Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry

Abstract

Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Delta32/Delta32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.

FIG. 1.

Growth of HIV-1 isolates passaged through PBL in the presence of maraviroc. Filled diamonds represent p24 values in wells containing virus passaged with maraviroc, open triangles represent p24 values in untreated control wells, and filled squares represent the concentrations of maraviroc in the treated cultures. The arrows indicate the passages at which virus supernatants were harvested for further analysis. Virus appeared to escape maraviroc inhibition in cultures of CC1/85 (A), RU570 (B), and SF162 (C), as shown by the fact that p24 levels in the maraviroc-containing cultures approximately matched the p24 levels in the drug-free control cultures by the end of the experiment. In contrast, escape variants were not recovered from cultures of 92BR017, 92BR018, and 92BR023. Results for 92BR018 are shown as an example in panel D. Although virus appeared to break through in the presence of 16 nM maraviroc at passage 8, this virus subsequently failed to replicate and there was no detectable p24 by passage 20 (not shown). A frozen aliquot of passage 10 supernatant was recovered and passaged in 8 nM maraviroc. This culture showed evidence of breakthrough again (passage 11) but was subsequently unable to replicate in higher concentrations of maraviroc.

FIG. 2.

Susceptibility of pseudoviruses derived from CC1/85 to maraviroc (A), SCH-C (B), and enfuvirtide (C) in the PhenoSense HIV Entry assay (U87CD4+ cells expressing CCR5). Similar results were obtained for RU570-derived viruses.

FIG. 3.

Representative susceptibility curves for MVCsens, MVCres, and SDM Env-recombinant NL4-3 clones with maraviroc tested in PBL (A) and in the PhenoSense HIV Entry assay (B) and with aplaviroc (C) and enfuvirtide (D) tested in PBL. For PBL assays (A, C, and D), PBL were infected with a standardized input of virus in the presence of various concentrations of inhibitor. Supernatant p24 levels were determined after 7 days. Data points represent the means of triplicate wells within the same assay, and the bars represent the standard error of the mean. PBL assays were repeated between two and five times; mean IC₅₀ are given in Table 6. For the PhenoSense HIV Entry assay (B), luciferase activity was measured to quantify infection of U87CD4+CCR5+ cells by pseudoviruses derived from the Env-recombinant NL4-3 clones in the presence of various concentrations of inhibitor.

FIG. 4.

Susceptibility of MVCres Env-recombinant NL4-3 clone to aplaviroc (A) and enfuvirtide (B) in the absence (open squares, solid line) or presence (open circles, dotted line) of 400 nM maraviroc in PBL.