Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: Virus-Host Interplay in SARS-CoV-2 Infection: Gaining Insights into Innate Immune Responses Induced by Variants of Concern and Mechanisms of Immunopathogenesis Using a Human Peripheral Blood Mononuclear Cell-Based In Vitro Model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus that emerged in late 2019 and caused the COVID-19 pandemic. Due to its high mutational plasticity, SARS-CoV-2 has given rise to multiple Variants of Concern (VOC), each displaying distinct replication efficiencies and immune-modulatory properties. Disease severity is linked to the host immune response to infection; therefore, elucidating the mechanisms connecting viral sensing to innate immune activation is crucial for identifying and characterizing aberrant responses elicited by the virus. The aims of this PhD project were: (1) to investigate the innate immune response to SARS-CoV-2 VOC, Alpha, Beta, Gamma, Delta, and Omicron (including sub-Omicron lineages), using a human peripheral blood mononuclear cell (PBMC)-based in vitro model focused on the release of pro-inflammatory and antiviral cytokines, and (2) to identify variant-specific innate immune signatures through a single-cell multi-omic analysis of the innate immune compartment upon SARS-CoV-2 BA.1 stimulation. Cytokine and chemokine release was quantified by multiplex bead array in PBMC supernatants collected 24 h post-stimulation. A strong IFN-α response was detected upon exposure to D614G and Alpha variants, while Beta, Gamma, and Delta failed to induce IFN-I, suggesting the acquisition of distinct immune evasion strategies. The Omicron BA.1 variant induced robust IFN-α, IL-6, and TNF-α production, together with increased secretion of IL-8 and Rantes, whereas Beta was the weakest inducer of cytokine and chemokine release. We further extended our analysis to the post-Omicron recombinant variant XBB.1 and compared it with BA.1. BA.1 stimulation elicited higher IL-6 levels, while XBB.1 triggered a stronger IFN-α response and was uniquely capable of inducing IL-12p70 production, a cytokine not observed with any other tested variant. Flow cytometric profiling of the innate immune compartment revealed functional differences between the two variants: XBB.1 promoted plasmacytoid and conventional dendritic cell maturation toward more antiviral phenotypes and enhanced CD8⁺ T-cell activation, whereas BA.1 reduced the frequency of CD14lowCD16high inflammatory monocytes, which increased following XBB.1 exposure. To dissect these mechanisms at higher resolution, integrated single cell transcriptomic and proteomic profiling was applied to HLA-DR-enriched PBMC stimulated with Omicron BA.1. Viral exposure induced extensive remodeling of monocyte physiology, affecting differentiation state, expression of adhesion molecules, and antigen-presentation capacity. Differentially expressed genes including CD16, CD11b, CD86 and CD29, and, were validated in independent donors at both transcriptional and protein levels, confirming their functional involvement. Mapping of viral RNA across innate subsets revealed preferential accumulation in CD14⁺ and CD16⁺ monocytes. In virus positive monocytes, transcriptional programs related to stress responses, inflammation, metabolic reprogramming, and cytokine/chemokine signaling were up-regulated, whereas IFN α/β signaling and antigen-presentation pathways were suppressed. Quantitative PCR and intracellular flow cytometry for dsRNA detection demonstrated that BA.1 transiently replicates within a subset of monocytes without achieving productive infection, selectively modulating genes that influence monocyte phenotype and differentiation. Altogether, these findings indicate that SARS-CoV-2 VOC differentially manipulate host intracellular pathways to tune inflammatory and antiviral responses in innate immune cells. Single-cell multi-omic data reveal that SARS-CoV-2 specifically targets monocytes to reprogram host immunity, providing mechanistic insight into how viral evolution shapes the interplay between immune evasion and innate immune pressure.