Exploiting innate immune markers and 2D and 3D cell-based in vitro systems to emulate the early blood signature associated with protection in anti-COVID-19 immunized subjects.

Protective responses raised by vaccination result from the coordinated action of immune cells in multiple locations across the body. In this context, system vaccinology studies provide an integrated picture of vaccine-driven molecular networks that are associated with later adaptive vaccine-protective responses. Blood modules, elicited early after vaccination, belong to innate immunity and include pro-inflammatory cytokines, interferon (IFN)-stimulated genes and transcripts associated with metabolic alteration critical for antiviral responses, antigen presentation and B cell activation. In particular, the cytokines IL-15, IL-6, TNF-α, IFN-γ, the chemokines IP-10 and MCP-1 and the IFN-inducible genes MX1 and IRF1 were induced early in subjects that get vaccinated with BNT162b2 mRNA COVID-19 vaccine and were found to correlate with vaccine-specific humoral protective responses (Severa M 2023, Severa M 2024). In the attempt to reproduce this scenario in immune-relevant in vitro models, here we simulated the mRNA vaccine intramuscular administration in a heterologous system composed of vaccine-injected 3D muscle-like tissue (3D-MT) and peripheral blood mononuclear cells (PBMC), representing the immune compartment. Moreover, to dissect stroma contribution in shaping early immune response to BNT162b2 vaccine, an in vitro system consisting of vaccine exposed human primary fibroblasts and PBMC was also used. Vaccine-injection in the 3D-MT stimulates the recruitment and phenotypical modulation of major antigen presenting cells, particularly monocytes and macrophages while factors from stromal compartment mainly account for dendritic cell expansion and activation. In these two heterologous systems, PBMC express the early innate immune module previously identified exvivo in sera of subjects that get vaccinated with BNT162b2 vaccine (Severa M 2023, Severa M 2024). In particular, tissue resident muscle cells prompt PBMC to generate an immunostimulatory soluble milieu, while stromal compartment equipes PBMC to express innate immune mediators belonging to IFN signature. Our results suggest that cell-based systems composed of PBMC, stromal and tissue specific cells can be exploited to recapitulate early blood signatures associated with protection during anti-COVID-19 immunization. These in vitro models might represent also valuable tools for the validation of in silico system vaccinology findings aimed at identifying panels of early regulated innate immune biomarkers predictive of vaccination outcome.