Longitudinal Analysis of HIV-2 Proviral DNA Reveals Archived Protease Inhibitor Resistance and Reservoir Evolution over Eight Years

Protease inhibitors (PIs) remain important components of HIV-2 treatment, but resistance genotyping is frequently challenging in individuals with low or undetectable plasma viremia. Proviral DNA sequencing may provide access to archived viral variants and improve understanding of long-term resistance and clinical evolution. In this retrospective longitudinal study, 27 individuals with HIV-2, both ART-experienced and ART-naïve, followed at a hospital in Lisbon, were analyzed. The HIV-2 protease gene was amplified from peripheral blood mononuclear cell-derived proviral DNA, cloned, and sequenced (Sanger sequencing) at baseline and, for ART-treated participants, after eight years of follow-up. Resistance profiles were interpreted using the Stanford HIVdb, HIV-2EU, and Rega algorithms. Clinical data, including ART history, CD4 counts, and plasma viral load, were collected longitudinally. Amino acid diversity was assessed using Shannon entropy, and longitudinal CD4 dynamics were evaluated using mixed-effects models with time-varying ART exposure. Sensitivity analyses were performed using generalized estimating equations (GEE). A total of 222 clonal HIV-2 protease sequences clustered within group A. Major PI resistance mutations were detected in 21.4% of ART-experienced and 23.1% of ART-naïve individuals at baseline. Longitudinal resistance trajectories varied across participants, including persistence, apparent emergence, and non-detection of previously identified mutations. Mixed-effects modeling revealed substantial inter-individual variability in CD4 trajectories, with no statistically significant associations observed between CD4 evolution and ART status, time, or their interaction. GEE analyses yielded consistent results, supporting robustness across modeling frameworks. Entropy analysis identified localized sequence diversity changes restricted to a small number of protease residues, with positions 60 and 75 differing between groups at baseline and position 21 showing longitudinal variation among treated participants. This study demonstrates that proviral DNA sequencing captures archived HIV-2 protease diversity and reveals persistent and dynamic resistance patterns within the viral reservoir. While no population-level association between ART exposure and CD4 trajectory was observed, marked inter-individual variability highlights the complexity of longitudinal immune recovery in HIV-2 infection. These findings support the value of proviral sequencing as a complementary research tool for characterizing long-term viral evolution in settings where plasma-based genotyping is limited.