Evolution of HIV-1 tropism at quasispecies level after 5 years of combination antiretroviral therapy in patients always suppressed or experiencing episodes of virological failure

Received 27 February 2014; returned 15 April 2014; revised 29 May 2014; accepted 12 June 2014Objectives:Tropism evolution of HIV-1 quasispecies was analysed by ultra-deep pyrosequencing (UDPS) inpatients on first-line combination antiretroviral therapy (cART) always suppressed or experiencing virologicalfailure episodes.Methods:Among ICONA patients, two groups of 20 patients on cARTfor ≥5 years, matched for baseline viraemiaand therapy duration, were analysed [Group I, patients always suppressed; and Group II, patients experiencingepisode(s) of virological failure]. Viral tropism was assessed by V3 UDPS on plasma RNA before therapy (T0) andon peripheral blood mononuclearcell proviral DNA before–after therapy (T0-T1), using geno2pheno false positiverate (FPR) (threshold for X4: 5.75). For each sample, quasispecies tropism was assigned according to X4 variantfrequency: R5, ,0.3% X4; minority X4, 0.3%–19.9% X4; and X4, ≥20% X4. An R5–X4 switch was defined as achange from R5/minority X4 in plasma/proviral genomes at T0 to X4 in provirus at T1.Results: At baseline, mean FPR and %X4 of viral RNA were positively correlated with those of proviral DNA. Aftertherapy, proviral DNA load significantly decreased in Group I; mean FPR of proviral quasispecies significantlydecreased and %X4 increased in Group II. An R5–X4 switch was observed in five patients (two in Group I andthree in Group II), all harbouring minority X4 variants at T0.Conclusions:UDPS analysis revealsthat the tropism switch isnot an ‘on–off’ phenomenon, but may result fromaprofound re-shaping of viral quasispecies, even under suppressive cART. However, episodes of virological failureseem to prevent reduction of proviral DNA and to accelerate viral evolution, as suggested by decreased FPR andincreased %X4 at T1 in Group II patients.Keywords: cART, HIV quasispecies, co-receptor usage, ultra-deep pyrosequencing, tropism switch