CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

420

Molecular Mechanisms Underlying Anti-HIV-1 Fc-Effector Functions of Human IgA Allelic Variants Marek K. Korzeniowski 1 , William D. Tolbert 1 , Haleigh Conley 2 , Cordelia Manickam 2 , Souradip Dasgupta 3 , Maurice Bukenya 2 , Andrew Hudson 2 , Robin Flinko 3 , Monika Chandravanshi 1 , George K. Lewis 3 , Georgia D. Tomaras 2 , Krishanu Ray 3 , R. Keith Reeves 2 , Justin Pollara 2 , Marzena Pazgier 1 1 Uniformed Services University of the Health Sciences, Bethesda, MD, USA, 2 Duke University School of Medicine, Durham, NC, USA, 3 University of Maryland, Baltimore, MD, USA Background: IgA is the most abundant antibody in humans, yet its function in viral infections, including HIV-1, is elusive. Humans have two IgA subclasses: IgA1 (with 1 allelic variant) and IgA2 (with 3 allelic variants), differing in sequence, hinge length, and glycosylation, and expressed as monomers (mIgA), dimers (dIgA), or secretory forms (sIgA). Neutralization at mucosal surfaces is believed to be the primary anti-viral mechanism of IgA. Yet, while all IgA forms have an Fc region, only mIgA is known to bind FcαRI (CD89) and mediate Antibody-Dependent Cellular Phagocytosis (ADCP) and Antibody-Dependent Cellular Cytotoxicity (ADCC). dIgA and sIgA are believed to lack significant Fc effector function due to steric hindrance of accessible CD89 binding sites. Here we aim to provide a comprehensive structure-anti-HIV-1 Fc-function analysis of all known variants of human IgA. Methods: mIgA, dIgA and sIgA forms of IgA1 and IgA2 were generated using the variable regions of anti-HIV-1 IgG1 antibodies: the ADCC-potent A32 and the broadly neutralizing N49P9.6. CD89 binding was evaluated by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS). CD89 mediated signaling through Syk and ERK kinases phosphorylation was analyzed using multiplex Luminex assay. ADCP of virions and ADCC of gp120-coated cells were measured using neutrophils or PBMCs as effector cells, respectively. Structures of the Fc-FcαRI complexes were solved using X-ray crystallography. Results: We engineered and expressed all human IgA subclasses and allelic variants, along with their oligomeric forms using the variable regions of A32 and N49P9.6 IgG1. All IgA variants bound to FcαRI, with dissociation constants in the low uM range (2-5 x10 -7 M). All mIgA variants were capable of signaling through CD89 and mediating Fc-dependent effector functions, including ADCP and ADCC, with IgA1 showing slightly enhanced potency. Notably, we also observed evidence of ADCC activity with dimeric IgA2(m1) and ADCP activity for dIgA2(mn). Crystal structures of the Fc-FcαRI complex for mIgA1 and mIgA2(m1) revealed slight differences in the formation of the binding interfaces. Conclusions: Monomeric IgA is not the only form of human IgA capable of binding to and signaling through the FcαRI (CD89) receptor. Our findings indicate that oligomeric forms of IgA can also interact with CD89 and mediate Fc-effector activities, providing new insights into biological roles of IgA, the most abundant Ab isotype in the human immune system. Sensitivity of HIV-1 CRF01_AE Envelopes to Broadly Neutralizing Antibodies VRC07-523 and PGDM1400 Gabriel Smith 1 , Sebastian Molnar 1 , Michelle Zemil 1 , Suteeraporn Pinyakorn 2 , Indie Showell-De Leon 1 , Diana Wasson 1 , Carlo Sacdalan 3 , Nittaya Phanuphak 4 , Sandhya Vasan 1 , Julie Ake 2 , Lydie Trautmann 1 , Morgane Rolland 2 , Victoria Polonis 2 , Shelly Krebs 2 1 Henry M Jackson Foundation, Bethesda, MD, USA, 2 US Military HIV Research Program, Bethesda, MD, USA, 3 SEARCH, Bangkok, Thailand, 4 Institute of HIV Research and Innovation, Bangkok, Thailand Background: Understanding the sensitivity of viral envelopes to broadly neutralizing antibodies (bNAbs) encoded within the reservoir is of major importance in evaluating efficacy in analytical treatment interruption (ATI) trials using bNAbs as an intervention in people living with HIV (PLWH). BNAbs have been shown to impact viral load setpoint during interruption of antiretroviral therapy (ART) for viruses sensitive to bNAb neutralization. In this study, we assessed the sensitivity of viral envelopes (Envs) sequenced from RV254 participants who started ART during acute infection for sensitivity to the bNAbs VRC07-523LS and PGDM1400LS. VRC07-523LS and PGDM1400LS target two separate sites on the HIV Env, the CD4 binding site and the V2 apex, respectively, increasing the likelihood of sensitivity of these viral Envs when they are combined together. Methods: Following sequencing and cloning of 119 representative Envs from 116 RV254 participants, pseudoviruses (pSVs) harboring functional Envs were produced via transfection. Env sensitivity was measured in neutralization assays in which several concentrations of VRC07-523LS alone, PGDM1400LS alone, and a combination of VRC07-523LS and PGDM1400LS were tested against uniform volumes of each candidate-derived pSV. IC80s and IC50s of each mAb were

determined and compared between the single mAbs and the combination of PGDM1400LS and VRC07-523LS. IC50s or IC80 <1mg/mL were considered sensitive to bNAb neutralization. Results: Of the 119 Envs, 59% were sensitive to PGDM1400LS (potency=0.116μg/mL), 77% were sensitive to VRC07-523LS (0.324 μg/mL), and 87% were sensitive to the combination using IC50 <1mg/mL (Fig. 1a). When assessing IC80, 47% were sensitive to PGDM1400 (potency=0.202 μg/mL), 49% were sensitive to VRC07-523LS (0.483 μg/mL), and 63% were sensitive to the combination (Fig. 1b). The majority of Envs (85%) tested were CFR01_AE. Conclusions: A key component evaluating efficacy in ATI trials using bNAbs is the sensitivity of the glycoprotein envelope to antibody neutralization. Combining VRC07-523LS and PGDM1400LS bNAbs provided increased coverage across viral Envs. Furthermore, PGDM1400LS demonstrated more potent neutralization across the screened candidates and required lower concentrations to achieve a higher level of viral inhibition in comparison to VRC07-523LS. These data provide evidence that CRF01_AE Envs sequenced from viral Env are sensitive to VRC07-523LS and PGDM1400LS and antibodies may be used as candidate bNAbs in future ATI trials.

Poster Abstracts

422

Transcriptional Analysis of Intestinal Immune Cells in People With HIV Receiving bNAbs Connie A. Zhao 1 , Divya Jha 1 , Katrina Millard 2 , Darwin D'souza 1 , Vladimir Roudko 1 , Francesca Cossarini 1 , Marina Caskey 2 , Saurabh Mehandru 1 1 Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2 The Rockefeller University, New York, NY, USA Background: Much of the HIV reservoir resides in tissues such as the gut associated lymphoid tissue (GALT). Broadly neutralizing antibodies (bNAbs) are under investigation for their potential to aid elimination of latently infected cells; however, little is known about their effect in key tissues such as the GALT. Here, we aim to study the effects of bNAbs on intestinal immune cells. Methods: Study participants with suppressed HIV infection on antiretroviral treatment (ART) receiving a single infusion of two long-acting bNAbs (3BNC117 LS and 10-1074-LS) were prospectively enrolled and underwent colonoscopy at baseline and at 6-8 weeks post-infusion. Freshly processed ileal and colonic tissue biopsies were analyzed using single-cell RNA sequencing (scRNA-seq). The R-based package, Seurat, and the Enrichr platform were used for data analysis. Results: Tissue samples were obtained from five study participants at baseline. Post-infusion tissue was available for two participants, yielding an aggregated dataset of 85,899 cells with 28,504 unique molecular identifiers (UMIs). Median (range) participant age was 55 (30-63) years old. Participants had been diagnosed with HIV for 17 (5-30) years with nadir and enrolment CD4+ T cell count of 249 (193-400) and 681 (440-1,113) cells/uL, respectively. Baseline CD4+ T cells in the ileum and colon were characterized by expression of pathways involved in regulatory T lymphocyte development ( IL2 , IL2RA , CTLA4 , FOXP3 ) and tumor necrosis factor (TNF)/nuclear factor kappa B (NF-kB) signalling ( LTA , LTB , TNFSF ). Baseline CD8+ T cells were characterized by expression of pathways involved in chemokine receptor signalling ( CCL4 , CCR9 ) and lymphoid and non-lymphoid cell immunoregulatory interactions ( CD247 , ZAP70 ). Post-infusion CD4+ and CD8+ T cells showed significantly lower expression of pathways related to TNF/NF-kB and interferon signalling ( NFKBIA , TNFAIP3 , IFNG , STAT1 ).

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