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HIV-1 predisposed to acquiring resistance to maraviroc (MVC) and other CCR5 antagonists in vitro has an inherent, low-level ability to utilize MVC-bound CCR5 for entry
© Roche et al; licensee BioMed Central Ltd. 2011
Received: 25 August 2011
Accepted: 7 November 2011
Published: 7 November 2011
Maraviroc (MVC) and other CCR5 antagonists are HIV-1 entry inhibitors that bind to- and alter the conformation of CCR5, such that CCR5 is no longer recognized by the viral gp120 envelope (Env) glycoproteins. Resistance to CCR5 antagonists results from HIV-1 Env acquiring the ability to utilize the drug-bound conformation of CCR5. Selecting for HIV-1 resistance to CCR5-antagonists in vitro is relatively difficult. However, the CCR5-using CC1/85 strain appears to be uniquely predisposed to acquiring resistance to several CCR5 antagonists in vitro including MVC, vicriviroc and AD101.
Here, we show that Env derived from the parental CC1/85 strain is inherently capable of a low affinity interaction with MVC-bound CCR5. However, this phenotype was only revealed in 293-Affinofile cells and NP2-CD4/CCR5 cells that express very high levels of CCR5, and was masked in TZM-bl, JC53 and U87-CD4/CCR5 cells as well as PBMC, which express comparatively lower levels of CCR5 and which are more commonly used to detect resistance to CCR5 antagonists.
Env derived from the CC1/85 strain of HIV-1 is inherently capable of a low-affinity interaction with MVC-bound CCR5, which helps explain the relative ease in which CC1/85 can acquire resistance to CCR5 antagonists in vitro. The detection of similar phenotypes in patients may identify those who could be at higher risk of virological failure on MVC.
Human immunodeficiency virus type 1 (HIV-1) entry is initiated by the interaction of the viral gp120 envelope (Env) glycoproteins with cellular CD4 and a coreceptor, either CCR5 or CXCR4 . Maraviroc (MVC) and other CCR5-antagonists such as vicriviroc (VVC, also known as SCH-D), AD101 (a preclinical precursor of VVC), and aplaviroc (APL) are HIV-1 entry inhibitors that bind to- and alter the conformation of CCR5, such that CCR5 is no longer recognized by gp120 . Thus, CCR5-antagonists are allosteric inhibitors of HIV-1 entry [2–4]. MVC has been approved for use in treatment-experienced and antiretroviral therapy (ART)-naïve HIV-1-infected adults who have no evidence of CXCR4-using virus in plasma . As with other antiretrovirals, treatment with CCR5-antagonists can result in drug resistance leading to virological rebound. Although virological failure can arise from the emergence of CXCR4-using HIV-1 strains that were present at very low levels prior to initiation of a CCR5-antagonist , genuine resistance to CCR5-antagonists results from adaptive alterations in gp120 enabling recognition of the drug-bound conformation of CCR5 [7–15].
Being allosteric inhibitors of virus entry, resistance to CCR5-antagonists is evident by plateaus in virus inhibition curves below 100% inhibition . The magnitude of the reduction in plateau height can be quantified as the maximal percent inhibition (MPI), which reflects the ability of HIV-1 gp120 to recognize the drug bound conformation of CCR5. For example, MPIs can be high (> 80%)  signifying a relatively inefficient ability of gp120 to utilize the drug-bound conformation of CCR5, or low (< 20%)  signifying relatively efficient utilization of drug-bound CCR5. However, MPIs can be influenced by differences in the level of CCR5 expression on target cell populations [9, 11, 12]. Generally, in cell lines, there is an inverse relationship between the MPI achieved by a given virus with resistance to a CCR5-antagonist, and the level of CCR5 expression. Clinically, MPIs of HIV-1 have been reported using the PhenoSense™ Entry assay , which uses the U87-CD4/CCR5 cell line. These cells express comparatively lower levels of CCR5 than other commonly used indicator cells such as TZM-bl, JC53 and NP2-CD4/CCR5 cells  and therefore, are likely to provide a relatively conservative measure of resistance to CCR5-antagonists. Consistent with this view, results from the clinical trials of MVC in treatment-experienced subjects (MOTIVATE) showed that most MVC-resistant viruses in subjects failing therapy had relatively high MPIs within the range of 80-95%, when tested using the PhenoSense™ Entry assay ( and references within).
The in vitro generation and characterization of HIV-1 variants with resistance to antiretroviral drugs is vital for elucidating resistance mechanisms. However, selecting for HIV-1 resistance to CCR5-antagonists is relatively difficult . One particular HIV-1 strain, CC1/85 , has been used in a number of independent studies for the in vitro generation of HIV-1 resistance to different CCR5-antagonists including MVC, VVC and AD101 (for example, [16, 18–20]). In fact, the published in vitro CCR5-antagonist resistance studies are heavily biased towards the characterization of resistant variants derived from CC1/85. The CC1/85 strain of HIV-1 may therefore be predisposed to acquiring resistance to CCR5- antagonists in vitro. Here, we sought to elucidate the phenotypic features of CC1/85 that underlie this predisposition. A better understanding of these mechanisms has the potential to identify subjects with increased risk of developing resistance to MVC and other CCR5-antagonists.
MVC-Sens and MVC-Res plasmids contain the env gene of CC1/85 virus and a derivative with MVC-resistance, respectively, cloned into the pSVIII-Env expression vector [15, 16]. Single-round luciferase reporter viruses pseudotyped with MVC-Senv or MVC-Res Envs, or with the CCR5-using (R5) YU2, JRCSF, NB6-C3 or NB8-C4 Envs as controls were produced as described previously . The characterization and maintenance of TZM-bl, JC53, U87-CD4/CCR5, NP2-CD4/CCR5 and the dually CD4- and CCR5-inducible 293-Affinofile cells, and the preparation of peripheral blood mononuclear cells (PBMC) has been described previously [15, 21]. Maraviroc resistance assays were conducted using Env-pseudotyped luciferase reporter viruses, or replication competent viruses carrying MVC-Res or MVC-Sens env genes, as described previously [15, 16]. For experiments using 293-Affinofile cells, populations expressing CD4 together with different levels of CCR5 ranging from relatively low to high were generated by inducing the cells with 2.5 or 5 ng per ml of minocycline and either 15.6, 31.2, 62.5, 125, 250, 500, 1000 or 2000 nM of ponasterone A, as described previously . Alterations in drug sensitivity were assessed by reductions in the MPI as described previously [15, 16].
Discussion and conclusions
Our results suggest that MVC-Sens Env, which was derived from the CC1/85 strain of HIV-1, has an inherent ability to recognize the MVC-modified conformation of CCR5. These results provide a plausible explanation as to why CC1/85, but not most other R5 HIV-1 strains, appears to be predisposed to acquiring resistance to MVC and other CCR5-antagonists in vitro, and why CC1/85 has been frequently used for this purpose in a number of independent studies [16, 18–20]. The disclosure of the ability of MVC-Sens Env, but not other R5 Envs, to recognize drug-bound CCR5 in cell lines engineered to overexpress CCR5 distinguishes CC1/85 from other R5 viruses as being predisposed to develop a genuinely resistant profile when cultured in the presence of increasing concentrations of CCR5-antagonist. However, despite this predisposition, 17 virus passages and multiple Env mutations were required for CC1/85 to acquire resistance to MVC, compared to only 1 passage for this virus to acquire resistance to 3TC , suggesting that even CC1/85 has a relatively high genetic barrier to acquiring resistance to CCR5-antagonists.
The results of this study suggest that a similar baseline ability of HIV-1 to interact with drug-modified CCR5 may exist in certain subjects prior to commencing MVC or other CCR5-antagonists. To this end, in a longitudinal study of 21 ART-naïve subjects with HIV-1 subtype C, we have identified two subjects whose viruses exhibit plateaus of incomplete inhibition by MVC in NP2-CD4/CCR5 cells and 293-Affinofile cells expressing high levels of CCR5, but which are completely inhibited by MVC in cell lines expressing lower levels of CCR5, in a strikingly similar fashion as CC1/85 (, manuscript in preparation). In addition, retrospective analysis of 11 subjects who developed APL-resistance during the CCR100136 (EPIC) clinical trial of APL showed that baseline viruses of 8 individuals (73%) had some evidence of partial APL-resistance prior to therapy . Further analysis of one of these baseline viruses confirmed that the Env glycoproteins had a low-affinity interaction with APL-bound CCR5 . The clinical significance of viral variants with low-level basal recognition of drug-bound CCR5 in the setting of MVC and other CCR5-antagonist therapies remains to be determined by more extensive in vivo studies. To this end, we have shown that the likelihood of developing resistance to CCR5-antagonists in vivo is influenced also by the activity of the patient's optimized background therapy (B. Jubb and M. Westby, unpublished data). Nonetheless, our results suggest that certain individuals could be at increased risk of drug failure on MVC and other CCR5-antagonists due to predisposition for development of resistance.
One reason why U87-CD4/CCR5 cells are justifiably used by the PhenoSense™ Entry assay for the measurement of HIV-1 resistance to CCR5-antagonists is because the CCR5 expression levels on these cells more closely reflects CCR5 levels on primary CD4+ T-cells . However, should future in vivo studies demonstrate that patients with baseline HIV-1 strains possessing inherent low-level ability to recognize drug-bound CCR5 are at greater risk of drug failure due to a predisposition to develop resistance, this phenotype is likely to be masked or only weakly exposed in U87-CD4/CCR5 cells. Pre-screening candidates for CCR5-antagonist therapy by a modified drug susceptibility assay using NP2-CD4/CCR5 cells or 293-Affinofile cells could potentially identify these individuals.
Acknowledgements and funding
We thank J. Sodroski for providing pSVIII-YU2 Env plasmid and for providing pCMVΔ P1Δ envpA and pHIV-1Luc plasmids. We also thank D. Kabat for providing JC53 cells, N. Shimizu and H. Hoshino for permission to use NP2-CD4/CCR5 cells, and D. Mosier and R. Nedellec for supplying the NP2-CD4/CCR5 cells. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl cells from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc.; U87-CD4/CCR5 cells from Dr. HongKui Deng and Dr. Dan R. Littman.
This study was supported by a grant by the Australian Center for HIV and Hepatitis Virology Research (ACH2) to PRG and MJC, and by a grant from NIH/NIAID to BL (R21 AI092218). MR is supported by a Monash University Postgraduate Research Scholarship. PRG is the recipient of an Australian National Health and Medical Research Council (NHMRC) Level 2 Biomedical Career Development Award. SRL is the recipient of a NHMRC Practitioner Fellowship. The authors gratefully acknowledge the contribution to this work of the Victorian Operational Infrastructure Support Program received by the Burnet Institute.
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