ABSTRACT Coronaviruses and influenza A viruses (IAV) can cause severe respiratory disease and have pandemic potential. Both viruses depend on priming of their glycoproteins by host cell proteases for the acquisition of infectivity, and the responsible enzymes represent potential targets for intervention. Initial studies suggested that these viruses may exploit redundant proteolytic systems. However, research conducted over the last two decades has pointed to a key role for a single enzyme in coronavirus and IAV priming, the transmembrane protease serine 2 (TMPRSS2). Interest in TMPRSS2 as a host dependency factor and therapeutic target intensified during the COVID-19 pandemic, prompting extensive investigation into its biology, substrate specificity, and pharmacological inhibition. Here, we review recent efforts to define the role of TMPRSS2 in coronavirus infection and to target this protease for antiviral intervention.

Common marmosets (Callithrix jacchus) are valuable non-human primate (NHP) animal models in biomedical research, including infectious diseases modelling. However, for in vitro studies only a few immortalized cell lines have been generated, and additional lines are needed to comply with the 3R principles of replacement, reduction and refinement. Here, we present the generation and characterization of three cell lines derived from kidney tissue, which were immortalized by transduction of SV40 large T antigen. The cell lines display an epithelioid morphology, show differential podoplanin expression and are likely of pericyte origin, as deduced from expression profiles of marker genes obtained by RNA sequencing analysis (RNA-seq). All cell lines had a functional interferon (IFN) system, as shown by responsiveness to human IFNβ and marmoset IFNα14 and the induction of interferon-stimulated genes (ISG). Infection with retroviral pseudotypes demonstrated susceptibility to entry driven by glycoproteins from a wide range of human pathogenic viruses. Finally, these cell lines are highly permissive for Zika virus, for which marmosets are a model organism, and Herpes simplex virus 1, which causes a deadly disease in marmosets. We believe that these cell lines are a valuable resource for in vitro studies on marmosets.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a pre-pandemic coronavirus that is transmitted from camels, the natural reservoir, to humans and can cause severe disease. MERS cases have been documented in Arabia but not Africa, although the virus is circulating in both Arabian and African camels. Further, evidence has been provided that viruses in African camels might have a reduced capacity to cause disease. However, the underlying determinants are incompletely understood. Here, employing pseudotyped particles as model systems for MERS-CoV entry into cells, we compared cell entry of viruses from African and Arabian camels and its inhibition. We show that viruses found in Arabian camels and recent human cases are less susceptible to inhibition by human soluble DPP4 (sDPP4) than viruses from African camels, although both enter human cells efficiently and are comparably sensitive to inhibition by interferon-induced transmembrane (IFITM) proteins and neutralizing antibodies. Furthermore, relative resistance to sDPP4 was linked to mutation Q1020R, present in the spike proteins of recent Arabian but not African viruses. Finally, indirect evidence was obtained that sDPP4 in human plasma can inhibit MERS-CoV cell entry. These results support the concept that soluble DPP4 might constitute a natural barrier against human infection that is more efficiently overcome by viruses currently circulating in Arabian camels than those in African camels.IMPORTANCEMiddle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that can cause severe lung disease, MERS, and is transmitted from camels to humans. Although MERS-CoV infects camels in both Africa and Arabia, MERS cases have only been documented in Arabia for reasons that remain incompletely understood. Here, we provide evidence that viruses recently circulating in Arabian camels and causing human infections are less susceptible to inhibition by human soluble DPP4 (sDPP4)-a secreted version of the viral receptor that is present in various bodily fluids. Furthermore, we link sDPP4 resistance to mutation Q1020R in the spike protein of these viruses. These results suggest that viruses currently circulating in Arabian camels are better equipped to overcome a natural barrier to infection, sDPP4, than those circulating in African camels.

Multivalent lectin-glycan interactions (MLGIs) are widespread and vital for pathogen infection, cell-cell communication, and immune regulation, making them attractive therapeutic targets. Despite significant efforts, research progress in MLGI targeting therapeutics remains limited, due to our incomplete understanding of the structural and biophysical mechanisms of some key MLGIs, which has hampered the design of spatially matched multivalent therapeutics. Moreover, the overlapping glycan specificities of various lectins make it difficult to target MLGIs with high potency and selectivity. To address this challenge, we have recently developed polyvalent glycan nanoparticles (glycan-NPs) as biophysical probes for MLGIs. The NPs' unique, size-dependent optical properties are exploited as sensitive readouts for quantifying MLGI affinities and thermodynamics, while their nanoscale size and high electron microscopy contrast are exploited for probing binding modes and binding site orientation. Despite this success, how design features such as glycan type, density, and linker flexibility govern glycan-NP MLGI properties remains underexplored. In this work, we coated gold nanoparticles (GNPs) with varying densities of a lipoic acid-oligo(ethylene glycol)-α-manno-α-1,2-biose (DiMan) or fucose (Fuc) ligand of varying linker lengths and studied their MLGIs with DC-SIGN, an important tetrameric lectin viral receptor found on dendritic cells. Using our recently established GNP fluorescence quenching assay, we reveal that displaying DiMan or Fuc polyvalently on a GNP surface greatly enhances their DC-SIGN affinity, with low nanomolar apparent Kds, ∼480 000-fold tighter than the corresponding monovalent binding. Their binding is driven by enthalpy, with favorable enthalpic but unfavorable entropic terms, and their absolute values depend on linker flexibility and glycan density. At high glycan densities, a short and less flexible linker is favored by maximizing enthalpic gains while minimizing entropic penalties, whereas at low glycan densities, a long and flexible linker is favored by increasing the reach and adaptivity of terminal glycans to maximize favorable enthalpic gains. These results reveal a delicate balance between glycan density and flexibility in controlling glycan-NP MLGI properties and their underlying thermodynamic mechanisms. Finally, we demonstrate that GNP-glycans potently block DC-SIGN-augmented viral entry into host cells with subnanomolar IC50s, which are positively linked to their DC-SIGN MLGI affinity.

The furin motif in the SARS-CoV-2 spike (S) protein is important for lung cell entry. It is embedded in an extended loop structure and preceded by a highly conserved QTQTX motif that is required for efficient furin cleavage of the SARS-CoV-2 WA-1 S protein. BA.3.2 is an emerging SARS-CoV-2 saltation variant that is spreading globally in April 2026 and encodes a highly mutated S protein. Here, we analyzed whether the QTQTX motif is also required for spike protein cleavage and lung cell entry of BA.3.2. We report that two patient-derived spike sequences of the BA.3.2 subvariant BA.3.2.2 lack the first QT repeat of the QTQTX motif and show that this motif is largely dispensable for both cleavage and lung cell entry of BA.3.2.2, which we found to depend on TMPRSS2. Our results suggest that the reconfiguration of the BA.3.2 S protein during persistent infection may have significantly altered the determinants of furin cleavage.IMPORTANCEThe furin motif in the SARS-CoV-2 spike (S) protein is unique among sarbecoviruses and constitutes a virulence determinant. A QTQTX motif located immediately upstream of the furin motif is required for furin cleavage of the S protein of the virus that circulated early in the pandemic. Here, we show that the QTQTX motif is largely dispensable for S protein processing and S protein-driven lung cell entry of the emerging saltation variant BA.3.2, which is currently spreading globally. Thus, BA.3.2 evolution within immunocompromised individuals may have relaxed the requirements for furin processing of the spike protein.

The antigenic drift of SARS-CoV-2 toward the JN.1 lineage has prompted the development of variant-adapted COVID-19 booster vaccines. However, these boosters are thought to primarily recall pre-existing memory B cells (MBC), raising concerns about their ability to realign the immune response in highly pre-exposed populations. Here we analyze antibody and B cell responses in pre-exposed individuals (n = 42; median 4.5 prior COVID-19 vaccinations; 90% with at least one prior SARS-CoV-2 infection) following vaccination with a JN.1-adapted mRNA vaccine. Vaccination is associated with increased IgG binding and enhanced neutralization of JN.1 and related descendant variants. Longitudinal profiling of antigen-specific MBC shows that Wu01-only and Wu01/JN.1 cross-reactive cells remain dominant, while JN.1-only cells modestly increase by day 21. Single-cell RNA-sequencing of antigen-specific MBC in a representative sub-cohort (n = 7), combined with functional monoclonal antibody analyses, demonstrates that somatic hypermutation (SHM) drives intra-clonotype specialization toward improved JN.1 binding and neutralization. These findings indicate maturation of pre-existing, class-switched MBC rather than substantial de novo recruitment of naïve B cells. In conclusion, JN.1-adapted booster vaccination is associated with refinement of pre-existing MBC repertoires toward the JN.1 antigenic space and with enhanced neutralization of contemporary and antigenically proximate variants.

In 2025, a mutated H3N2 lineage, subclade K, emerged, showing high activity in many regions. Mutations in the hemagglutinin (HA) may affect cell entry and antibody-mediated neutralisation. Using pseudovirus particles, we show that subclade K-HA drives augmented entry into certain cell lines and displays significant antibody evasion. Both phenotypes were linked to mutation A186D. Influenza vaccination significantly boosted H3N2 subclade K neutralisation, suggesting that current vaccines may provide considerable protection.

The recently detected Omicron BA.2.86 lineage contains more than 30 amino acid mutations relative to BA.2. BA.2.86 and its JN.1 derivative evade neutralization by serum antibodies of fully vaccinated individuals. In this study, we elucidate epitopes driving the immune escape of BA.2.86 and JN.1 via pseudovirus neutralization. Here we generate 33 BA.2.86 mutants, each reverting a single mutation back to BA.2. We use this library in an approach that we call reverse mutational scanning to define distinct neutralization titers against each epitope. Mutations within the receptor binding domain at K356T, V483Δ, and to a lesser extent N460K, A484K, and F486P enhance immune escape. Interestingly, 16insMPLF within the spike N-terminal domain and P621S within S1/S2 also significantly contribute to antibody escape of BA.2.86. Upon XBB.1.5 booster vaccination, neutralization titers against JN.1 and BA.2.86 improve considerably, and residual immune escape is driven by 16insMPLF, N460K, E554K, and to a lesser extent P621S, and A484K. SARS-CoV-2 Omicron lineage BA.2.86 has over 30 mutations compared to the parental BA.2 lineage. Here Bdeir and colleagues apply reverse mutational scanning to determine which among these mutations present in Omicron BA.2.86 are epitopes linked to immune escape from antibody recognition.

In 2024, a clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) emerged in dairy cattle in the United States and spread rapidly to over 1,000 herds across multiple states. At least 41 human infections have occurred through contact with infected cattle, though no fatalities have been reported so far. This raises questions about whether the human innate immune system provides a barrier to bovine H5N1 HPAIV and whether seasonal influenza vaccines offer cross-protection. To address these questions, we used pseudoviruses bearing hemagglutinin (HA) and neuraminidase (NA) from seasonal influenza A or various H5Ny HPAIV strains (from cattle, duck, and seal). Pseudoviruses bearing H5N1 HPAIV HA and NA entered a wide range of mammalian and avian cell lines, including multiple cell lines from the human respiratory tract, while entry into A549 human lung cells was reduced when IFITM proteins were expressed. Additionally, preincubation of pseudovirus particles bearing H5N1 HPAIV HA and NA with plasma from individuals vaccinated with seasonal influenza vaccines inhibited viral entry. Collectively, these results suggest that the human innate immune system imposes a barrier against bovine H5N1 HPAIV infection and that seasonal influenza vaccines can induce cross-neutralizing activity against bovine H5N1 HPAIV.

AbstractTo assess the effect of the updated mRNA JN.1 omicron vaccine (bretovameran, BioNTech/Pfizer, Mainz, Germany) in an immunocompromised and elderly population, we measured humoral immune responses after mRNA omicron JN.1 vaccination in 37 haemodialysis patients before and 21 days after vaccination.We observed a 3-fold change in anti-S IgG, and a 4·7-fold change in anti-S omicron IgG. Memory B cells (MBC) exclusively binding the receptor binding domain (RBD) of JN.1 displayed a median frequency of 0·11% before vaccination and changed significantly 3·9-fold to a median of 0·43%. Cross reactive JN.1 RBD and Wuhan-Hu-1 S-binding MBCs and MBCs only binding to Wuhan-Hu-1 S changed 2·3-fold and 1·8-fold, respectively. Using a vesicular stomatitis virus-based pseudovirus particle (pp) neutralisation assay, baseline response rates were 86% for XBB.1.5pp, 78% for JN.1pp, 73% for and KP.2pp, 65% for KP.2.3ppand KP.3pp, and 68% for LB.1pp. After vaccination, the response rates for all pseudoviruses increased significantly, and we observed a mean increase in neutralisation of XBB.1.5pp, JN.1pp, KP.2pp, KP.2.3pp, KP.3pp, and LB.1ppof 8·3-fold, 18·7-fold, 22·5-fold, 18·7-fold, 25·5-fold, and 23·5-fold, respectively. In summary, our report provides first evidence for a firm humoral immune response in dialysis patients after mRNA omicron JN.1 vaccination.Our data suggest that the vaccine could be highly effective at enhancing protection of vulnerable populations against evolving SARS-CoV-2 variants.

New SARS-CoV-2 variants continue to emerge and may cause new waves of COVID-19. Antibody evasion is a major driver of variant emergence but variants can also exhibit altered capacity to enter lung cells and to use ACE2 species orthologues for cell entry. Here, we assessed cell line tropism, usage of ACE2 orthologues and antibody evasion of variant MC.10.1. This variant arose from the highly prevalent KP.3.1.1 variant, reached a prevalence of 10-15 % in certain countries in the spring of 2025 and contains a single amino acid mutation in the spike (S) protein, A435S, relative to the KP.3.1.1 S protein. We found that MC.10.1 and the parental KP.3.1.1 S protein show similar expression and similar capacity to fuse cells and to use ACE2 orthologues from different species for entry. In contrast, MC.10.1 S protein-driven entry into Calu-3 lung cells was reduced as compared to the KP.3.1.1 S protein. Finally, MC.10.1 S protein-bearing particles were less susceptible to neutralization by antibodies induced upon vaccination with the JN.1 booster vaccine as compared to their counterparts bearing KP.3.1.1 S protein. Collectively, our results indicate increased antibody evasion but reduced cell entry efficiency of variant MC.10.1.

Multivalent lectin–glycan interactions (MLGIs) are vital for viral infection, cell-cell communication and regulation of immune responses. Their structural and biophysical data are thus important, not only for providing insights into their underlying mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information remains to be limited for some important MLGIs, significantly restricting the research progress. We have recently demonstrated that functional nanoparticles, including ∼4 nm quantum dots and varying sized gold nanoparticles (GNPs), densely glycosylated with various natural mono- and oligo- saccharides, are powerful biophysical probes for MLGIs. Using two important viral receptors, DC-SIGN and DC-SIGNR (together denoted as DC-SIGN/R hereafter), as model multimeric lectins, we have shown that α-mannose and α-manno-α-1,2-biose (abbreviated as Man and DiMan, respectively) coated GNPs not only can provide sensitive measurement of MLGI affinities but also reveal critical structural information (e.g., binding site orientation and mode) which are important for MLGI targeting. In this study, we produced mannuronic acid (ManA) coated GNPs (GNP-ManA) of two different sizes to probe the effect of glycan modification on their MLGI affinity and antiviral property. Using our recently developed GNP fluorescence quenching assay, we find that GNP-ManA binds effectively to both DC-SIGN/R and increasing the size of GNP significantly enhances their MLGI affinity. Consistent with this, increasing the GNP size also significantly enhances their ability to block DC-SIGN/R-augmented virus entry into host cells. Particularly, ManA coated 13 nm GNP potently block Ebola virus glycoprotein-driven entry into DC-SIGN/R-expressing cells with sub-nM levels of EC50. Our findings suggest that GNP-ManA probes can act as a useful tool to quantify the characteristics of MLGIs, where increasing the GNP scaffold size substantially enhances their MLGI affinity and antiviral potency.

Multivalent lectin-glycan interactions (MLGIs) are vital for viral infection, cell-cell communication and regulation of immune responses. Their structural and biophysical data are thus important, not only for providing insights into their underlying mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information remains to be limited for some important MLGIs, significantly restricting the research progress. We have recently demonstrated that functional nanoparticles, including ∼4 nm quantum dots and varying sized gold nanoparticles (GNPs), densely glycosylated with various natural mono- and oligo- saccharides, are powerful biophysical probes for MLGIs. Using two important viral receptors, DC-SIGN and DC-SIGNR (together denoted as DC-SIGN/R hereafter), as model multimeric lectins, we have shown that α-mannose and α-manno-α-1,2-biose (abbreviated as Man and DiMan, respectively) coated GNPs not only can provide sensitive measurement of MLGI affinities but also reveal critical structural information (e.g., binding site orientation and mode) which are important for MLGI targeting. In this study, we produced carboxyl mannose (ManA) coated GNPs (GNP-ManA) of two different sizes to probe the effect of glycan modification on their MLGI affinity and antiviral property. Using our recently developed GNP fluorescence quenching assay, we find that GNP-ManA binds effectively to both DC-SIGN/R and increasing the size of GNP significantly enhances their MLGI affinity. Consistent with this, increasing the GNP size also improves their antiviral potency considerably. Particularly, ManA coated 13 nm GNP potently blocks both DC-SIGN-mediated pseudo–Ebola virus cellular entry with an EC50 of ∼1 nM. Our findings suggest that GNP-ManA probes can act as a useful tool to quantify the characteristics of MLGIs, where increasing the GNP scaffold size substantially enhances their MLGI affinity and antiviral potency.

Drug screening resembles finding a needle in a haystack: identifying a few effective inhibitors from a large pool of potential drugs. Large experimental screens are expensive and time-consuming, while virtual screening trades off computational efficiency and experimental correlation. Here we develop a framework that combines molecular dynamics (MD) simulations with active learning. Two components drastically reduce the number of candidates needing experimental testing to less than 20: (1) a target-specific score that evaluates target inhibition and (2) extensive MD simulations to generate a receptor ensemble. The active learning approach reduces the number of compounds requiring experimental testing to less than 10 and cuts computational costs by ∼29-fold. Using this framework, we discovered BMS-262084 as a potent inhibitor of TMPRSS2 (IC50 = 1.82 nM). Cell-based experiments confirmed BMS-262084’s efficacy in blocking entry of various SARS-CoV-2 variants and other coronaviruses. The identified inhibitor holds promise for treating viral and other diseases involving TMPRSS2.

COVID-19, caused by SARS-CoV-2, has led to significant morbidity and mortality worldwide. The pandemic has sparked extensive efforts to develop therapeutic strategies targeting either the virus itself or human proteins involved in the infection, resulting in hundreds of potential drugs and numerous clinical trials. In the search for new anti-SARS-CoV-2 drug candidates, we focussed on natural products. Using the cyanobacteria as a source of new structures due to their unique metabolism, two spumigins were isolated from the Baltic cyanobacterium Nodularia spumigena and tested for their activity against SARS-CoV-2. These compounds efficiently reduced SARS-CoV-2 infection in human A549ACE2/TMPRSS2 cells and fully differentiated primary human airway epithelium cell systems, in which viral entry depends on activation of the viral spike protein by the cellular serine protease, such as TMPRSS2. In contrast, the compounds did not inhibit viral replication in Vero cells, which lack TMPRSS2 but have high levels of cysteine protease cathepsin L, which may serve as an alternative for spike protein activation. Biochemical assays showed that spumigins inhibit TMPRSS2 with an EC50 ranging from 17 to 85 nM at which no toxicity is observed. Good parameters of identified inhibitors prove that cyanobacteria may serve as a rich source for new scaffolds reaching beyond the canonical chemical combinatorial space.