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Emergence in vivo of Vicriviroc Resistance in HIV-1 Subtype C: Role of V3 Loop and Susceptibility to Other CCR5 Antagonists
Athe M.N. Tsibris*1,2, M Sagar2,3, Z Su4, C Flexner5, W Greaves6, P Skolnik7, E Coakley8, M Subramanian9, R Gulick10, and D Kuritzkes2,3
1Massachusetts Gen Hosp, Boston, US; 2Harvard Med Sch, Boston, MA, US; 3Brigham and Women`s Hosp, Boston, MA, US; 4Harvard Sch of Publ Hlth, Boston, MA, US; 5Johns Hopkins Univ, Baltimore, MD, US; 6Schering-Plough Res Inst, Kenilworth, NJ, US; 7Boston Med Ctr, MA, US; 8Monogram Biosci, South San Francisco, CA, US; 9Human Genome Sci, Rockville, MD, US; and 10Weill Med Coll of Cornell Univ, New York, NY, US
Background: We previously identified a CCR5-using
(R5) HIV-1 isolate with high-level resistance to the CCR5 antagonist vicriviroc
(VCV) in 1 of 29 VCV-treated subjects with virologic failure in AIDS Clinical
Trials Group (ACTG) 5211, a phase IIb study of this drug in triple-class
experienced subjects. Emergence of VCV resistance was associated with
accumulation of mutations in the V3 loop stem of gp120. We sought to determine
the role of these V3 loop mutations in VCV resistance, and their effect on
HIV-1 susceptibility to other entry inhibitors.
Methods: VCV susceptibility (PhenoSense Entry
Susceptibility assay), cloning, and sequencing of full-length env (gp160) were
performed on plasma HIV-1 samples obtained at entry, confirmation of virologic
failure (week 19), and at week 28. Using yeast gap repair homologous
recombination, we incorporated the predominant subject-derived full-length env
clone observed at each time point into an NL4-3 backbone to generate infectious
recombinant HIV. Viruses were grown in 293T cells, titered, and used to infect
a CCR5-expressing reporter cell line (TZM-bl) in the presence and absence of
increasing concentrations of VCV, TAK779, enfuvirtide, or a CCR5 monoclonal
antibody. We then constructed chimeric env molecules incorporating
segments (gp120, gp41, V1-V3 segment, or V3 loop alone) of the week 28 env
into a week 0 env backbone to determine which env domain(s) were
responsible for the observed VCV resistance.
Results: Sequence and phylogenetic analysis showed
the envelope from this subject clustered with HIV-1 subtype C genotypes. Susceptibility
testing of recombinant chimeric viruses showed that insertion of V3 loop alone
from the week 28 virus conferred resistance to VCV and cross-resistance to
TAK779. Infection by VCV-resistant recombinants was enhanced by increasing VCV
and TAK779 concentrations. No effect on enfuvirtide susceptibility was noted.
VCV resistance sensitized virus to inhibition by the competitive CCR5
antagonist HGS004, resulting in a 5.4-fold decrease in HGS004 IC50.
Conclusions: These findings demonstrate that
mutations in the V3 loop stem of a clade C virus are sufficient to confer VCV
resistance and cross-resistance to at least one other small-molecule CCR5
antagonist. Enhanced susceptibility to the competitive CCR5 antagonist HGS004
may be due to decreased ability of VCV-resistant env to bind the native
CCR5 receptor.
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