Resistance to the CCR5 Antagonist Maraviroc Is Characterised by Dose-Response Curves that Display a Reduction in Maximal Inhibition
M Mosley1, C Smith-Burchnell1, J Mori1, M Lewis1, M Stockdale1, W Huang2, J Whitcomb2, C Petropoulos2, M Perros1, and Mike Westby*1
1Pfizer Global R&D, Sandwich, UK and 2Monogram Biosci, South San Francisco, CA, US
Background: We previously generated R5
HIV-1 variants by serial passage that are resistant to the CCR5 antagonist maraviroc (MVC, UK-427,857). To characterize
the molecular mechanism of this resistance we performed site-directed
mutagenesis on envelope (Env) derived from an
MVC-resistant (MVCres) variant of HIV-1 strain CC1/85
and assessed the susceptibility of the mutants to MVC, both in PHA-stimulated peripheral blood lymphocytes (PBL)
and an Env pseudovirus
Methods: Sequencing analysis of Env
following serial passage of the MVCres CC1/85 strain
in the absence of compound suggested that the molecular determinants of
resistance were localised in the V3 loop. A 2.56-kb fragment encompassing Env was amplified from the MVCres
variant and site-directed mutagenesis was performed on the region encoding the
V3 loop using QuickChangeXL. Mutated Env fragments were subsequently cloned into pNL4-3. Virus
was recovered in the tissue culture supernatant of HEK 293T cells, 48 hours
after transfection with the Env
recombinant NL4-3 site-directed mutagenesis. Virus susceptibility to MVC,
aplaviroc (GW873140), enfuvirtide and efavirenz was assessed in PHA-stimulated
PBL, using p24 levels in the cell culture media 7 days after acute infection.
The susceptibility of Env-pseudoviruses to MVC was
also measured in a single cycle replication assay using U87CD4+CCR5+ cells.
Results: The MVCres Env recombinant NL4-3 clone exhibited high-level resistance
to MVC but remained sensitive to aplaviroc, confirming that it required CCR5 for entry into PBL. Back mutation of either
316T or 323V (numbering from HxB2), to A and I, respectively, resulted in a
virus that gave an incomplete dose response curve (plateau). When both
mutations were replaced, dose response curves similar to the MVC-sensitive
parental virus were obtained. A high concentration of MVC blocked the activity
of aplaviroc on MVCres variants, consistent with the
compounds binding to overlapping sites on CCR5
and suggesting that MVCres viruses have acquired the
ability to use MVC-occupied receptors for entry.
Conclusions: Reduced MVC susceptibility
was characterised by dose response curves that did not reach 100% inhibition.
These results support the view that resistance to CCR5
antagonists is characterised by selection of variants that recognize inhibitor:receptor complexes, and indicate
that parameters other than fold changes in IC50 may be important for
identifying resistance in patients.