Daniele Spalletta

Background: Lenacapavir (LEN) is a first-in-class, long-acting HIV-1 capsid inhibitor, approved for the treatment of heavily treatment-experienced (HTE) people with HIV (PWH) with multidrug resistance (MDR). To date, few data are available on resistance profiles in real life. Methods: Paired plasma and PBMC samples from nine HTE PWH with MDR enrolled in the PRESTIGIO Registry receiving LEN in combination with an optimized background regimen were collected before (baseline) and during LEN treatment. Next-generation sequencing was performed using in-house protocols. LEN susceptibility was assessed by single-cycle assay. Intra-patient viral evolution was evaluated through quasispecies distance analysis. Results: At baseline, no major LEN resistance-associated mutations were detected; the accessory capsid polymorphism T107A was identified as a minority variant (5.9%) in one case, with phenotypic assays confirming full LEN susceptibility (mean fold-change: 1.2). Participants received LEN for a median [IQR] of 19.8 [18.5–23.2] months. Two individuals started LEN with undetectable viremia and maintained suppression. The other seven participants were viremic; six achieved viral suppression, with one experiencing a blip. One low-adherent participant exhibited persistent viremia with Q67K and K70H as the majority variants in both plasma (98.5%; 97.8%) and PBMCs (75.7%; 75.6%), conferring a 2081-fold reduction in LEN susceptibility. Intra-host variability in the capsid and pol regions remained low over time and across compartments. Conclusions: In this case series of HTE PWH with MDR, LEN was highly associated with maintenance or achievement of virological suppression and limited virus evolution, with only one participant remaining viremic and showing a majority highly LEN-resistant variant.

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.

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 home-made 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). Accordingly, 60 samples (41 HIV-1 RNA, 19 HIV-1 DNA) with various HIV-1 subtypes and viremia levels were tested. Molecular amplification was successful in 83.3% of cases (75.6% HIV-1 RNA and 100% HIV-1 DNA), with sequencing achieved in 92.0% of amplified samples (87.1% HIV-1 RNA and 100% HIV-1 DNA). The protocol showed high NGS performance with HIV-1 RNA samples with viremia >500 copies/mL (92.6%) and a slight impact of subtype-associated variability (subtype B: amplification and sequencing 86.0% and 91.9%; non-B: 76.5% and 92.3%, respectively). 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 for personalized HIV-1 treatment with CAI-based strategies, enabling efficient characterization of LEN resistance mutations in the CA region across different HIV-1 subtypes, using both DNA and RNA samples.