CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

quantifiable baseline VL and ≥1 follow up timepoint (SNG001 n=75, 180 timepoints; placebo n = 61, 150 timepoints). There was lower amino acid diversity in the inhaled interferon recipients (SNG001 vs placebo, median APD 7.03x10 -5 vs 10.40x10 -5 , p=0.056). Individuals receiving SNG001 had significantly lower numbers of emerging mutations than the placebo group for the ORF1a (SNG001 vs placebo, median 1.51x10 -4 vs 4.54x10 -4 mutations per Kb, p=0.04), ORF3a (1.21x10 -3 vs 2.42x10 -3 per Kb, p=0.03), and Spike (5.23x10 -4 vs 11.77x10 -4 per Kb, p=0.04) genes. No specific emerging SARS-CoV-2 amino acid changes were enriched in the SNG001 recipients. Conclusions: Treatment with inhaled SNG001 was associated with lower viral sequence diversity and less frequent emerging amino acid polymorphisms in SARS-CoV-2 isolated from nasal swabs. These results provide some of the first in vivo data on the virologic effects of exogenous interferon exposure in COVID-19 and provide intriguing evidence that interferon may prevent SARS-CoV-2 diversification and escape. HIV-1 Env Exhibits Distinct Conformations and Neutralization Profiles in CSF and Blood Madeline M. Broghammer, Aaron N. Gillman, Cassian K. Birler, Rohith R. Vujjini, Samuel A. McCarthy-Potter, Hillel Haim University of Iowa, Iowa City, IA, USA Background: HIV-1 replicates in different body compartments and sub-compartments characterized by distinct compositions of small solutes and proteins. It is often assumed that virus proteins maintain the same conformation, function and sensitivity to inhibitors in different molecular environments, in vivo and in vitro . The protein albumin shows remarkable variability in concentration between the blood and brain compartments – 35-50 mg/mL in blood versus 0.2 mg/mL in cerebrospinal fluid (CSF) and interstitial fluid of the brain. Given such differences, and the promiscuous interactions of albumin with other molecules, we examined the effects of this protein on conformation of Env and HIV-1 sensitivity to antibodies. Methods: Pseudoviruses and replicative HIV-1 that contain the Env of strain AD8 were produced and tested for their sensitivity to antibodies in whole CSF and serum from HIV-negative donors. To determine the isolated effects of albumin, viruses were also tested in media containing CSF- or serum-like levels of this protein, and in albumin-supplemented CSF. Antibodies that target the major neutralizing and non-neutralizing epitopes of gp120 and gp41 were tested. Conformation of Env was determined by binding of the antibodies to the membrane-bound form of Env expressed on HOS cells. Structural stability of Env was analyzed in a ligand-independent manner by virus sensitivity to cold inactivation in the different fluids. Results: The interaction between HIV-1 and specific antibodies was markedly affected by the nature of the fluid. In low-albumin fluids, including CSF, Env exhibited a unique open-at-the-base conformation, with increased exposure of epitopes at the gp120-gp41 interface and membrane-proximal external region. Epitopes in the gp120 ectodomain were unaffected by composition of the fluids. Neutralization assays of HIV-1 in the different synthetic and whole biological fluids confirmed that albumin reduces exposure of epitopes at the trimer base. Interestingly, sensitivity of HIV-1 to cold inactivation was increased in the high-albumin fluids, indicating a more closed but structurally unstable form of this protein. Conclusions: These studies suggest that Env assumes unique conformations in different body compartments. Such changes result in distinct exposure profiles of epitopes that are considered as targets for therapeutics and vaccine development. These understandings may contribute to tailoring therapeutics to the conformational properties of HIV-1 proteins in each compartment. Modifying PF74 Improves Anti-HIV-1 Activity Against the Resistance Associated Capsid Mutation N74D William M. McFadden 1 , Karen A. Kirby 1 , Lei Wang 2 , Haijuan Du 1 , Zachary C. Lorson 1 , Huanchun Zhang 1 , Andres Emanuelli Castaner 1 , Savannah Brancato 1 , Atsuko Hachiya 3 , Shreya M. Ravichandran 1 , Carolyn M. Highland 4 , Philip R. Tedbury 1 , Robert A. Dick 1 , Zhengqiang Wang 2 , Stefan G. Sarafianos 1 1 Emory University, Atlanta, GA, USA, 2 University of Minnesota, Minneapolis, MN, USA, 3 National Hospital Organization Nagoya Medical Center, Nagoya, Japan, 4 Cornell University, Ithaca, NY, USA Background: Lenacapavir (LEN) is an FDA-approved drug for highly treatment experienced individuals living with multidrug-resistant HIV-1 infection. LEN inhibits HIV-1 replication with sub-nanomolar potency by targeting the capsid protein (CA) at the “FG-binding pocket” (FGBP). Clinical trials report the

emergence of CA mutations like M66I and N74D after LEN treatment. N74D is also selected by PF74, the first reported antiviral to bind the FGBP with sub micromolar potency. PF74 contains three aromatic moieties, R1, R2, and R3; our structure-based studies have developed improved compounds with unique chemistry at R1 and R3. Methods: The EC 50 s of FGBP-targeting compounds ZW-1260, ZW-1261, ZW-1514, ZW-1517, and ZW-1559 were determined against N74D NL4-3 HIV-1 in TZM-bl cells and compared to reported wild-type (WT) potency. X-ray crystallography solved novel structures of FGBP compounds complexed with a native WT CA (PDB: 9DTM) or with only the N74D mutation (PDB: 9DTN, 9DTO) and these were compared to reported WT or N74D structures (PDB: 4XFZ, 7M9F, 7MKC). Thermal shift assays, analyzed with TSAR , determined the melting temperature (T m ) and biolayer interferometry determined binding kinetics to disulfide-stabilized CA hexamers (CA HEX ). Transmission electron microscopy and in vitro assembly assays determine the compounds’ effects on WT or N74D CA lattice. Results: While N74D virus caused 6-fold PF74 resistance, modifications at R1 and R3 improved inhibition (Table 1). Compounds with a para -Cl on R1 and/or N-ethyl on R3 decreased N74D resistance; the R1 p -Cl of ZW-1261 interacts with N74 while R3 N-ethyl of ZW-1514 interacts with Q67. Additional interactions between modified R1 and/or R3 with CA HEX increase the T m for both WT and N74D CA HEX , more so than PF74. CA rapidly polymerized in vitro , even in the absence of salt, after the addition of compounds with R3 C5-OH modifications, likely due to a H-bonding network involving the modified indole and the adjacent CA monomer of CA HEX . Conclusions: Understanding the interactions responsible for increased potency and decreased resistance is essential for improved drug design. We report modifying R1 and R3 of PF74 as distinct strategies to overcome N74D resistance. One modification, R3 C5-OH, forms intermolecular interactions within and between CA subunits, which, unlike PF74, enables rapid lattice assembly in vitro . Overall, these data provide a structure-guided approach to improve antiviral activity and the resistance profile of FGBP-targeting compounds.

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Viral Escape From Broadly Neutralizing Antibodies in Non-Subtype B Viruses Teresa Murphy 1 , Dennis Copertino Jr 2 , Gabriel Galeotos 1 , Neha Shah 3 , Julie Ake 4 , Ajay P. Parikh 4 , Abdulwasiu Tiamiyu 5 , Zahra Parker 6 , Hannah Kibuuka 7 , Valentine Sing’Oei 8 , John Owuoth 8 , Jonah Maswai 9 , Rebecca M. Lynch 1 , for the African Cohort Study (AFRICOS) Study Group 1 George Washington University, Washington, DC, USA, 2 Weill Cornell Medicine, New York, NY, USA, 3 Walter Reed Army Institute of Research, Silver Spring, MD, USA, 4 United States Military HIV Research Program, Bethesda, MD, USA, 5 Walter Reed Program–Nigeria, Abuja, Nigeria, 6 Walter Reed Project– Lagos, Lagos, Nigeria, 7 Walter Reed Program–Makerere University, Kampala, Uganda, 8 Walter Reed Project–Kisumu, Kisumu, Kenya, 9 Walter Reed Project–Eldoret, Eldoret, Kenya Background: Broadly neutralizing antibodies (bNAbs) provide a useful tool for HIV cure strategies because of their ability to target conserved regions on the envelope (Env) protein in the context of both virions and infected cells. Multiple clinical trials of single and combination bNAb therapies in people living with HIV demonstrated transient viral suppression after infusion. The ease of viral escape, however, is a major obstacle to durable suppression by bNAbs. We are investigating common escape pathways from bNAbs to inform future bNAb combination trials. Methods: We designed an in vitro viral escape assay to test bNAb and Env combinations in a high throughput manner. From PBMC, we isolated single non-subtype B viruses from Nigerian, Ugandan and Kenyan participants in the African Cohort Study (AFRICOS). Each virus stock was sequenced, subtyped and characterized for sensitivity to 9 bNAbs. Virus E01-0085.V2 was a subtype

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CROI 2025 210