CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

functions, and are potentially useful in HIV eradication. Understanding how different ICs kill cells will guide their clinical application.

HIV-infected cells to cell death. Retinoids are derivatives of Vitamin A that target retinoic receptors causing antiproliferative and proapoptotic activity. Several are FDA-approved or in clinical trials for different etiologies. The aim of this study was to evaluate the ability of retinol, 3 of its natural metabolites and 9 synthetic derivatives to sensitize HIV infected CD4 T cells to NK cell killing. Methods: Naïve CD4 T cells isolated from PBMCs from male and female donors were activated, expanded and then infected with the replication competent HIV molecular clone NL43. CD4 T cells were cocultured overnight with autologous NK cells in the presence of 1μM of a retinoid and antiretroviral therapy, with or without 100ng/mL of IL15. We used three retinoids: alitretinoin, tazarotene acid and AM80 to assess their ability to enhance Antibody-Dependent Cell Cytotoxicity (ADCC) by using the same procedure with the broadly neutralizing antibody N6 (1μg/mL). To assess the mechanisms by which these retinoids enhance NK killing of HIV-infected cells, we measured changes of MHC Class I receptor expression on CD4 T cells and cytotoxicity markers on NK cells. Results: None of the retinoids were toxic alone or with IL15. Without IL15, none of the retinoids influenced NK natural cytotoxicity. With IL15, alitretinoin (n=8, p<0.001), tazarotene, tazarotene acid and AM80 (n=8, p<0.05), significantly enhanced natural cytotoxicity of HIV-infected cells compared to DMSO control. Mechanistically, these four retinoids increased NK degranulation upon target recognition in the presence of IL15 (n=8, p<0.05). In initial studies, all three retinoids enhance HLA-F expression on CD4 T cells, previously known to enhance recognition by NK cells. Furthermore, these three retinoids significantly enhanced the ability of N6 to promote ADCC compared to DMSO control regardless of IL15 by increasing CD16 expression on NK cells (n=8, p<0.0001). Conclusion: We identified three retinoids capable of enhancing HIV-infected cells to NK natural cytotoxicity and ADCC. We are conducting experiments to confirm the mechanism/s associated with this phenotype. Our studies may provide further evidence of small molecules that could be used clinically to reduce persistent reservoirs. Comparison of Anti-HIV ADC and Immunotoxin Shows Clinically Relevant Differences in Cytotoxic Effect Seth H Pincus 1 , Tami Peters 1 , Megan Stackhouse 1 , Kelli Ober 1 , Frances Cole 1 , Hans-Peter Kiem 2 , Robert Harrington 3 , Xinyi Wang 2 , Anne-Sophie Kuhlmann 2 , Valérie Copié 1 1 Montana State University, Bozeman, Montana, 2 Fred Hutchinson Cancer Center, Seattle, WA, USA, 3 University of Washington, Seattle, WA, USA Background: The persistent reservoir of cells carrying a functional provirus is the barrier to HIV eradication. One approach to reservoir depletion is sequential activation of latent virus, followed by purging of HIV-expressing cells. initially it was hoped that once cells were activated, viral cytopathic effect or host immune responses would clear the reservoir, but recent studies using mAbs have shown greater effects. We have armed antibodies with cytotoxic agents to enhance killing of HIV-infected cells, independent of ADCC or complement activity. We screened >200 mAbs to identify those most effective at delivering the toxic moiety, and found that the anti-gp41 mAb 7B2, conjugated to ricin A chain, is safe and effective in SHIV-infected macaques. However, it was immunogenic, limiting its utility. We therefore sought to develop less immunogenic immunotoxins (ITs) and antibody drug conjugates (ADCs). Methods: We screened drugs for cytotoxicity on resting lymphocytes . ADCs were prepared by chemical conjugation to 7B2. Cytotoxicity of ADCs was compared to ITs on Env-expressing cells. Antiviral effects were measured in tissue culture. The effect of ADCs and ITs on metabolism and transcription in HIV-infected cells was studied by NMR-based metabolomics and RNAseq. Results: PNU-159682, an anthracycline, killed resting cells. We compared 7B2-PNU (ADC) to 7B2-ricin A (IT) in vitro. Cytotoxicity of the IT was 10X greater than the ADC on Env+ target cells, whereas neither IT nor ADC killed Env- cells. The ADC required >24 hr to initiate cytotoxicity; IT killing began within 6 hr. The ADC elicited bystander killing of ENV- cells when mixed with Env+ cells, the IT did not. At 6 hours, metabolomic analyses of IT-treated cells, but not ADC treated or untreated cells, demonstrated elevated amino acids and choline and depressed taurine, creatine, and fumarate. At 24 hr the metabolic profiles of all three groups diverged. Similarly, the transcriptome of IT-treated cells diverged at 6 hr, while at 24 hours, the three transcriptional profiles were distinct. Pathway analysis indicated significant up-regulation of transcriptional control and death pathways in both IT and ADC treated cells Conclusion: Cytotoxic immunoconjugates (ICs) targeted by anti-gp160 mAbs are potent killers of cells expressing HIV Env, work independently of Fc-effector

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Type I IFN Signaling and Regulation in Vesatolimod-Treated Virally Suppressed Adults With HIV-1 Susie S Huang , Liao Zhang , Donovan Verrill , Christiaan R. de Vries , Elena Vendrame, Devi SenGupta , Jeff J. Wallin , Yanhui Cai Gilead Sciences, Inc, Foster City, CA,USA Background: Vesatolimod (VES) is a well tolerated and selective toll-like receptor-7 agonist under development as part of an HIV cure regimen. VES treatment increases interferon-stimulated gene (ISG) expression and immune cell activation in people with HIV (PWH); additionally, VES monotherapy promotes a modest delay in HIV rebound in HIV controllers, and VES pharmacodynamic response is higher with 8 mg compared with 10-12 mg doses in PWH treated during chronic infection. We used mRNA-Seq to further investigate the immune mechanism and regulation of type I interferon (IFN) signaling pathways in response to VES in a phase 1b, double-blind, placebo controlled trial (NCT02858401; GS-US-382-1450). Methods: The study enrolled virally suppressed PWH (age ≥18 years, plasma HIV-1 RNA <50 copies/mL) and randomized 6:2 to receive VES or placebo biweekly for 10 doses. Whole blood mRNA collected pre-dose and 24 hours after doses 1 and 10 from participants who received VES 6 mg, 8 mg, or 10-12 mg (n=24) was used for mRNA-Seq (Illumina Stranded mRNA Prep; NovaSeq 6000). A linear mixed-effects and placebo-adjusted model was used to test for differentially expressed genes (DEGs) associated with type I IFN regulation and regulators of pattern recognition receptors (FDR<0.1). Results: VES consistently upregulated DEGs associated with type I IFN signaling after doses 1 and 10 in all 3 doses evaluated (Figure). After dose 1, the highest number of DEGs, including those associated with activation and inhibition, was observed with VES 8 mg (38 genes), followed by VES 6 mg (21 genes) and VES 10/12 mg (15 genes); type I IFN activation was most pronounced with VES 8 mg versus the other doses. After dose 10, VES 6 mg induced the highest type I IFN activation and numbers of genes (49), followed by VES 8 mg (31) and VES 10/12 mg (27), respectively. Also, a lower induction in type I IFN activation was observed by post dose 10 versus post dose 1 in all but the 6 mg dose group, with the greatest reduction in the 8 mg group. VES-mediated type I IFN activation was greatest after dose 1 at 8 mg, followed by dose 10 at 6 mg. Conclusion: Whole blood transcriptome analysis identified pronounced differences in gene expression based on VES dose and administration order. The results suggest that a cumulative effect of multiple VES doses could modulate VES pharmacodynamic response, which should be taken into account in future combination studies with VES.

Poster Abstracts

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