CROI 2025 Abstract eBook

Abstract eBook

Oral Abstracts

Conclusions: These findings demonstrate that mutations in multiple regions in the HIV-1 genome – Env, NC, and IN – collectively contribute to INSTI resistance. These results further our understanding of INSTI resistance and support the need for genotypic analysis outside of IN in individuals failing INSTI-containing regimens. Selection of Nucleocapsid Mutations With Virologic Failure of Tenofovir/ Lamivudine/Dolutegravir Kerri J. Penrose 1 , Yuta Hikichi 2 , Rahil Sethi 1 , B. Jay Goetz 1 , Lauren O. Siffert 1 , Sujith A. Valiyaparambil 3 , Carole L. Wallis 4 , Caitlyn McCarthy 5 , Uma R. Chandran 1 , Cissy Kityo 6 , Charles W. Flexner 7 , Michael Hughes 5 , Eric O. Freed 2 , John W. Mellors 1 , Urvi M. Parikh 1 , for the ACTG A5381 Study Team 1 University of Pittsburgh, Pittsburgh, PA, USA, 2 National Cancer Institute at Frederick, Frederick, MD, USA, 3 Frontier Science & Technology Research Foundation, Inc, Amherst, NY, USA, 4 Lancet Labs and BARC SA, Johannesburg, South Africa, 5 Harvard TH Chan School of Public Health, Boston, MA, USA, 6 Joint Clinical Research Centre, Kampala, Uganda, 7 The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Tenofovir-lamuvidine-dolutegravir (TLD) is highly effective as ART but the causes of virologic failure (VF) in individuals on TLD without integrase (IN) resistance mutations are not well understood. Off-target mutations in nucleocapsid (NC) have been selected in vitro and are associated with decreased DTG susceptibility in vitro . Samples from ACTG study A5381 were examined to assess the potential contribution of NC mutations to VF in individuals on TLD. Methods: A5381 was an observational cohort study enrolling individuals aged >10y with viral load >1000 c/ml at start of TLD as 1st, 2nd or 3rd-line ART. Paired study entry and VF samples from 57 participants with confirmed VF on ≥6 months of TLD were sequenced by whole genome Illumina MiSeq. Mutations were identified with HyDRA Web (IN) and a pipeline using reference alignment to HXB2 RefSeq (NC_001802.1) with BWA v0.7.17, LoFreq 2.1.5 and SNPeff 5.0 for putative variant calling (NC) with a mutation threshold of 20% frequency. Site directed NC and integrase (IN) mutants were phenotyped for DTG sensitivity with TZM-bl assays. Results: Mutations in NC at a frequency of ≥20% of the virus population at VF but not before TLD start were detected in 10 of 57 (17%) participants (Table). Of these 10, two participants had both NC and IN mutations at VF but not at study entry, suggesting dual selection. Participant 1 selected A30T (57% frequency) and M46I (69% frequency) in NC and the accessory mutation H51Y (9% frequency) in IN. Participant 2 selected N27I (77% frequency) in NC and the major mutation R263K (100% frequency) and polymorphism M50I (100% frequency) in IN. Laboratory-generated infectious mutant clones of NC-N27I and IN-M50I/R263K show 1.8- and 2.4-fold resistance to DTG, respectively. When they are combined, the triple mutant showed 5.8-fold resistance. IN-M50I/R263K mutants show lower infectivity than wild-type (WT), but NC-N27I did not further affect viral infectivity in the context of IN-WT and or IN-M50I/R263K. Conclusions: This work provides the first clinical evidence of selection of NC mutations with TLD failure and of decreased DTG susceptibility from the combination of NC and IN mutations. Our findings support further investigation of the potential contributions of mutations outside of IN to VF of TLD. Optimizing On-Demand Tenofovir Disoproxil Fumarate/Emtricitabine Dosing in Women for HIV Prevention Julie B. Dumond, Craig Sykes, Amanda Schauer, Angela Kashuba, Mackenzie Cottrell University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Background: Despite availability of long-acting products for HIV pre-exposure prophylaxis (PrEP), some women may prefer on-demand oral PrEP. Using an existing PK/PD model (Cottrell JID 2016), we previously predicted protective drug exposure would be sustained in the female genital tract (FGT) for 5 days after sex for >80% of the population using the 2-1-1 IPERGAY regimen (#2 tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) tablets pre-sex then #1 tablet 24 and 48 hours after), and >90% of the population regularly taking 4 tablets/week on Tuesday, Thursday, Saturday, and Sunday. Here, we sought to identify the optimal on-demand TDF/FTC regimen to achieve protective drug exposure in the FGT over 5-10 days post-sex. Methods: TDF/FTC tissue PK/PD predictions were generated via the aforementioned published in vivo/in vitro model with the EC 90 derived from an in vitro synergy model of TDF/FTC’s active metabolites vs competing endogenous nucleotides. The proportions of 1000 predicted profiles achieving EC 90 in the FGT at 5, 7, and 10 days post-sex were calculated for all simulated dosing scenarios. These were extensions of the 2-1-1 regimen and included: 2-2-1, 2-2-2, 2-1-1-1,

week of injection. Pretreatment for approximately 3-5 minutes with an ice pack substantially reduced pain. Conclusions: Following administration of IM LEN formulation 1, mean C trough after 12M was above mean SC LEN Q6M C trough , confirming the potential of Q12M dosing. Comparison with LEN Q6M Phase 3 PrEP will be conducted as PK data become available. Both formulations of Q12M LEN were safe and well tolerated. These data support future development of IM Q12M LEN for HIV PrEP, which has the potential to significantly improve PrEP uptake and persistence. Elucidating the Mechanism by Which Nucleocapsid Mutations Confer Resistance to InSTIs Yuta Hikichi 1 , Ryan C. Burdick 1 , Sean C. Patro 2 , Kerri J. Penrose 3 , Erin Clark 1 , Sherimay D. Ablan 1 , John W. Mellors 3 , Urvi M. Parikh 3 , Xiaolin Wu 4 , Vinay K. Pathak 1 , Eric O. Freed 1 1 National Cancer Institute at Frederick, Frederick, MD, USA, 2 Leidos Biomedical Research, Inc, Frederick, MD, USA, 3 University of Pittsburgh, Pittsburgh, PA, USA, 4 National Institutes of Health, Bethesda, MD, USA Background: People living with HIV (PLWH) receiving HIV integrase (IN) strand transfer inhibitors (INSTIs) have been reported to experience virological failure (VF) in the absence of resistance mutations in IN. To elucidate INSTI resistance mechanisms and pathways, we performed long-term (~ one year) passaging of several viral isolates with an escalating concentration of the INSTI dolutegravir (DTG). Independent of viral strain and cell type, HIV-1 became resistant to DTG by sequentially acquiring mutations in Env, nucleocapsid (NC), and, in some cases, IN. This study aims to elucidate the role of NC in HIV-1 resistance to INSTIs. Methods: We evaluated the impacts of the selected NC mutations on DTG sensitivity and viral infectivity. qPCR and time-of-addition (TOA) assays were conducted to examine the impacts of mutations on post-entry events in the virus replication cycle. Results: The NC mutations selected in the presence of DTG confer modest (3-5-fold) resistance to INSTIs, a level of resistance comparable to that observed with clinically relevant IN mutations. These NC mutations do not appreciably affect viral RNA packaging, viral assembly, virus release, or infectivity. qPCR and TOA analyses with RT inhibitors and INSTIs revealed that the NC mutations accelerate the kinetics of integration. Notably, the shortened time frame between reverse transcription and integration correlates with reduced sensitivity to DTG, suggesting that NC mutations limit the window of opportunity for INSTIs to bind intasomes and block integration. Examination of the sequence at 2-LTR junctions showed no significant difference, suggesting that NC mutants preserve viral DNA ends, which are important for integration and INSTI binding. During DTG selection in primary PBMCs, we identified NC-N27I and IN-S153Y, which conferred

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2.5-fold and 3.4-fold resistance to DTG, respectively. We observed that mutations in NC and IN acted in concert to confer 8.5-fold resistance to DTG, suggesting that NC mutations engage in epistatic interactions with IN resistance mutations. Significantly, we identified some of the in vitro-selected NC mutations in PLWH who experienced VF with DTG-containing regimens.

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CROI 2025