Background:  Resistance to small molecule CCR5 antagonists cannot be reliably predicted genotypically or by measurement of IC₅₀ shifts. In contrast, resistance can manifest as a reduction in the maximal percentage of inhibition (MPI) or plateau of dose-response curves. Studies have shown that the magnitude of response measured can be influenced by both cell type and assay format (single- vs multi-cycle). However, the effect of virus concentration on MPI has not been previously described. Here we assess the effect of virus concentration on the measurement of resistance to vicriviroc (VCV).

Methods:  Pseudoparticles and replication competent viruses containing cloned chimeric envelope sequences from a VCV sensitive and resistant virus (RU570) were generated and standardized by p24 content. U-87-CCR5 cells were treated with VCV and infected with decreasing 2-fold dilutions of virus. Replication was assessed in both single- and multi-cycle infection assays using luciferase or p24 antigen detection. Resistance to VCV was quantified as MPI.

Results:  In both the single and multi-round infection assays, the efficacy of VCV (MPI) was related to the concentration of resistant virus. Infection of cells with high titer inocula produced flattened dose response curves with MPI values <50%, indicative of resistance. However, reduction in virus concentration resulted in progressively increased MPI values that approached 100% at low viral inocula (0.2 ng p24/mL). Control viruses were fully inhibited by VCV (MPI = 100%) regardless of virus concentration or assay format.

Conclusions:  The VCV-resistant virus used in this study displayed a range of susceptibilities to VCV, depending on the virus concentration used in the assay. At high viral input, viruses appeared resistant with MPI values below 50%, however, decreasing virus input manifested as increased susceptibility to VCV. This behavior was observed in either single or multi-round assay formats. The dependence of virus concentration on MPI is consistent with predictive models of allosteric inhibition of receptor-agonist interactions and is likely explained by an increase in affinity of resistant viruses for VCV-bound CCR5. These results demonstrate the impact of virus concentration on the measurement of resistance and should be considered when comparing the susceptibility profiles of viral isolates and assigning cutoff values for resistance to CCR5 antagonists.