Christian Callebaut

Lenacapavir (LEN) is the first-in-class capsid inhibitor that inhibits HIV capsid function in vitro with picomolar activity. LEN has been approved for the treatment of HIV in combination with other antiretroviral agents (ARVs), as well as for the prevention of acquisition of HIV-1 in people who may benefit from pre-exposure prophylaxis (PrEP). In vitro investigations of the resistance profile of LEN have identified resistance-associated mutations (RAMs) at six residues in the HIV-1 capsid protein (CA), all found in the CA structural pocket where LEN binds and conferring LEN-resistance with various degrees of loss of susceptibility. LEN was initially evaluated in a clinical study (CAPELLA) of heavily treatment-experienced (HTE) people with HIV (PWH), in which 14 of 72 participants had emergence of in vitro-predicted LEN RAMs. Despite the presence of LEN RAMs in these participants with viral rebound, treatment with LEN led to viral suppression in a large majority of HTE PWH in CAPELLA. In treatment-naïve participants receiving subcutaneous LEN + asynchronous oral ARVs (CALIBRATE) 4 of 157 participants had emergence of LEN RAM after >2 years of study. In contrast, no cases of viral rebound with resistance to LEN have been observed in virologically suppressed PWH switching to the once-daily single-tablet regimen (STR) combining LEN with the integrase strand-transfer inhibitor (INSTI) bictegravir after up to 48 weeks in two Phase 3 studies, and for >3 years in Phase 2. Similarly, no resistance to LEN has been observed after up to 96 weeks in the Phase 2 study of once-weekly regimen of the deoxyadenosine analog RT inhibitor islatravir + LEN. Finally, in the Phase 2 study of the 6-monthly injectable LEN + 2 broadly neutralizing antibodies (bNAbs) combination, only one instance of resistance to LEN was observed in conjunction with loss of susceptibility to one of the bNAbs through 1 year of treatment. Overall, resistance to LEN in regimens using synchronous dosing (daily or weekly oral, or 6-monthly injectable) has been rare in clinical studies. As the use of LEN is predicted to increase in the near future with these potential new combinations, capabilities to test for capsid resistance are being developed through global commercial options as well as through regional health institutions.

Background: Islatravir (ISL), a nucleoside reverse transcriptase translocation inhibitor, and lenacapavir (LEN), a capsid inhibitor, have pharmacokinetic profiles supporting once-weekly (QW) oral dosing. In a Phase 2 study, QW ISL+LEN maintained a high rate (94.2%) of viral suppression (VS; HIV-1 RNA <50 copies/mL) at Week 48. We report resistance analyses through Week 48. Methods: Virologically suppressed participants received ISL (2 mg QW) plus LEN (600 mg Days 1+2, then 300 mg QW; n=52) or once-daily bictegravir/emtricitabine/tenofovir alafenamide (50/200/25 mg; n=52). Proviral DNA sequencing of HIV-1 protease, reverse transcriptase, and integrase was performed at screening; available historical genotypes were collected. Participants with primary nucleoside reverse transcriptase inhibitor (NRTI) or non-NRTI (NNRTI) resistance-associated mutations (RAMs) were excluded. Post-baseline resistance analyses were performed for participants with HIV-1 RNA ≥200 copies/mL at virologic failure (≥50 copies/mL at two consecutive visits) or ≥50 copies/mL at study drug discontinuation/last visit. Results: Of 104 enrolled participants, five randomized with historical genotypes were subsequently found to have primary NRTI and/or NNRTI RAMs; all five maintained VS, including two participants with M184V/I (one/group). One participant receiving ISL+LEN with low-level viremia on Day 1 (HIV-1 RNA 251 copies/mL) met criteria for resistance analysis; no emergent resistance was observed, and they subsequently resuppressed on ISL+LEN. Longitudinal analysis of plasma virus detected identical viruses with no evidence of viral evolution. Conclusions: ISL+LEN maintained high rates of VS, including in one participant with pre-existing M184V. No participants developed drug resistance. These findings support the ongoing evaluation of QW oral ISL+LEN for HIV-1 treatment.

Lenacapavir (LEN) is a long-acting HIV-1 capsid inhibitor that binds to the HIV-1 capsid protein with picomolar antiviral activity, disrupting its function and inhibiting viral replication. Here, we identified capsid mutations in samples from individuals treated with LEN across two clinical trials that were considered potential LEN resistance–associated mutations. The gag encoding regions of clinical isolates with capsid mutations, as well as associated site-directed mutants, were cloned into the infectious molecular clone pXXLAI and pNL4-3-JRFL-secNLuc, encoding replication-competent HIV-1. Their effects on LEN susceptibility, replication kinetics, and three-dimensional capsid structure were investigated. Phenotypic analyses of the HIV-1 clinical isolates and site-directed mutants revealed that all resistance-associated mutations decreased LEN susceptibility to various degrees but were frequently associated with substantial replication defects. Structural modeling confirmed that LEN binding in the binding pocket was altered in the presence of capsid mutations, with predicted binding affinity changes correlating with observed potency shifts. These findings provide insights into LEN-resistance mechanisms and underscore the unusually high fitness costs associated with treatment-emergent capsid mutations.

Background: Lenacapavir (LEN) is an HIV-1 capsid inhibitor being evaluated for pre-exposure prophylaxis (PrEP). The PURPOSE trials assessed the efficacy of LEN and emtricitabine/tenofovir disoproxil fumarate (F/TDF) in cisgender women (PURPOSE 1; P1) and in cisgender men, transgender women, transgender men and gender non-binary persons (PURPOSE 2; P2). Emtricitabine/tenofovir alafenamide (F/TAF) was also assessed in P1. Both studies demonstrated the superiority of LEN to F/TDF. We describe resistance analyses from P1 and P2, which provide the first data regarding emergent drug resistance in the context of LEN for PrEP. Methods: HIV testing was performed at screening, baseline, and every study visit. Participants who acquired HIV-1 with a viral load of ≥200 copies/mL were evaluated for resistance by genotyping of the HIV-1 capsid, protease, reverse transcriptase and integrase genes at HIV diagnosis. Adherence in the F/TDF and F/TAF groups was measured by dried blood spot. Results: Resistance to LEN was detected in 0 of 2134 participants (P1) and 2 of 2179 participants (P2); both developed N74D. Four participants in each study receiving LEN were found to have unrecognized HIV-1 at baseline; 4/8 participants developed N74D. In P1, 2/37 participants analyzed for resistance in the F/TAF group had M184I ± K65R. In the F/TDF groups, M184M/I/V was detected in 1/16 participants (P1) and 1/9 participants (P2). Conclusions: Acquisition of HIV while receiving LEN and resistance to LEN in the context of unrecognized HIV was rare but associated with the emergence of the N74D LEN resistance-associated substitution in this population.

The global fight against HIV/AIDS has been significantly bolstered by the development and implementation of pre-exposure prophylaxis (PrEP), yet innovation in PrEP interventions, improved adherence and greater access are still needed to maximize its benefit. Nonhuman primate (NHP) infection with simian immunodeficiency virus (SIV) has served as an instrumental animal model in advancing HIV PrEP research. This review comprehensively examines the utility of NHP models in evaluating the efficacy, pharmacokinetics, and safety of diverse PrEP strategies, including oral, injectable, implantable, and topical formulations. It discusses the development of diverse challenge models that simulate human transmission routes and the advantages of NHPs in enabling controlled and mechanistically informative studies. It also highlights the successful translation of pivotal NHP studies evaluating tenofovir-based regimens as well the long-acting agents, cabotegravir and lenacapavir, into the clinical settings, emphasizing the consistently high predictive power of the NHP models for the HIV PrEP clinical efficacy. Finally, it underscores the importance of species-specific pharmacologic considerations and the value of NHP data in informing clinical trial design. As the global community strives to end the HIV epidemic as a public health threat in the absence of an efficacious prophylactic vaccine, NHP models make a critical contribution in the development of next-generation HIV prevention tools.

Background: In the phase 3 ALLIANCE study, both bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) and dolutegravir plus emtricitabine/tenofovir disoproxil fumarate (DTG + F/TDF) achieved high rates of HIV-1 RNA suppression through week 96 in adults with HIV-1 and hepatitis B virus initiating treatment (NCT03547908). In this study, we quantify preexisting HIV-1 resistance, evaluate its effect on HIV-1 virologic suppression, and describe postbaseline HIV-1 resistance through week 96. Methods: Preexisting HIV-1 resistance was assessed by historical and/or screening genotyping. HIV-1 RNA suppression to <50 copies (c)/mL at week 96 was assessed by the preexisting resistance category. Postbaseline resistance was assessed in participants with HIV-1 RNA ≥200 c/mL through week 96. Results: Primary nucleoside/nucleotide reverse transcriptase inhibitor, nonnucleoside reverse transcriptase inhibitor, and protease inhibitor resistance substitutions were present at baseline in 4 (1.7%), 19 (7.9%), and 5 (2.1%) of 241 participants, respectively. Virologic suppression rates were high, irrespective of preexisting primary resistance substitutions, including M184I. Six participants (3 per group) had confirmed HIV-1 RNA ≥200 c/mL and did not resuppress to <50 c/mL while on study drugs; none of the 5 with postbaseline resistance data had treatment-emergent primary resistance substitutions. One participant on DTG + F/TDF with multiple virologic failures and documented nonadherence by pill count had treatment-emergent K70E and M184V/I, and subsequently resuppressed. Conclusion: In people with HIV-1 and hepatitis B virus treated with first-line B/F/TAF or DTG + F/TDF, preexisting HIV-1 resistance was uncommon and did not affect virologic suppression. No treatment-emergent HIV-1 resistance occurred with B/F/TAF, further supporting the high barrier to resistance of this regimen.

Long-acting antiretroviral agents for preexposure prophylaxis (PrEP) represent a promising new alternative to daily oral regimens for HIV prevention. Lenacapavir (LEN) is a first-in-class long-acting capsid inhibitor approved for the treatment of HIV-1 infection. Here, we assessed the efficacy of LEN for PrEP using a single high-dose simian-human immunodeficiency virus (SHIV) rectal challenge macaque model. In vitro, LEN showed potent antiviral activity against SHIV, as it did for HIV-1. In macaques, a single subcutaneous administration of LEN demonstrated dose proportional increases in and durability of drug plasma levels. A high-dose SHIV inoculum for the PrEP efficacy evaluation was identified via virus titration in untreated macaques. LEN-treated macaques were challenged with high-dose SHIV 7 weeks after drug administration, and the majority remained protected from infection, as confirmed by plasma PCR, cell-associated proviral DNA, and serology testing. Complete protection and superiority to the untreated group was observed among animals whose LEN plasma exposure exceeded its model-adjusted clinical efficacy target at the time of challenge. All infected animals had subprotective LEN concentrations and showed no emergent resistance. These data demonstrate effective SHIV prophylaxis in a stringent macaque model at clinically relevant LEN exposures and support the clinical evaluation of LEN for HIV PrEP in humans.

Background: HIV-1 pre-exposure prophylaxis (PrEP) has focused predominantly on protective efficacy in receptive sex, with limited research on the dosing requirements for insertive sex. We pre-clinically assessed the ex vivo pharmacokinetic–pharmacodynamic (PK–PD) profile of tenofovir (TFV) and tenofovir alafenamide (TAF) in foreskin tissue. Methods: Inner and outer foreskin explants were exposed to serial dilutions of TFV or TAF prior to addition of HIV-1_BaL at a high (HVT) or a low viral titer (LVT). Infection was assessed by measurement of p24 in foreskin culture supernatants. TFV, TAF and TFV–diphosphate (TFV–DP) concentrations were measured in tissues, culture supernatants and dosing and washing solutions. Results: Dose–response curves were obtained for both drugs, with greater potency observed against LVT. Inhibitory equivalency mimicking oral dosing was defined between 1 mg/mL of TFV and 15 µg/mL of TAF against HVT challenge. Concentrations of TFV–DP in foreskin explants were approximately six-fold higher after ex vivo dosing with TAF than with TFV. Statistically significant negative linear correlations were observed between explant levels of TFV or TFV–DP and p24 concentrations following HVT. Conclusions: Pre-clinical evaluation of TAF in foreskin explants revealed greater potency than TFV against penile HIV transmission. Clinical evaluation is underway to support this finding.

Tenofovir alafenamide (TAF) is a phosphonoamidate prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV). TAF is approved for the treatment of HIV-1 infection as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. When dosed once-daily, TAF results in approximately 90% lower levels of plasma TFV and a 4-fold increase in intracellular TFV-diphosphate (TFV-DP) in PBMCs compared with the TFV prodrug tenofovir disoproxil fumarate (TDF). Several antiretrovirals, including TDF, have been associated with bone mineral density decreases in patients; the effect of clinically relevant TAF concentrations on primary osteoblast viability was therefore assessed in vitro. Studies in PBMCs determined that a 2-hour TAF exposure at concentrations similar to human plasma C_max achieved intracellular TFV-DP levels comparable to those observed after the maximum recommended human dose of 25 mg TAF. Comparable intracellular TFV-DP levels were achieved in primary osteoblasts with 2-hour TAF exposure daily for 3 days at concentrations similar to those used for PBMCs (100–400 nM). No change in cell viability was observed in either primary osteoblasts or PBMCs. The mean TAF CC₅₀ in primary osteoblasts after 3 days of daily 2-hour pulses was >500 μM, which is >1033 times higher than the TAF maximum recommended human dose plasma C_max. In summary, primary osteoblasts were not preferentially loaded by TAF compared with PBMCs, with comparable TFV-DP levels achieved in both cell types. Furthermore, there was no impact on osteoblast cell viability at clinically relevant TAF concentrations.