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 ⌬32/⌬32 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.

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4 + T Cells

“…Addressing the implications of resistance should make it possible to determine if virologic failure in the face of CCR5 antagonist therapy will affect viral tropism and disease progression in any meaningful way. Passaging HIV-1 in the presence of increasing concentrations of a CCR5 antagonist in vitro can result in the development of mutations in Env that enable it to recognize the drugbound conformation of CCR5 (22,33,51,56). A similar resistance pathway can occur in vivo or, as we have shown, can even occur in the absence of drug selection (48,53).…”

“…A similar resistance pathway can occur in vivo or, as we have shown, can even occur in the absence of drug selection (48,53). This resistance pathway often, but not always, results in a virus that is resistant to multiple CCR5 antagonists (22,33,39,53,56). The CCR5 antagonists that have been used clinically all seem to bind to a hydrophobic region in the transmembrane helices of CCR5 and induce conformational alterations in the extracellular domains of the coreceptor that inhibit recognition by gp120 (13,31,32,46,52).…”

“…Because all Env clones isolated from this patient were resistant to complete suppression by aplaviroc, we performed a protein BLAST search on the Env clone demonstrating the greatest resistance to aplaviroc (clone pre5.2) and identified JRFL as a closely related, commonly studied HIV strain that is sensitive to inhibition by aplaviroc and other CCR5 antagonists. Since mutations in the V3 loop are commonly associated with resistance to CCR5 antagonists (22,33,53,56), we constructed chimeric viruses in which the V3 loops of pre5.2 and JRFL were exchanged. Pseudotype infection assays were performed on NP2/CD4/CCR5 cells in the presence or absence of 10 M aplaviroc, a level empirically determined to be 3 log units higher than required to saturate CCR5 receptors on this cell line.…”

“…A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1,2,22,33,36,51,56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21,48).…”

Polymeric Anti‐HIV Therapeutics

“…[30] In addition, it has been reported that laboratory adapted maraviroc-resistant HIV mutants can still bind to the coreceptor while maraviroc is bound to CCR5, suggesting that the virus binding site and the maraviroc binding site are independent of each other and that maraviroc works by an allosteric mechanism. [31] Since its approval, maraviroc has shown to be very effective in patients carrying R5 tropic HIV and has shown little occurrence of maraviroc resistant HIV strains. However, maraviroc produces an immunomodulary effect characterized by an increase in CD8þ T-cells, of which the effect of long term use has not yet been determined.…”