Baseline Lenacapavir Resistance and Capsid Variability in Bulgaria: A Next-Generation Sequencing Study of Newly Diagnosed HIV-1 Infections

Lenacapavir, the first approved HIV-1 capsid inhibitor, is now being introduced for both treatment and pre-exposure prophylaxis, yet the capsid-coding region lies outside the pol fragment examined in routine genotypic resistance testing, and baseline data on capsid variability remain scarce, particularly in epidemics dominated by non-B lineages. We applied next-generation sequencing of the HIV-1 gag gene to 103 lenacapavir-naive individuals newly diagnosed in Bulgaria between 2023 and 2025. Nine lineages were identified. Subtype B accounted for 46.6% of the sequences, whereas non-B lineages together predominated (53.4%), led by sub-subtype F1 (13.6%) and CRF01_AE (8.7%); a further 10.7% remained unassigned and grouped predominantly with BF1 recombinant references. The distribution of these lineages differed significantly according to the probable route of transmission (p = 0.007). No major lenacapavir resistance-associated mutation was detected (0 of 103; 95% CI 0.0–3.6). Substitutions at position 107 were the only changes observed at a resistance-associated position: T107S in two individuals and a T107T/S mixture in one (2.9% combined). Notably, the two T107S sequences formed a CRF01_AE pair supported by a bootstrap value of 100, indicating that this accessory polymorphism can persist in closely related viruses. All viruses were genotypically predicted to remain susceptible to lenacapavir. These findings establish a molecular baseline for capsid surveillance in Bulgaria, a South-Eastern European country with a genetically diverse HIV-1 epidemic.