Development of a Next‐Generation Sequencing Protocol for Assessing Lenacapavir Resistance in HIV‐1 Capsid

Lenacapavir (LEN) is a first-in-class capsid inhibitor (CAI) that targets multiple stages of the HIV-1 lifecycle, showing efficacy in heavily treatment-experienced (HTE) individuals with multidrug-resistance (MDR) and in pre-exposure prophylaxis (PrEP). This study aimed to characterize a novel in-house next-generation sequencing (NGS) protocol targeting HIV-1 capsid (CA) region using both HIV-1 RNA from plasma and HIV-1 DNA from peripheral-blood-mononuclear-cells (PBMCs). A total of 60 samples (41 HIV-1 RNA and 19 HIV-1 DNA) with various HIV-1 subtypes and viremia levels were tested. Overall, molecular amplification was successful in 83.3% of cases (75.6% for HIV-1 RNA and 100% for HIV-1 DNA), while high quality sequences were obtained in 76.7% of samples (65.9% for HIV-1 RNA and 100% for HIV-1 DNA). Among RNA samples with viremia ≥ 500 copies/mL, sequencing success reached 92.6%, showing a statistically significant association with viral load. Subtype-specific analysis showed amplification and sequencing rates of 86.0% and 79.1% for subtype B, and 76.5% and 70.6% for non-B subtypes, with no significant difference. Reproducibility was fully confirmed by pairwise similarity analyses at 10% and 20% frequency cutoff, upon reprocessing 13 HIV-1 RNA samples. This protocol provides an important tool, primarily for subtype B, for personalized HIV-1 treatment with CAI-based strategies, enabling efficient characterization of LEN resistance mutations in the CA region, using both DNA and RNA samples.