CROI 2025 Abstract eBook

Abstract eBook

Oral Abstracts

138

Lenacapavir Directs Specific Proteasome-Mediated Degradation of Gag Proteins in HIV-Infected Cells Clayton Faua 1 , Serena Bernacchi 2 , Amélia Aeschelmann 2 , Axel Ursenbach 1 , Christine Cheneau 1 , Baptiste Hoellinger 1 , David Rey 1 , François Danion 1 , Christiane Moog 3 , Samira Fafi-Kremer 1 , Matteo Negroni 2 , Pierre Gantner 1 1 University Hospital of Strasbourg, Strasbourg, France, 2 Strasbourg University, Strasbourg, France, 3 French National Institute of Health and Medical Research (Inserm), Paris, France Background: Due to its high binding affinity to HIV capsid (p24), the first-in class capsid inhibitor Lenacapavir (LEN) might display a post-integration effect on capsid production. However, this effect and its underlying mechanism, as well as its clinical impact on LEN-treated individuals, have yet to be characterized. Methods: The post-integration effect of increasing concentrations of LEN was measured in vitro (8E5, ACH2, U1, transfected 293T cells) and ex vivo (PBMCs from n=12 viremic HIV-infected participants enrolled in the APRIL study [NCT05752318]) by analysing HIV transcripts and proteins expression by flow cytometry and p24 and HIV RNA levels in supernatants. Results: LEN induces a rapid dose-dependent decrease of intracellular p24 detection at therapeutic concentrations in HIV-1 infected cell lines (reaching top plateau within 15 minutes exposure of 50nM LEN), whereas HIV-2 infected cell line was less sensitive. The latently-infected cell line also displayed a p24 decrease on LEN independently of the activation pathway targeted. The intracellular matrix (p17), but not the envelope (Env), detection decreased with the same magnitude as p24, suggesting that LEN targets the Gag polyprotein (Pr55). These decreases further translated in lower levels of viral particle release in supernatants on LEN. Intracellular HIV Gag and Nef transcripts levels were unchanged, and maturation inhibition did not affect the LEN-induced p24 decrease, suggesting a post-transcription but pre-maturation effect. The p24 detection in oligomerization mutants (W184D+M185D, P38E+A42E+E45R) and LEN-resistant mutants (N74D and Q67H+N74D) was less affected by LEN, suggesting that LEN binding requires specific capsid oligomers and residues. Proteasome inhibition by bortezomib partially constrained the LEN-induced p24 decrease, suggesting a proteasome-mediated p24 degradation. LEN also impaired p24 detection ex vivo in productively-infected CD4+ T cells from viremic HIV-infected individuals (decreased intracellular p24 [but not envelope] detection associated with decreased viral particle release). Conclusions: Altogether, LEN appears to be causing a rapid and specific proteasome-mediated degradation of not only the capsid but also other gag proteins in vitro and ex vivo , which echoes in virions production. Reduced Gag proteins expression by infected cells in LEN-treated individuals could impact recognition of these cells by the immune system and thus the dynamics of HIV reservoirs and should be further studied. RhCMV Expands CCR5 Memory CD4 T Cells and Increases Acute-Phase Seeding of SIV DNA in the Gut Mucosa Chrysostomos Perdios 1 , Naveen Suresh Babu 2 , Celeste Coleman 1 , Anna T. Brown 1 , Matilda Mostrom 1 , Carolina Allers 1 , Lara Doyle-Meyers 1 , Christine Fennessey 3 , Amitinder Kaur 1 , Brandon Keele 3 , Michael L. Freeman 4 , Joseph C. Mudd 2 1 Tulane National Primate Research Center, Covington, LA, USA, 2 Tulane University, Metairie, LA, USA, 3 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 4 Case Western Reserve University, Cleveland, OH, USA Background: Cytomegalovirus (CMV) is a prevalent beta-herpesvirus that persists asymptomatically in immunocompetent hosts. More than 90% of people with HIV are co-infected with CMV. During long-term ART, HIV-1/CMV-coinfection is associated with inflammatory-related co-morbidities and persistence of the HIV-1 reservoir, yet the precise degree to which CMV contributes to these pathologies remains unclear. Methods: We utilized specific pathogen-free rhesus macaques (RMs) that are seronegative for rhesus CMV (RhCMV) throughout life. Adult RhCMV- RMs (n=8) were age and gender matched to animals that naturally acquired RhCMV in early life (n=10) through co-housing. Animals were infected intravenously with 5000IU SIVmac239M and after 28 days received daily suppressive ART for 2 years. Plasma RNA and cell-associated SIV DNA were quantitated by PCR, and immunophenotype by flow cytometry was assessed in blood, lymph node (LN), and gut tissues at baseline and acute SIV (14dpi). Results: Overall, natural coinfection with RhCMV led to a more severe SIV burden. RhCMV+ RMs had higher peak viremia (p=0.016), and near log-fold increases in seeding of SIV DNA at 14dpi in the colon and duodenum (p<0.0001, p=0.0008, respectively; Fig. A). RhCMV+SIV+ RMs took significantly longer to fully suppress viremia following ART initiation (p=0.031). At 14dpi,

RhCMV+SIV+ animals exhibited higher frequencies of activated (CD169+) monocytes in blood (p=0.003) and HLA-DR+CD4+ T-cells in LNs (p=0.033). Prior to SIV, RhCMV+ RMs had higher percentages of CCR5+CD4+ memory T-cells in the blood (p=0.012), LN (p=0.0014), and colon (p=0.01), suggesting that RhCMV systemically increases SIV target cells (Fig. B). At baseline, target cell frequencies in LNs, but not other anatomical sites, were associated with higher peak viremia (p=0.0016, R=0.673). Notably, in RhCMV+ RMs, most CCR5+CD4+ T-cells did not respond to an RhCMV lysate ex vivo (p=0.002), suggesting target cell expansion by antigen-independent mechanisms. We also observed that HIV-CMV+ human donors (n=6) exhibited higher CCR5 expression on terminal effector memory CD4+ T-cells when compared to those of HIV-CMV- donors (n=6; p=0.041). Conclusions: RhCMV exacerbates SIV burden, and this is associated with body-wide expansion of CCR5+ target cells, most of which do not react to RhCMV peptides. RhCMV may promote seeding of the SIV reservoir in a diverse array of CD4+ clonotypes. Targeting CMV may be therapeutic during periods of active reservoir seeding. The figure, table, or graphic for this abstract has been removed. Differential Replication, Innate Immune Control, and Virulence of Global and Endemic Clade II MPXV Rebecca P. Sumner 1 , Lucy Eke 1 , Bruno Hernaez 2 , Alasdair Hood 1 , Telma Sancheira Freitas 1 , Hannah Ashby 1 , Ailish Ellis 1 , Marine Petit 1 , Sian Lant 1 , Preetam Parija 1 , Bryan Charleston 3 , Geoffrey Smith 4 , David Ulaeto 5 , Antonio Alcami 2 , Carlos Maluquer de Motes 1 1 University Surrey, Guildford, UK, 2 Centro de Biología Molecular Severo Ochoa, Madrid, Spain, 3 The Pirbright Institute, Pirbright, UK, 4 University of Oxford, Oxford, UK, 5 Defence Science and Technology Laboratory, Salisbury, UK Background: Monkeypox virus (MPXV) is a zoonotic virus endemic to Africa that has recently spread globally, becoming the first human orthopoxvirus (OPXV) since smallpox eradication. The MPXV global outbreak strain (Clade IIb, lineage B) derives from Clade IIb lineage A strains that circulate and remain endemic to Africa with limited human-to-human transmission. Clinically, lineage A-infected individuals exhibit more severe disease including substantial viral dissemination (>100 lesions per individual) relative to lineage B patients (<10 localised anogenital lesions). Lineages A and B differ by 51 nucleotides, most of which are compatible with host APOBEC3-mediated mutation. The consequences of these mutations to the biology of the virus remain unclear. Methods: Here we used comparative virology to determine the phenotypic properties of MPXV lineage B relative to the closest ancestral lineage A and related OPXV. Results: We found that both global and endemic MPXV replicated to the same extent in multiple cell types, but the global lineage B virus produced lower extracellular virus titres and showed defects in long-range spread. Both viruses had extensive capacity to suppress human innate antiviral responses, with a major role for the viral cGAMP nuclease (poxin). Consistent with this, RNAseq in infected fibroblasts and macrophages showed no significant induction of interferon (IFN) or antiviral-related KEGG pathways with either MPXV strain. Both viruses however induced strong inflammatory cytokine responses that have also been observed in patients and, interestingly, were absent with other OPXV. Importantly, comparative transcriptomics revealed defects in innate immune control by global MPXV, including IFNβ induction, increased innate immune signalling gene expression and enhanced MAP kinase activation. Furthermore, global MPXV had reduced capacity to suppress interleukin-1β-mediated immune responses. Consistent with the above, the global strain had reduced virulence in a mouse model of MPXV infection relative to the ancestral endemic strain. Conclusions: Our findings reveal phenotypic differences between these two clade IIb viruses and demonstrate attenuation of globally circulating MPXV concomitant with human transmission. Our data may help explain clinical observations that lineage B is associated with milder disease. Finally, our work reveals that the APOBEC3-like mutational footprint is not only a signature of MPXV transmission amongst humans but also drives phenotypic changes.

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CROI 2025