity of gp120 for CD4 in the presence of ibalizumab. However, this mechanism is inconsistent with data from previous studies demonstrating comparable CD4-gp120 binding affinities for ibalizumab-susceptible and -resistant variants (3
) and the murine antibody precursor 5A8 (13
). Based on existing observations, the most probable explanation for ibalizumab escape is the ability of ibalizumab-resistant variants to facilitate CD4-induced conformational changes in the CD4-gp120 complex, which enable coreceptor engagement despite bound ibalizumab. Further investigation will be required to confirm this hypothesis or determine whether the inhibitory mechanism involves events further downstream in the HIV entry process. Either way, the reestablishment of requisite conformational changes may be enabled by the elimination of steric hindrances imposed by one or more V5 glycosylation moieties.
Fig. 6. V5 glycans are situated in close proximity to the gp120-CD4 interface. The interface of the gp120 core structure (orange) with CD4 (blue) is adjacent to the V5 region (red) and the V5 site 1 glycan located at amino acid position 460 (green). gp120 glycans (more ...)
From a clinical standpoint, the lack of detectable cross-resistance between ibalizumab and other entry inhibitors such as maraviroc and enfuvirtide suggests that there exists a potential to prescribe ibalizumab irrespective of prior entry inhibitor therapy or coreceptor tropism. Consistent with the observed lack of cross-resistance, critical determinants of maraviroc and enfuvirtide resistance have been mapped to the V3 region and gp41, respectively, and do not appear to overlap with the determinants of ibalizumab resistance reported in this study. In vitro
drug combination studies have demonstrated synergistic antiviral activities for ibalizumab and enfuvirtide (44
) and additive-to-synergistic activity for ibalizumab and maraviroc (S. P. Weinheimer et al., unpublished data), providing further support for ibalizumab as a potentially valuable addition to antiretroviral drug regimens. However, more studies are needed to further evaluate the potential for cross-resistance between ibalizumab and other entry inhibitors and the effects of combination therapy in the clinical setting.