SARS-CoV-2 3C-like protease (3CL^pro or M^pro) cleaves the SARS-CoV-2 polyprotein and >300 intracellular host proteins to enhance viral replication. By lytic cell death following gasdermin (GSDM) pore formation in cell membranes, antiviral pyroptosis decreases 3CL^pro expression and viral replication. Unexpectedly, 3CL^pro and nucleocapsid proteins undergo unconventional secretion from infected cells via caspase-activated GSDMD/E pores in the absence of cell lysis. Bronchoalveolar lavage fluid of wild-type SARS-CoV-2-infected mice contains 3CL^pro, which decreases in Gsdmd^-/-Gsdme^-/- mice. We identify new 3CL^pro cut-sites in GSDMD at LQ²⁹↓³⁰SS, which blocks pore formation by 3CL^pro cleavage at LH²⁷⁰↓N lying adjacent to the caspase activation site (NFLTD²⁷⁵↓G). Cleavage inactivation of GSDMD prevents excessive pore formation, thus countering antiviral pyroptosis and increasing 3CL^pro secretion. Extracellular 3CL^pro retains activity in serum, dampens platelet activation and aggregation, and inactivates antiviral interferon-λ1. Thus, in countering gasdermin pore formation and pyroptosis in SARS-CoV-2 infection, 3CL^pro is secreted with extracellular pathological sequelae.

Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.

The Pfizer-BioNTech coronavirus vaccine (BNT162b2) was among the first nanoparticle-based vaccines approved by the World Health Organisation (WHO) and demonstrated 95% efficacy against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Despite its success, the precise immune mechanism behind its effectiveness remains poorly understood. This study investigated the early immune responses occurring in the draining lymph node (dLN) following vaccination. A well-established murine vaccination model was used to investigate the distribution and immunological effects of BNT162b2 in vivo. Vaccine trafficking to the dLN, cellular uptake, and spike protein expression were assessed following immunisation. Single-cell transcriptomic analyses and functional in vivo experiments were performed to identify the immune cell populations involved in orchestrating vaccine-induced responses and to characterise their interactions with adaptive immune cells. BNT162b2 was rapidly transported to the dLN, where it was predominantly captured by leukocytes that subsequently expressed the SARS-CoV-2 spike protein. Among these cells, plasmacytoid dendritic cells (pDCs) emerged as regulators involved in both inflammatory and humoural immune responses. Single-cell transcriptomic profiling revealed active interactions between pDCs and CD8⁺ T cells. Functional in vivo studies further demonstrated that pDCs promoted CD8⁺ T-cell activation and expansion, indicating an involvement in shaping adaptive immunity following vaccination. These findings identify pDCs as antigen-presenting cells involved in coordinating the immune response to BNT162b2. By regulating both innate and adaptive immune pathways and facilitating CD8⁺ T-cell activation, pDCs appear to play a functional role in the efficacy of this mRNA vaccine. This study provides additional insights into the immunological processes underlying mRNA vaccine-induced protection and may inform the future design and optimisation of mRNA-based immunotherapies and vaccines. Swiss National Science Foundation, Leonardo Foundation.

Histopathology and immunohistochemistry (IHC) are central to COVID-19 tissue evaluation. However, conventional manual scoring is limited by its semiquantitative nature and potential observer variability. In this retrospective study of experimentally infected mice, we implemented a semi-automated digital pathology workflow using commercially available software to quantitatively assess SARS-CoV-2 nucleocapsid (N) immunolabeling in lung (n = 135) and brain (n = 67) tissues. The performance of digital quantification was evaluated against conventional manual scoring, and its association with virological and pathological parameters was assessed across different stages of disease progression. Digital IHC quantification demonstrated strong agreement with manual scoring in both lung [R = 0.94, p < 0.0001, concordance correlation coefficient (CCC) = 0.95] and brain [R = 0.98, p < 0.0001; CCC = 0.977], demonstrating concordance between the two approaches. In addition, digital SARS-CoV-2 N immunolabeling quantification showed significant positive correlations with viral RNA levels, infectious viral titers, and histopathological scores, supporting its association with independent measures of SARS-CoV-2 infection and lesions. Although computational image analysis requires additional infrastructure, technical expertise, and increased analysis time, it provides an objective continuous quantitative measure of SARS-CoV-2 N immunolabeling that complements conventional manual assessment and may reduce observer-dependent variability.

Histopathology and immunohistochemistry (IHC) are central to COVID-19 tissue evaluation. However, conventional manual scoring is limited by certain subjectivity and its semiquantitative nature. In this retrospective study of experimentally infected mice, we implemented a deep learning-based digital pathology workflow using com-mercially available software to quantitatively assess SARS-CoV-2 antigen burden in lung (n=135) and brain (n=67) tissues. The performance of digital quantification was evaluated against conventional manual scoring, and its biological relevance was as-sessed by correlation with established virological and pathological parameters across different stages of disease progression. Digital IHC quantification demonstrated near-perfect agreement with manual scoring in both lung [R=0.94, p< 0.0001, concord-ance correlation coefficient (CCC)=0.969] and brain (R=0.98, p< 0.0001; CCC=0.98) in-dicating high reproducibility and accuracy. In addition, digital antigen quantification showed significant positive correlations with viral RNA levels, infectious viral titers, and histopathological scores, indicating that it provides biologically meaningful readout of SARS-CoV2 infection. Although computational image analysis requires ad-ditional infrastructure, technical expertise, and increased analysis time, these invest-ments provide a more objective and reproducible alternative to the traditional manual gold standard while generating quantitative data that enable a more precise assess-ment of SARS-CoV-2–associated disease.