CROI 2025 Abstract eBook

Abstract eBook

Oral Abstracts

141

Nef Modulates Actin to Prevent Post-Integration Sensing of HIV-1 Alexandre Laliberté 1 , Caterina Prelli Bozzo 2 , Dhiraj Acharya 3 , Maximilian Hirschenberger 1 , Meta Volcic 1 , Oliver T. Fackler 4 , Michaela Gack 3 , Konstantin M. J. Sparrer 1 , Frank Kirchhoff 1 1 Ulm University Medical Center, Ulm, Germany, 2 Yale University, New Haven, CT, USA, 3 Cleveland Clinic Florida Research and Innovation Center, Port St. Lucie, FL, USA, 4 Heidelberg University, Heidelberg, Germany Background: HIV-1 replication is associated with the generation of various nucleic acid products that may act as ligands for cytosolic innate immune sensors including RIG-like receptors (RLRs). However, to induce an effective innate immune response, RLRs need to be primed by actin disturbances, sensed by R12C (Acharya et al., Cell 2022). While mounting evidence suggests that HIV-1 is sensed by RLRs, whether HIV-1 is capable of antagonizing this sensing mechanism is currently unclear. Methods: Using wild-type and mutant clade B and C HIV-1 constructs, we quantified the impact of Nef-dependent actin remodeling on viral replication and the induction of antiviral and pro-inflammatory responses in macrophages, dendritic cells and CD4+T cells. The impact of immune sensing on viral replication was further examined by blocking IFN signaling. To define the key sensors and underlying mechanisms, we knocked-out MAVS in human primary macrophages (MDMs) and treated cells with HIV-1 inhibitors. Results: We found that a single R191A or F191A mutation in the C-terminus of Nef, which specifically prevents Pak2-mediated phosphorylation of the actin modulator cofilin, doubled the level of MX1, CXCL10 and CCL5 induction in primary MDMs and dendritic cells (DCs) upon HIV-1 infection. Strikingly, infection of DCs with this mutant is also associated with an up to 10-fold increase in production of pro-inflammatory cytokines such as IP-10, IL-6 and IL-8. Using HIV inhibitors, we show that this response is dependent on integration. Furthermore, CRISPR-Cas9 KO of MAVS in primary human macrophages abrogated innate immune activation. We also support a role for the sensor MDA-5 as it is activated and ISGylated upon infection with the mutant virus. Finally, we show that Nef prevents recruitment of R12C to RLRs and thus maintains both MDA-5 and RIG-I in a phosphorylated, inactive state, similar to that of uninfected cells. Conclusions: Our results present HIV-1 Nef as the first viral counter-mechanism of actin disturbances critical for effective immune sensing. Profiling Therapeutic Vaccine-Driven HIV-Specific CD8 T Cells With Single-Cell TCR Sequencing Assays Rafael Tiburcio 1 , Lily Zemelko 1 , George Gruenhagen 1 , Alaa Abdellatif 1 , Kara Chew 2 , David B. Weiner 3 , Steven G. Deeks 1 , Gabriela K. Fragiadakis 1 , Rachel Rutishauser 1 1 University of California San Francisco, San Francisco, CA, USA, 2 University of California Los Angeles, Los Angeles, CA, USA, 3 The Wistar Institute, Philadelphia, PA, USA Background: Generating robust virus-specific CD8+ T cell responses is the major objective of most HIV therapeutic vaccines. However, standard T cell assays fail to distinguish whether vaccine-expanded T cell populations are boosted pre-existing responses and/or newly-recruited clonotypes. Here, we utilized two single cell T cell receptor (TCR) sequencing approaches to characterize CD8+ T cell clonotypes expanded in the peripheral blood of five people living with HIV two weeks after receiving a multiclade consensus HIV gag/pol ± env DNA vaccine/IL-12 regimen (n=3) versus placebo (n=2) in the PENNVAX trial (NCT03606213). Methods: First, we sequenced single paired TCR alpha and beta chains from enriched CD8+ T cells using the high-throughput Omniscope OS-T assay (per sample sequenced: median 1.3e6 [1.7e5-1.9e6] cells and 1.1e5 [6.1e4-1.6e5] unique clonotypes, defined as matching CDR3b amino acid [aa] sequence plus V/J usage). To investigate clonal dynamics between pre- and post-vaccination, expanded clones were defined as having a log2 fold change of ≥2 post- vs pre vaccination (FDR adj. p-value <0.05, Fisher’s exact test). Clusters of clonotypes with similar CDR3 beta sequences were identified using TCRdist3. Additionally, we performed 10X single cell (sc)RNA/TCR sequencing from sorted MHC class I multimer+ HIV-specific CD8+ T cells from the same participants and timepoints. Results: Among the three vaccinated individuals, we observed a significant expansion of 133 (15% newly detected), 415 (49% new), and 445 (12.5% new) CD8+ T cell clonotypes. Network graphs of the expanded clonotypes revealed hubs containing both boosted and newly detected clonotypes. The CDR3 beta aa sequence of 3.4% of boosted and 6.4% of newly detected clonotypes matched publicly available sequences reported to be HIV-specific. Expansion dynamics of clonotypes sequenced by 10X from multimer+ HIV-specific CD8+ T cells matched their expansion dynamics in the high-throughput assay.

Conclusions: By performing scTCRseq on approximately 1 million CD8+ T cells per sample, we successfully identified coordinated expansion following an HIV therapeutic vaccine of both boosted clonotypes as well as what appear to be newly expanded clonotypes with similar TCRs. Our data provide a benchmark for understanding how scTCRseq assays can be used to study vaccine-elicited HIV-specific T cells and lay the groundwork for future studies aimed at optimizing therapeutic strategies for HIV. Efficacy of Bivalent Versus Monovalent COVID-19 Vaccines Among People With HIV, Ubuntu Trial, 2022-24 Sufia Dadabhai 1 , Bo Zhang 2 , Aaron Hudson 2 , Asa Tapley 2 , Taraz Samandari 3 , Penny L. Moore 4 , Ethel Kamuti 5 , Harriet Nuwagaba-Biribonwoha 6 , Nonhlanhla N. Mkhize 4 , Margaret Yacovone 7 , Phillip Kotze 8 , Yunda Huang 2 , Nigel Garrett 9 , Glenda Gray 10 , Lawrence Corey 2 , for the CoVPN 3008 Ubuntu Study Team 1 The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA, 2 Fred Hutchinson Cancer Center, Seattle, WA, USA, 3 COVID-19 Prevention Network, Seattle, WA, USA, 4 National Institute for Communicable Diseases, Johannesburg, South Africa, 5 Center for Infectious Disease Research in Zambia, Lusaka, Zambia, 6 ICAP at Columbia University, New York, NY, USA, 7 National Institutes of Health, Bethesda, MD, USA, 8 Qhakaza Mbokodo Research Clinic, Ladysmith, South Africa, 9 Centre for the AIDS Programme of Research in South Africa, Durban, South Africa, 10 South African Medical Research Council, Cape Town, South Africa Background: Prospective trials have not previously compared the efficacy of boosting with the ancestral/monovalent mRNA vaccine (WA-1) versus a bivalent ancestral plus BA.4/BA.5-targeted vaccine in African countries, with high HIV and SARS-CoV-2 seroprevalence. Methods: The second phase of CoVPN 3008 (Ubuntu trial) was a double-blind randomized trial in 7 countries, among people with HIV (PWH) or another comorbidity linked to severe Covid-19. It activated during the BA.4/5 omicron wave, progressing through to XBB.1. At month 6 (M6), previously-vaccinated volunteers received 100 mcg of either a monovalent or bivalent boost (1:1), then monthly PCR testing until M18. We compared the risk of symptomatic Covid-19 between arms using cumulative incidences and Cox regression overall and by HIV status, SARS-CoV-2 serology (SARS+/SARS-), CD4 count and HIV viremia. We assessed neutralizing anti-Spike antibody ID50 titers against BA.4/5 and XBB.1.5 (dominant strain during the trial) before boost and 1-month after, in 100 PWH per arm. Results: From 10/2022-03/2023, 3988 participants (3086 PWH; 3491 SARS+) received the monovalent (n=1976) or bivalent (n=2012) boost and were followed until 05/2024. At M6, characteristics were balanced by arm with 68% born female and median age 37 years overall; among PWH, median CD4 count was 653 cells/mm 3 , 18% had HIV viremia and 95.8% were on antiretroviral therapy. At M12 (6 months after boosting), we observed 113 Covid-19 cases (all Omicron) including 1 non-hospitalized severe case. Incidence was 5.58 per 100 person-years, with no difference in efficacy overall (HR=1.00, 95% CI [0.69,1.45], Fig 1); by HIV status (PWH HR=0.99 [0.64,1.51]; PWoH HR=1.03 [0.50,2.14]); by prior SARS-CoV-2 infection (SARS+ HR=0.98 [0.66,1.46]; SARS- HR=1.17 [0.42,3.47]); or by CD4 count or HIV viremia (not shown). HRs were similar at M18. Geometric mean titers to BA.4/5 increased from 835 at M6 to 2544 at M7 in the monovalent arm vs. 773 to 3639 in the bivalent; and to XBB.1.5 from 135 to 559 in the monovalent arm vs. 156 to 1191 in the bivalent (Fig 1). The <2 fold higher titers among bivalent recipients were not associated with discernable clinical benefit. Severe disease was rare based on daily O 2 readings, temperatures and symptom checks following each positive PCR. Conclusions: Among PWH, despite modestly higher neutralizing titers to circulating strains of Omicron after bivalent boosting, the bivalent boost provided no added protection against symptomatic Covid-19 over the monovalent boost.

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Oral Abstracts

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CROI 2025