Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Monkeypox virus (MPXV): induction and sensitivity to Interferon antiviral effects

Introduction Monkeypox (mpox) is a zoonotic viral disease caused by the monkeypox virus (MPXV), which belongs to the Orthopoxvirus genus. This genus also includes the variola virus (VARV), which causes smallpox. In May 2022, an unprecedented multi-country outbreak of MPXV clade II primarily affected men who have sex with men (MSM), though not exclusively. Despite decades of human infection, the interaction between MPXV and innate immunity remains poorly understood. Interferons (IFNs) represent the first line of host antiviral defense. Members of the Poxviridae family have demonstrated the capacity to counteract type I IFNs (IFN-I, e.g. IFN-α and IFN-β) and IFN-γ. In light of these considerations, the objective of this PhD thesis was to analyze the expression of type I and II IFNs in clinical samples obtained from various anatomical regions of MPXV-infected patients, as well as the kinetics of type I IFN induction and the activation of the downstream IFN pathway following MPXV infection in different cellular models that are susceptible to MPXV infection. Furthermore, to better characterize the interplay between MPXV infection and the IFN response, the sensitivity of MPXV to treatment with different IFN-I/II preparations was evaluated. Material and methods Skin lesions (SL, n=9), anal canal brushing (ACB, n=5), nasopharyngeal swabs (NPS, n=4) and peripheral blood mononuclear cells (PBMC, n=3) were collected from male patients infected with MPXV. MPXV DNA was quantified in SL, ACB and NPS using the Bioperfectus Monkeypox Virus Real-Time PCR Kit (BioPerfectus Technologies Co., Jiangsu, China). PBMCs were obtained from sex- and age-matched healthy donors (HDs). The induction of IFN-I and the expression of IFN-related genes (STAT1, STAT2, ISG15, ISG56, IDO and PKR) were analyzed in the IFN-competent, MPXV-permissive adenocarcinoma epithelial cell line (A549). The sensitivity of MPXV to IFN-I/II was evaluated in A549 cells, as well as in Henrietta Lacks (HeLa) cells and African green monkey kidney epithelial (VeroE6) cells, which are known to be susceptible to MPXV infection in vitro. IFN-I induction was evaluated through the stimulation of A549 cells with UV-inactivated vesicular stomatitis virus (VSV) of the Indiana strain, which is known for its ability to induce IFN. IFN preparations were also tested against well-established IFN-sensitive viruses: VSV and encephalomyocarditis virus (EMCV). All MPXV experiments were conducted in the Biosafety Level 3 facility at INMI “Lazzaro Spallanzani” in Rome using isolate hMPXV/Italy/UN-INMI-Pt2/2022 (GenBank accession number ON745215.1). MPXV growth kinetics in A549 cells was determined using real-time PCR and gene expression analysis using RT-real-time PCR. Statistical analyses were performed using IBM SPSS Statistics 27.0.1 and graphs were created using GraphPad Prism 8.0.1. Results The expression of IFN-α, IFN-β, IFN-ω, and IFN-γ was analyzed in different clinical samples taken from male patients infected with MPXV. The expression of IFNs was compared between PBMC from MPXV patients and healthy donors (HD), while the results for other anatomical districts are descriptive. IFN-I were expressed at higher levels in PBMC from MPXV patients than in HD, while IFN-γ expression was comparable between the two groups. Levels of IFN-I mRNA varied depending on the anatomical district, with higher levels observed in the ACB and SL [p<0.05 (IFN-α: NPS vs SL; NPS vs ACB; IFN-β: NPS vs SL; ACB vs PBMC; IFN-ω: NPS vs SL; NPS vs ACB; SL vs PBMC), and p<0.01 (IFN-β: SL vs PBMC). Furthermore, IFN-γ expression was higher in SL (*p<0.05, ACB vs PBMC; ***p<0.001, NPS vs SL; SL vs ACB). Furthermore, levels of IFN-I transcripts were inversely related to MPXV DNA cycle threshold (Ct) levels. Conversely, there was a trend towards higher IFN-γ expression in samples with a higher MPXV-DNA Ct value, suggesting a potential anti-MPXV activity. In vitro, MPXV infection induces delayed expression of IFN-I between 48 and 72 hours, compared to UV-inactivated VSV. Furthermore, their expression was not associated with decreased MPXV replication. We then investigated MPXV sensitivity to the antiviral effects of various IFN preparations, including the IFN-α2b subtype, the natural IFN-αN1 preparation (comprising all IFN-α subtypes), IFN-ω, IFN-β, and IFN-γ. Our results showed that MPXV was resistant to both IFN-α preparations and IFN-ω, and displayed moderate sensitivity to IFN-β. The strongest efficacy was obtained with IFN-γ, with a reduction in viral titer observed at lower concentrations compared to IFN-I To further understand the interaction between MPXV and the IFN pathway, we analyzed the expression of selected genes in the downstream IFN pathway that are known to be involved in controlling and causing poxvirus infection. These genes include STAT1, STAT2, ISG15, ISG56, IDO and PKR. We examined their expression in MPXV-infected cells, in cells treated with IFN and in cells infected with MPXV and treated with IFN. As expected, all the IFN preparations induced significant expression of all the examined genes. Conversely, MPXV infection did not result in a significant increase in gene expression. Furthermore, treatment of MPXV-infected cells with IFNs did not induce high levels of expression of the analyzed genes, with only a few exceptions: ISG15 for IFN-I, and high induction of IDO for IFN-γ. Conclusions In conclusion, these results suggest that MPXV can counteract IFN production and interfere with downstream signaling pathways. MPXV also displays resistance to the antiviral effects of IFN-I, but not IFN-γ. The protective role of IFN-γ is further supported by its inverse correlation with MPXV DNA levels in clinical samples, as well as its ability to induce IDO expression in MPXV-infected cells in vitro. These findings shed light on the unique role of IFN-γ in host defense against MPXV and suggest its potential as a therapeutic agent for managing MPXV infection.