Evidence of HLA-mediated immune response driving the maintenance of CTL-escape variants in patients with undetectable viral load

Evidence of HLA-mediated immune response driving the maintenance of CTL-escape variants in patients with undetectable viral load.

Dario A Dilernia, L Lourtau, L Jones, S Rodriguez, C Bautista, M Gomez-Carrillo, M Losso, and H Salomon.

Immune response drives the selection of CTL-escape mutations during the course of HIV infection. After initiation of HAART, antiviral drugs exert a stronger selective force leading to a higher limitation of viral replication. Our objective was to evaluate the effectiveness of immune response as a selective force in a context of extremely reduced viral population size.

Gag gene was sequenced and HLA-A and B genotyped over 108 samples from drug-naïve HIV-1 positive individuals. Associations between HLA alleles and viral polymorphisms were assessed by logistic regression. Multiple comparison corrections were addressed by the BH method (q-values). Phylogeny correction was performed by a Bayesian Markov-Chain Monte-Carlo method. Analysis of site-specific synonymous and non-synonymous substitution rate was assessed through the codon-based ML IFEL method. For four patients with viral load < 50 copies/ml (identified according to their HLA profile), RNA extraction was performed from 7 ml of plasma through an ultracentrifugation-based method and gag gene amplified through a Hi-Fi PCR. 20-40 clones were sequenced in samples obtained previously and during HAART.

We found that HLA-B57 and A03 were the most efficient alleles in forcing CTL-escape, targeting mainly the previously characterized epitopes TSTLQEQIGW(p=0.0002) and RLRPGGKKK(p<10E-7), respectively. Sites under significant positive selection (p<0.05) during HAART were position 20 (within A03-restricted RLRPGGKK) for patient A02A3B35B39, position 385 (within A3-restricted RGNFRNQRK) for patient A3A31B7B45 and position 84 (within A2-restricted SLYNTVATL) for patient A2A3B39B57. Epitopes sequences of RLRPGGKKK and TSTLQEQIGWF were in the escape state for patients harboring the selective alleles.

In spite of the low viral diversity achieved during HAART, we detected sites under positive selection. Our results show that during successful HAART, targeted CTL-epitopes are still forced to evolve adaptively, suggesting that CTL-mediated immune response would be able to keep driving the evolution of HIV variants even in viral population with a remarkable low replication rate.