CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

453

Type I Interferons Drive Sustained Dominance of CCR5-Tropic Variants During HIV Infection In Vivo Priya Pal , Sara Nicholson, Hongbo Gao, Liang Shan Washington University in St Louis, St Louis, MO, USA Background: A curious phenomenon is observed in people living with HIV (PLWH) who are not on antiretroviral therapy. Despite there being no difference in viral fitness between CXCR4(X4)-tropic and CCR5(R5)-tropic HIV viruses in vitro, R5-tropic viruses typically dominate in the early clinical stages. While R5 tropic viruses are selected during transmission events, it is surprising that X4 tropic virus takes years to emerge, if at all. CXCR4, the co-receptor for X4 tropic viruses, is expressed by the vast majority of CD4+ T cells while CCR5 is expressed by ~15% of total CD4+ T cells. In addition, it only takes a few amino acid changes to switch tropism. Even under maraviroc treatment, a CCR5 antagonist, X4 tropic viruses rarely emerge. Instead, resistant viruses arise that can bind to the maraviroc bound CCR5, which requires generation of multiple simultaneous mutations. These studies suggest that X4 viruses are stably suppressed by the immune system in vivo beyond transmission. We aimed to understand the molecular mechanism of X4-specific immune control in vivo. Methods: In vitro models are not suited to explore the mechanism of R5 dominance. To better understand tropism specific immune control of HIV, an interferon-α/β receptor knockout (IFNAR-KO) human immune system was reconstituted in mice. Briefly, human CD34+ cells were transduced with Cas9 protein and guiding RNAs targeting the IFNAR coding gene or scrambled controls by electroporation before injection into 1-3 day old pups. Nine weeks post engraftment, these mice were infected with X4- or R5-tropic HIV isolates. Results: In humanized mice engrafted with unmodified CD34+ cells, X4- and R5-tropic viruses replicate equally well when present alone. In contrast, when mice were infected with both viruses, there was a near complete suppression of X4 viral replication and sustained dominance of R5 viral replication, recapitulating what is observed in patients. This restriction of X4 viral replication was lost in mice with an IFNAR-KO immune system. High levels of type 1 IFN production was observed in mice challenged with both X4- and R5- tropic viruses, which resulted in selective suppression of X4-tropic viruses. These results suggest that emergence of X4-tropic viruses post viral transmission is likely blocked by type 1 IFNs. Conclusion: Our work reveals that X4 tropic virus is selectively restricted by type I interferon (IFN) beyond the transmission event. We will explore how this restriction is lost in the later clinical stages. Crosstalk Between TLR-8 and RLR Enhanced Antiviral Immunity Against Acute HIV-1 and PWH Killian Vlaming 1 , John L. van Hamme 1 , Pien M. van Paassen 1 , Tanja M. Kaptein 1 , Karel A. van Dort 1 , Peter Reiss 1 , Annelies Verbon 2 , Casper Rokx 2 , Monique Nijhuis 3 , Jan M. Prins 1 , Neeltje Kootstra 1 , Godelieve J. de Bree 1 , Teunis B. Geijtenbeek 1 1 Academic Medical Center, Amsterdam, Netherlands, 2 Erasmus University Medical Center, Rotterdam, Netherlands, 3 University Medical Center Utrecht, Utrecht, Netherlands Background: Achieving an HIV-1 cure requires both reactivation of the viral reservoir and antiviral immunity. In HIV negative individuals we showed that crosstalk between TLR8 and RLR enhances antiviral immunity by increasing production of IL12, IL27 and type I IFNs, promoting Th1 and Tfh differentiation and boosting CD8+ cytotoxicity. Targeting TLR8 and RLR could elicit antiviral immunity required in a HIV-1 cure strategy. We investigated whether this crosstalk persists in the context of HIV-1, in acute infected, treated, individuals as well as individuals who started ART in the chronic phase. Methods: Four distinct participant groups were included: age matched HIV-negative (n=28), HIV-positive individuals treated in the chronic phase of infection (n=28) (ART initiated at CD4 <300), and individuals that initiated ART during the acute phase of infection (24-weeks (n=17) post-treatment initiation and three years (n=11) post-treatment initiation). PBMCs were stimulated with TLR8 and RLR agonists alone or in combination for 24 hours. Immune responses; cytokine levels, type I IFN responses, and co-stimulatory molecules, were assessed. Results: TLR8 agonist GS9688 induced pro-inflammatory cytokines IL6 and IL12. Poly(I:C)-lyovec, a RLR agonist, induced IL27 in PBMCs in all 4 groups. In HIV-negative individuals combination of the TLR8 agonist and RLR agonist resulted in a two-fold increase of IL12 and IL27, while IL6 secretion was decreased compared to single-stimulation. In the acutely treated individuals (24 weeks, 3 year post treatment initiation) crosstalk was largely preserved when

innate immune sensing and persistent type I IFN responses in macrophages. However, the signaling pathways downstream of HIV-1 icRNA sensing have not been fully elucidated. In this study, we show that IRF5 mediates type I IFN responses downstream of icRNA sensing in HIV-1 infected macrophages, and that persistent IRF5 activation in older PWH may contribute to inflammaging. Methods: THP-1/PMA macrophages or monocyte-derived macrophages (MDMs) with selective abrogation of MAVS signalosome members, IRF3, IRF5, IRF7, TRAF6, and IKK-β were infected with a single cycle HIV-1 reporter. MDMs were also differentiated from CD14+monocytes derived from a primary patient cohort, stratified by age into younger (<35 yo) and older (>50 yo) donors. Cells were harvested at 2-3 days post infection and analyzed for infection establishment by flow cytometry, RT-qPCR for ISG expression, while cell-free supernatants were harvested for type I IFN and IP-10 production by bioassay and ELISA, respectively. Infected macrophages were also fixed on coverslips for determining nuclear-cytoplasmic IRF5 localization using immuno-fluorescence microscopy. Results: Though initiation of MAVS signaling can activate transcription factors, IRF3, IRF5 and IRF7, disruption of IRF5 expression had the highest impact on HIV-1 icRNA-induced type I IFN and IP-10 expression. Furthermore, attenuation of TRAF6 (necessary for IRF5 ubiquitination and nuclear localization) expression, but not IKK-β, abrogated IP-10 production in macrophages. Interestingly, expression and nuclear localization of IRF5 was constitutively upregulated in older MDMs which translated to higher IFN-β and IP-10 expression, though infection establishment and HIV-1 icRNA expression was similar in MDMs from both younger and older donors. Conclusion: Collectively, these results elucidate the mechanism of icRNA sensing in HIV-1 infected macrophages and offer insight into potential targets for therapeutics to alleviate chronic immune activation in PWH. Expression of Unspliced HIV-1 RNA Induces NLRP1 Inflammasome Activation in Myeloid Cells Ivy Hughes , Sallieu Jalloh, Andrés Quiñones, Hisashi Akiyama, Rahm Gummuluru Boston University, Boston, MA, USA Background: Despite the success of combination antiretroviral therapy (ART), chronic HIV-1 infection in long-lived tissue-resident cells such as macrophages and CNS-resident microglia and associated chronic inflammation remain a major barrier to successful HIV-1 management strategies. Inflammasome activation is a critical checkpoint in innate immune responses and HIV-1 has been proposed to activate multiple inflammasome sensors, including NLRP3 and CARD8 (activated by HIV-1 protease). Recent work in our laboratory has shown that expression of HIV-1 intron-containing RNAs (icRNA) is sufficient to activate innate immune responses. As ART does not affect viral RNA expression in long-lived infected cells, persistent expression of HIV-1 icRNA thus presents a novel mechanism for inflammasome activation and chronic inflammation observed in people living with HIV (PWH). Methods: Primary human monocyte-derived macrophages (MDMs, differentiated from CD14+ monocytes) or phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages were infected with HIV-1. Supernatants from infected cells were collected and assayed by ELISA for IL-1β. Transfected siRNAs were used to knock down putative sensors in MDMs, while lentiviral constructs were used to knock down or overexpress putative sensors in THP-1/PMA macrophages. Results: We found that inflammasome activation in MDMs was dependent on the nuclear export of HIV-1 icRNA, similar to the activation of type I interferon. Surprisingly, unlike induction of type I IFN responses, MAVS-signaling was not required for inflammasome activation and IL-1β secretion. IL-1β secretion was dependent on caspase-1 and the inflammasome sensor NLRP1, but unlike HIV-1-mediated activation of CARD8 inflammasome, not dependent on viral protease or enzymatic activity. Furthermore, knockdown of NLRP1 did not affect the activation of type I IFN responses in MDMs, suggesting cytosolic icRNA expression engages divergent innate immune sensing pathways. Finally, HIV-1 infection also induced inflammasome activation and IL-1β secretion in NLRP1 overexpressing THP1/PMA macrophages, confirming that NLRP1 senses HIV-1 icRNA expression. Conclusion: Persistent expression of HIV-1 icRNA in long-lived infected cells may contribute to persistent immune activation in PWH via NLRP1 activation. Future studies in our laboratory will address the mechanism of HIV-mediated NLRP1 activation.

Poster Abstracts

452

454

CROI 2024 115