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

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Abstract
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.
Funding Information
  • Gilead Sciences

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