Background:  Nucleotide mixtures detected in HIV-1 population sequences are a reflection of sequence diversity. The frequency of such ambiguous nucleotides has been shown to correlate with length of HIV-1 infection. Here, we evaluate changes in ambiguous nucleotide frequency during an analytic treatment interruption (ATI) and in comparison to a pre-ART time point representative of chronic untreated infection.

Methods:  Plasma samples were available from patients undergoing a 16-week ATI in the A5197 study, a randomized, placebo-controlled trial of an HIV-1 gag therapeutic vaccine. Pre-ART plasma was available for 19 participants. Both gag and RT coding sequences were amplified using gene-specific primers. Population sequences were analyzed using Sequencher and nucleotide mixtures were detected by the presence of multiple fluorescent peaks at one position on the chromatogram. Changes in ambiguous nucleotide frequencies over time were analyzed using the Wilcoxon matched-pairs signed rank test. A multiple linear regression model was used to derive independent correlates of ambiguous nucleotide frequency.

Results:  HIV-1 gag and RT were successfully sequenced at the time of initial detectable viremia (ATI weeks 2 to 8) for 104 participants and at ATI week 16 for 102 participants. For both gag and RT, the frequency of ambiguous nucleotides was significantly higher at ATI week 16 than at the first detectable viremia (median 1.20% vs 0.46% for gag, p <0.001; 0.96% vs 0.67% for RT, p = 0.02). For the subset of participants with available pre-ART samples (N = 19), the frequency of ambiguous nucleotides in HIV-1 gag was significantly higher at ATI week 16 than pre-ART (0.86% vs 0.33% for gag, p = 0.007; 0.75% vs 0.58% for RT, p = 0.21). There was no significant difference in the frequency of ambiguous nucleotides in HIV-1 gag at ATI week 16 between those receiving vaccine and placebo (1.13% vs 1.20%, p = 0.98). ATI week 16 viral load, but not treatment arm or the number of CD4⁺ HIV-1 gag-specific interferon-γ-producing cells, was significantly correlated with the frequency of ambiguous nucleotides in gag at ATI week 16.

Conclusions:  The frequency of ambiguous nucleotides within HIV-1 gag and RT increased over the first 16 weeks of treatment interruption. Increased ambiguous nucleotide frequency during the ATI, as compared to chronic infection, may represent greater viral diversity and thus a higher barrier to success for a therapeutic vaccine as compared to a preventive vaccine.