18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

STATE OF THE ART PRESENTATION: V3-DEPENDENT AND -INDEPENDENT PATHWAYS TO SMALL MOLECULE CCR5 INHIBITOR RESISTANCE

Antivir Ther 2009; 14 Suppl 1:A13 (abstract no. 11)

JP Moore
Weill Medical College of Cornell University, New York, NY, USA

OBJECTIVES: The small molecule CCR5 inhibitor vicriviroc (VVC) inhibits HIV type-1 entry. Our aim is to understand the mechanisms of resistance to VVC and similar compounds.

METHODS: Cross-resistant viruses were generated from the R5 primary isolate, CC1/85, in PBMC cultures under the selection pressures of AD101 (a VVC precursor) and VVC. The resistant isolates CC101.19 (AD101) and D1/85.16 (VVC) were cloned, sequenced and characterized in PBMCs and engineered cell lines (for example, Tzm-bl) with and without inhibitors using fully infectious viruses or Env pseudotypes.

RESULTS: Both variants retained the R5 phenotype, although some low-efficiency D1/85.16 X4 clones were present, both used the inhibitor–CCR5 complex and free CCR5 for entry and neither reverted to sensitivity when cultured without inhibitor. However, to varying extents, the resistant viruses used the inhibitor–CCR5 complex less efficiently than free CCR5. This is the basis of the ‘plateau effect’ commonly observed in cell-line-based resistance assays (for example, Phenosense); plateau heights reflect the relative usage of the two CCR5 configurations by resistant variants. CC101.19 followed a pathway to resistance involving four sequential changes in the gp120 V3 region, the most commonly observed site of resistance mutations in other studies. However, the D1/85.16 variant contained no V3 changes; the three key resistance mutations were not even located in gp120, but in the gp41 fusion peptide. The sequence changes in both viruses alter how their Env complexes interact with the CCR5 N terminus and ECL-2 domains, but in different ways at the levels of both Env and CCR5. We also show that VVC resistance is manifested differently in PBMCs and cell lines with dose–response curves showing assay-dependent complexities. To explain them, we created a model for resistance and generated theoretical inhibition curves that closely mimic experimental data. The model proposes that distinct CCR5 forms with varying affinities for CCR5 inhibitors are present in different proportions on different cell types and used selectively by resistant variants when inhibitor-ligated. Finally, resistant variants can have increased sensitivity to neutralizing antibodies with implications for resistance development pathways in vivo.

CONCLUSIONS: Several unusual aspects of CCR5 inhibitor resistance merit further understanding to guide clinical use of these new drugs.

2009-06-09
11

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