Background:  A minority of children under treatment with antiretroviral therapy (ART) experience discordant responses to therapy (increase in CD4⁺ cells despite persistently detectable viremia). It is currently hypothesized that either impaired replicative capacity of viruses emerging during ART, or improved patients’ thymic function are involved in this response. Since the genetic bottleneck determined by ART can modify the distribution of env variants within the HIV-1 quasispecies, evolution of HIV-1 env was analyzed as an index of intra-host viral evolutionary potential of HIV-1, and correlated with the parameters of immune restoration.

Methods:  Eight HIV-1-infected children experiencing virological failure to ART were studied. Plasma and PBMC, collected at baseline and for a mean period of 14.5 months after ART initiation, were analyzed for HIV-1 viremia, DNA proviral burden, thymic output by measurement of TRECs,changes in CD4 and CD8 memory and naive cell subsets. Evolution of the env gene was analyzed in plasma samples collected at ART entry and after 12 to 16 months, by amplification,cloning and sequencing of gp120 C2-V5 region. Evolutionary relationships among the viral variants and positive selection for amino acid changes were evaluated by comparing baseline and follow-up sample values for each child (PAUP*, Phylip, MEGA). The co-receptor usage conferred by the prevailing V3 sequences was analyzed using a recombinant assay.

Results:  Despite persistent HIV-1 viremia, 5/8 children showed a significant increase in CD4⁺ counts, (mainly naive CD4⁺ cells). This increase was probably due to restoration of the thymic function as all 5 children also demonstrated an increase in TREC and naive CD8⁺ cells. Evolution of gp120 env gene was shown in 6/8 patients; 5 of these also showed distinct clustering of viral variants correlating with positive selection. Of note, the ratios of V3 loop to C2-V5 Ka/Ks values were higher in immunological responders than in nonresponders (p = 0.0253). Finally the impact of V3 loop evolution on viral phenotype was documented by the loss of X4-tropic variants in 2 immunological responders.

Conclusions:  The results suggest that the evolution of the V3 loop sequences in patients showing an increase in CD4⁺ cells despite detectable viremia is mainly driven by recovery of the immune function; and that the immune reconstitution is an important selective constraint forcing V3 loop evolution towards a less pathogenic viral phenotype.

Keywords: env; evolution; immune restoration