Lack of detectable HIV-1 molecular evolution during suppressive antiretroviral therapy

Abstract

A better understanding of changes in HIV-1 population genetics with combination antiretroviral therapy (cART) is critical for designing eradication strategies. We therefore analyzed HIV-1 genetic variation and divergence in patients' plasma before cART, during suppression on cART, and after viral rebound. Single-genome sequences of plasma HIV-1 RNA were obtained from HIV-1 infected patients prior to cART (N = 14), during suppression on cART (N = 14) and/or after viral rebound following interruption of cART (N = 5). Intra-patient population diversity was measured by average pairwise difference (APD). Population structure was assessed by phylogenetic analyses and a test for panmixia. Measurements of intra-population diversity revealed no significant loss of overall genetic variation in patients treated for up to 15 years with cART. A test for panmixia, however, showed significant changes in population structure in 2/10 patients after short-term cART (<1 year) and in 7/10 patients after long-term cART (1-15 years). The changes consisted of diverse sets of viral variants prior to cART shifting to populations containing one or more genetically uniform subpopulations during cART. Despite these significant changes in population structure, rebound virus after long-term cART had little divergence from pretherapy virus, implicating long-lived cells infected before cART as the source for rebound virus. The appearance of genetically uniform virus populations and the lack of divergence after prolonged cART and cART interruption provide strong evidence that HIV-1 persists in long-lived cells infected before cART was initiated, that some of these infected cells may be capable of proliferation, and that on-going cycles of viral replication are not evident.

Figure 1. Measurements of HIV-1 diversity calculated as APD before, during and/or after cART in all patients in (A) Group 1 - short-term cART (B) Group 2 - long-term cART (C) Group 3 - cART with treatment interruptions and (D) the average of all groups.

Duration of treatment is shown in years in parentheses above the bar with the diversity measurement. Overall, HIV-1 plasma diversity did not change with initiation of therapy.

Figure 2. HIV-1 plasma RNA copy numbers and diversity as calculated by APD in longitudinal samples prior to and during cART in selected patients on (A) short-term cART (Group 1) (B) long-term cART (Group 2) and (C) cART with treatment interruptions (Group 3).

We found no relationship between HIV-1 RNA copy number and viral diversity in the plasma.

Figure 3. Measurements of the probability of panmixia before and after cART in (A) Group 1 - short-term cART (B) Group 2 - long-term cART (C) Group 3 - cART with treatment interruptions.

We considered a panmixia p value of <0.001 to be statistically significant according to the original publication of the method (1). Significance reveals a shift in population over time on cART and was found primarily in patients on long-term cART.

Figure 4. Neighbor-joining trees of single-genome plasma sequences from select samples from (A) Group 1 - short-term cART with no significant divergence of virus population from pre-therapy virus (B) Group 1 - short-term cART with significant divergence of virus during suppression on cART (C) Group 2 – long-term cART and (D) Group 3 – long-term cART, resuppression after a brief treatment interruption.

Phylogenetic analyses reveal populations of identical plasma sequences after long-term cART suggesting virus release from a long-lived proliferating cell population.

Figure 5. Neighbor-joining trees of single-genome plasma sequences from rebound viremia in patients in Group 3.

Phylogenetic analyses reveal populations of identical plasma sequences in rebound viremia after interrupting long-term cART.

Figure 6. Evolutionary distances of each single-genome sequence from pre-cART and during and after cART compared to the consensus subtype B HIV-1 sequence and plotted over (A) short-term cART (B) long-term cART (C) long-term cART with brief treatment interruptions and (D) rebound viremia.

Positive slopes indicate the emergence of new variants and on-going replication during cART. Only one patient (PID 1) showed a positive slope and evidence of molecular evolution during cART.