Emerging Populations of Enfuvirtide Resistant HIV-1 Contain Multiple Variants That Compete for Dominance

W. Huang¹, L. Maroldo^1,2, S Fransen¹, J Toma¹, JM Whitcomb¹,  C. Chappey¹, E. Coakley¹, and CJ Petropoulos^1
1ViroLogic, Inc., South San Francisco, CA, ²West Midtown Medical Group, New York, NY

Recently, we described the rapid emergence of an HIV-1 variant containing a rare single amino acid substitution (V38E) that confers high level resistance to enfuvirtide (ENF).  In the current study, we conducted a detailed analysis of multiple env clones to characterize this rapidly emerging resistance virus population.

Heptad repeat 1 and 2 (HR1, HR2) envelope sequences were determined for ~40 envelope clones derived from virus populations at days 10 and 40 of ENF treatment.  Based on HR1 sequences, approximately 10-12 clones from each timepoint were selected for ENF susceptibility testing using a single cycle HIV-1 env pseudovirion assay.

Results

ENF susceptibilities (IC₅₀ fold-change) at d0, d10 and d40 were 0.4, 7.6 and 400, respectively. Population genotyping identified the wildtype G-I-V motif at amino acid positions 36-38 of HR-1 at d0, a mixed G/D-I-V/E motif at d10, and a resistant G-I-E motif at d40. HR1 sequences were used to classify molecular clones derived from the d10, and d40 viruses.

The relatedness of all clones was confirmed by phylogenetic analysis of gp160 sequences. D10 clones consisted of susceptible and resistant variants: G-I-V(14%), D-V-V(2%), D-I-V(18%), G-I-A(18%), G-I-E(46%). No other mutations in HR1 were observed. 

By d40, all clones were G-I-E.  No double mutants were observed at either time point and all clones were R5-tropic.  ENF susceptibility varied by genotype; G-I-V(FC=0.6), D-I-V(FC=27), G-I-A(FC=21), D-V-V(FC=49), G-I-E(FC=583), and consistent for multiple clones of like genotype.  Infrequent mutations in HR2 did not contribute to significant reductions in ENF susceptibility.

The infectivity of env clones varied, but consistent reductions in clones that contained ENF resistance mutations were not apparent in this dataset.  Additional analyses are ongoing to attempt to identify compensatory mutations outside of HR1.

Conclusions

ENF resistance was associated with the early emergence of multiple variants containing distinct mutations in HR1. G-I-E variants exhibiting the largest reductions in susceptibility were prevalent at d10 and predominated by d40. The apparent lack of differences in env infectivity suggest that selection for G-I-E variants was driven by ENF pressure, however the contribution of infectivity will require a more thorough analysis of potential compensatory mutations.

06/27/05

Reference
W Huang and others. Emerging Populations of Enfuvirtide Resistant HIV-1 Contain Multiple Variants that Compete for Dominance (poster). Abstract 151. XIV International Drug Resistance Workshop. June 7-10, 2005. Quebec City, Quebec, Canada.  [Antiviral Therapy 2005; 10:S166]

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