Antiviral drug discovery for respiratory viruses is hindered by the lack of scalable physiologically relevant systems. Here, we report the first high-throughput screen of 764 natural plant extracts against respiratory syncytial virus (RSV) using human primary airway organoids as a relevant model. A parallel screen conducted in A549 cells allowed the identification of 70 extracts with organoid-specific antiviral activity from which 45 active phytocompounds were purified. We identified early- and late-acting antiviral compounds and demonstrated a polarization-dependent activity for some of them. Collectively, our results establish the use of airway organoids as a scalable first-line platform for high-throughput antiviral discovery and exploit the plant-derived chemical space as an underexplored source of RSV inhibitors. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, NRP79 407940_206469, SNF Sinergia CRSII5_189921 Aclon foundation, F02-11471

The Institute for In Vitro Sciences (IIVS) has sponsored a series of workshops with the aim of developing recommendations for scientific and technical approaches to conducting in vitro assays to assess potential toxicity within and across tobacco and nicotine products. As well as providing a background overview of oral tobacco and nicotine products, this report focuses on the methods used to generate extracts from these products for in vitro testing, the recommended best testing practices, and suggested areas for future research. This publication was developed by a working group of workshop members, in conjunction with the ninth IIVS in vitro workshop entitled 'In Vitro Models for Testing Oral Tobacco and Nicotine Products and Continued Discussion of In Vitro Models of Toxicity and Disease', which was held in January 2024.

Highly pathogenic avian influenza H5N1 viruses of clade 2.3.4.4b have caused widespread avian mortality and sporadic mammalian infections, raising concerns about their potential for efficient replication in the human population. Efficient replication in the human upper respiratory tract is considered a key barrier to transmission. Here, we demonstrate that an H5N1 virus isolated from bovine milk in Texas in 2024 (H5N1Tex/24) replicates as efficiently as the 2009 pandemic H1N1 virus (H1N1HH4/09) in well-differentiated human nasal epithelial cells. These cells express both avian- and human-type influenza receptors, indicating receptor adaptation is unnecessary for entry. H5N1Tex/24 replicates effectively at 33 degrees Celsius, reflecting nasal cavity temperature, whereas earlier avian H5N1 strains require 37 degrees Celsius, suggesting that H5N1Tex/24 has acquired another key adaptive feature to the human upper respiratory tract. H5N1Tex/24 remains sensitive to interferon-λ (IFN-λ) despite inducing low cytokine levels. Notably, no known mammalian-adaptive mutations such as PB2-E627K were detected. These findings suggest that H5N1Tex/24 possesses intrinsic traits enabling efficient replication in the human upper airways, a critical step toward potential airborne transmission, underscoring the need for vigilant surveillance. ### Competing Interest Statement The authors have declared no competing interest. Federal Food Safety and Veterinary Office, https://ror.org/01hwpsz06, 1.24.m

Abstract In vitro pulmonary models are widely used for safety evaluations, simulating human lung responses to inhaled particles or chemicals. These models help detect toxicity early, reducing the need for animal testing. In this study, we used two models: the Alveolar Macrophage Assay (AMA), a simple model, and the tissue model MucilAir™, a more robust system, to evaluate the safety of commercial carbon-based spray lubricants. We also characterized the lubricants to identify their physico-chemical properties but encountered difficulties due to interference from the silicone-based lubricant matrix. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) analysis could not detect carbon-based materials, likely due to the presence of polysiloxanes. Confocal Raman analysis revealed graphite, with minor contributions from graphene, which was consistent with Scanning Electron Microscopy (SEM) findings showing graphite-like particles. Exposure assessment revealed that spray droplets approximately 2.20 µm in size are released during application, suggesting inhalation as a potential exposure route. Hazard assessment using AMA was challenging; direct spray administration onto MucilAir™ caused cell damage or death. The submerged approach with macrophages led to density-driven separation of compounds, but a black material was partially incorporated into cells, triggering a dose-dependent release of lytic enzymes. Despite challenges in characterising and testing graphene-based lubricants, MucilAir™ was shown to be a sensitive model for assessing the safety of such products. A tailored approach, considering specific properties of lubricants, is critical for accurate hazard assessment. The MACRAME project was funded by the EU Horizon Europe (GA no 101092686), Swiss SERI (no 23.00141), and UKRI Innovate (no 10066165).

The respiratory syncytial virus (RSV) has been identified as a major causative agent of bronchiolitis in children; nevertheless, available therapeutic options remain limited. Natural products (NPs) have been valued in the field of drug discovery and hold great promise for addressing this gap. However, there is a lack of methodologies that combine high-throughput screening (HTS) of natural extracts (NEs) with parallel metabolomic profiling to target the isolation of potential antivirals. This study presents an antiviral discovery approach that integrates HTS of 192 NEs against RSV in human lung adenocarcinoma cells (A549) with their systematic metabolite profiling. To optimize the identification of compounds with antiviral activity, we developed a scoring system, referred to as the "bioscore". This score correlates the antiviral activity of each NE with the normalized intensities of its constituents across the 192 NEs. Using this approach, triterpenes, including betulinic acid (BA), were frequently identified in active NEs, contributing to a high hit rate of 38 %. After isolating BA from Pilea plataniflora, we confirmed its antiviral activity in A549 cells, as well as in physiologically relevant models such as airway organoids and human airway epithelia cultured at the air-liquid interface. We further investigated its mechanism of action and found an effect at the early stage of viral replication. Protoapigenone, a species-specific flavonoid from this set of 192 NEs, was also identified as a potential antiviral NP, though limited by its cytotoxicity. This integrative workflow enables scalable antiviral screening of NEs by directly linking metabolomics with bioactivity through bioscore-driven prioritization.

Highly pathogenic avian influenza (H5N1) viruses of clade 2.3.4.4b have caused significant losses in bird populations worldwide and repeatedly infected mammals, including humans, without sustained human to human transmission. Here we show that an H5N1 virus (H5N1Tex/24) isolated from bovine milk in Texas in 2024 replicates just as efficiently in differentiated human nasal epithelial cells as a pandemic H1N1 virus strain from 2009 (H1N1HH4/09), at both 37 °C and 33 °C. The adaptive mutations PB2 M631L and PA K497R promoted replication at 33 °C but had no effect on replication at 37 °C. An H5N1 virus (H5N1BE/22) isolated from a pelican in 2022, which lacked these mutations, replicated efficiently at 37 °C but poorly at 33 °C, and this limitation was not overcome by the introduction of the PB2 M631L and PA K497R mutations. The differentiated nasal epithelial cell cultures expressed receptors for both human and avian influenza viruses. Accordingly, no HA mutations associated with altered receptor specificity were detected. H5N1Tex/24 was able to effectively suppress the production of interferon-λ, yet remained sensitive to the antiviral effects of this cytokine. These findings suggest that H5N1Tex/24 possesses intrinsic traits supporting efficient replication in differentiated human upper airway cell cultures.

Abstract Two-dimensional (2D) graphene-based advanced materials are increasingly utilized across a wide range of applications, including battery technology, superconductors, biomedical products, and catalysis. As their use expands, so does the potential for human exposure, particularly in occupational environments during production, as well as during end-of-life handling. Inhalation represents a major route of exposure for workers in these settings. Here, we aimed to investigate potential inhalation-related health hazards of few-layer graphene (FLG; Carbon Waters)-epoxy composites using alveolar macrophages (NR8383) and ALIsens, AlveolAirTM and MucilAirTM advanced in vitro lung cell culture models. The results demonstrate successful detection of FLG in the epoxy (product) matrix by using Raman spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) techniques. The alveolar macrophage assay demonstrated material-specific variations in bioresponse. Pristine FLG did not impact macrophage viability but induced a pronounced secretion of the pro-inflammatory cytokine TNFα. In contrast, particles collected after abrasion of FLG-epoxy composites showed no significant upregulation in TNFα response but induced dose-dependent cytotoxicity in alveolar macrophages. In the bronchial and alveolar airway epithelial cultures, pristine FLG and abraded particles did not decrease cell viability or compromise barrier integrity, but a moderate reduction of mucociliary clearance was observed at concentrations above 10 µg/cm2, which was further confirmed with scanning electron microscopy (SEM). Overall, our data reveals distinct health risks associated with particle inhalation during FLG-epoxy composite production and degradation, affecting multiple lung cell types. These findings provide valuable mechanistic insights to support health hazard assessment and safe-by-design development of industrial graphene-based composite materials.

Airway epithelium plays a major role as the primary interface between human body and the external environment, acting both as a physical and functional barrier. In vitro airway models that reproduce the epithelium architecture are therefore a valuable tool for studying infection, inflammation, and transport processes. In this work, we present a label-free, non-invasive method to visualize and measure mucociliary transport in air–liquid human models using third-harmonic generation (THG) microscopy with an optical parametric amplifier laser source at 1300 nm. By exploiting the intrinsic nonlinear contrast at optical heterogeneities, THG provides high resolution images of both epithelial structures and of the overlying mucus layer without the need for fluorescence staining or sample processing. Time–lapse THG imaging reveals depth–dependent transport dynamics within the mucus, offering new insights into mucociliary transport mechanism. Our approach offers a physiologically relevant way to assess mucociliary function in vitro and could support studies on respiratory diseases, drug delivery and efficacy, and epithelial remodeling.

The SARS-CoV-2 main protease (NSP5) is the target of the FDA approved Nirmatrelvir (NTV) antiviral. Substitutions in NSP5, such as the P252L mutation, have been reported in clinical isolates and raise questions regarding its potential role in drug resistance. Here, we show that the P252L effect on NTV susceptibility is dependent on the culture model. The mutation arises spontaneously in VeroE6 cells without treatment but does not appear in Calu-3 cells. It becomes enriched under NTV pressure in both VeroE6 cells and Human Airway Epithelium (HAE) cultures but leads to a significant increase in EC50 values only in VeroE6 cells. This discrepancy between allelic selection and functional resistance highlights the importance of using appropriate culture models when evaluating resistance mechanisms.

Abstract Mucociliary clearance in the upper respiratory tract serves as a critical component of the innate immune defence, protecting the lungs from inhaled environmental agents. Disruption of this function increases vulnerability to respiratory infections and chronic diseases. While certain nanomaterials are known to induce pulmonary fibrosis and inflammation, their potential to impair mucociliary function remains insufficiently understood. This study investigated the interactions of silicon- and carbon-based nanomaterials, including silicon carbide nanowires (SiC NWs), silicon dioxide (SiO2), quartz silica DQ12, multiwalled carbon nanotubes (MWCNTs), and graphene nanosheets, with human airway mucus and primary human bronchial epithelial cells. Among the evaluated nanomaterials, only SiC NWs were able to penetrate through the mucus barrier, due to their low silanol group density and hydrophobic surface, which reduced interactions with mucus components. Four consecutive days of repeated exposure of primary human bronchial epithelial cultures to SiC NWs resulted in significant (P < 0.05) impairment of mucociliary clearance, accompanied by abnormal ciliary morphology. Gene expression analysis revealed upregulation of FOXJ1, indicating dysregulated ciliogenesis. Additionally, SiC NWs compromised epithelial barrier integrity and induced pro-inflammatory and pro-fibrotic responses. These findings identify SiC NWs as uniquely capable of penetrating the mucus barrier and disrupting mucociliary clearance function, thereby highlighting a previously underrecognized mechanism of nanomaterial-induced respiratory toxicity. This work underscores the need for careful evaluation of nanomaterial physicochemical properties in relation to airway defence mechanisms.

Antibiotic-resistant respiratory infections can lead to treatment failure, highlighting the need for alternative strategies. FLAMOD, a recombinant flagellin, stimulates innate immunity via Toll-like receptor 5 when delivered intranasally. In mice, FLAMOD protects against bacterial pneumonia. The protection is associated with activation of airway epithelial cells. This study aimed to assess the tolerance of human primary respiratory epithelium to FLAMOD administered apically as liquid droplets or by nebulization, to measure the innate immune response, and the pharmacokinetics of FLAMOD. We used epithelia reconstituted from human nasal and bronchial (MucilAir™), small airways (SmallAir™) and alveolar (AlveolAir™) primary epithelial cells, cultured at the air-liquid interface. We report that daily administration of escalating doses of FLAMOD for 5 days was well tolerated by epithelia as barrier integrity, cilia motion and cell viability were not affected. FLAMOD was rapidly degraded without leakage into the basal compartment. Each epithelial model exhibited responses involving pathways of innate defense and immune cell infiltration, which were dose-dependent, with an effective concentration of FLAMOD in the picomolar range. Similar tolerance profile and immune responses were obtained with airway epithelium from cystic fibrosis and chronic obstructive pulmonary disease patients. In conclusion, this study supports the stimulation of epithelial Toll-like receptor 5 signaling to fight against infections of vulnerable patients.

The lung mucosa at birth and shortly after is particularly vulnerable to respiratory syncytial virus (RSV) infection. Respiratory disease severity is strongly influenced by the age at first exposure, which may also affect the trajectory of airway function. Early-life represents a critical window for lung development and initial microbiota colonization, both of which shape mucosal immune responses to RSV. The impact of the initial establishment of the lung microbiota by its primo-colonizing strains on susceptibility to RSV infection in neonatal mice remains poorly described. In the present study, we showed that early-life primo-colonizing bacterial strains in the mouse lung differentially induce innate immune responses and influence RSV susceptibility in ex vivo models using lung explants and alveolar macrophages (AMs). We identified a specific bacterial strain (strain 17) whose prior exposure enhances type I interferon (IFN-I) responses in AMs upon RSV infection and reduces viral replication both ex vivo and in vivo in lung tissues. Intranasal administration of this strain during early life prevented the development of immunopathological responses upon RSV reinfection in adult mice. Finally, using a translational human airway epithelium model, we demonstrated that pre-exposure to strain 17 restricts RSV spread without cytotoxicity, likely via enhanced β-defensin 2 production. These findings highlight the potential of early-life microbiota modulation as a promising intervention for preventing RSV disease and its long-term respiratory consequences.

IntroductionThis work evaluated a non-animal toolbox to be used within a next-generation risk assessment (NGRA) framework to assess chemical-induced lung effects using human upper and lower respiratory tract models, namely MucilAir™-HF and EpiAlveolar™ systems, respectively.MethodsA 12-day substance repeated exposure scheme was established to explore potential lung effects through analysis of bioactivity readouts from the tissue integrity and functionality, cytokine/chemokine secretion, and transcriptomics.ResultsEleven benchmark chemicals were tested, including inhaled materials and drugs that may cause lung toxicity following systemic exposure, covering 14 human exposure scenarios classified as low- or high-risk based on historical safety decisions. For calculation of bioactivity exposure ratios (BERs), obtained chemical-induced bioactivity data were used to derive in vitro points of departures (PoDs) using a nonlinear state space model. PoDs were then combined with human exposure estimates, i.e., predicted lung deposition for benchmark inhaled materials using multiple path particle dosimetry (MPPD) exposure computational modeling or literature maximum plasma concentration (Cmax) for systemically available benchmark drugs.DiscussionIn general, PoDs occurred at higher concentrations than the corresponding human exposure values for the majority of the low-risk chemical-exposure scenarios. For all the high-risk chemical-exposure scenarios, there was a clear overlap between the PoDs and lung deposited mass and Cmax for the benchmark inhaled materials and therapeutic drugs, respectively. Our findings suggest that combining computational and in vitro new approach methodologies (NAMs) informed by adverse outcome pathways (AOPs) associated with pulmonary toxicity can provide relevant biological coverage for chemical lung safety assessment.

Lower respiratory infections, mostly caused by viral or bacterial pathogens, remain a leading global cause of mortality. The differences between animal models and humans contribute to inefficiencies in drug development, highlighting the need for more relevant and predictive, non-animal models. In this context, AlveolAir™, a fully primary in vitro 3D human alveolar model, was characterized and demonstrated the sustained presence of alveolar type I (ATI) and type II (ATII) cells. This model exhibited a functional barrier over a 30-day period, evidenced by high transepithelial electrical resistance (TEER). These findings were further validated by tight junctions’ confocal microscopy and low permeability to Lucifer yellow, confirming AlveolAir™ as robust platform for drug transport assays. Additionally, successful infections with H1N1 and SARS-CoV-2 viruses were achieved, and antiviral treatments with Baloxavir and Remdesivir, respectively, effectively reduced viral replication. Interestingly, both viruses infected only the epithelial layer without replicating in endothelial cells. These findings indicate AlveolAir™ as a relevant model for assessing the toxicity and permeability of xenobiotics and evaluating the efficacy of novel antiviral therapies.

The lung mucosa at birth and shortly after is particularly vulnerable to respiratory syncytial virus (RSV) infection. Respiratory disease severity is strongly influenced by the age at first exposure, which may also affect the trajectory of airway function. Early-life represents a critical window for lung development and initial microbiota colonization, both of which shape mucosal immune responses to RSV. The impact of the initial establishment of the lung microbiota by its primo-colonizing strains on susceptibility to RSV infection remains largely unknown. In the present study, we showed that early-life primo-colonizing bacterial strains in the mouse lung differentially induce innate immune responses and influence RSV susceptibility in ex vivo models using lung explants and alveolar macrophages. We identified a specific bacterial strain that enhances IFN-I antiviral pathways and reduces RSV replication both in vitro and in vivo. Intranasal administration of this strain to neonatal mice prevented the development of immunopathological responses upon RSV reinfection in adulthood. These findings highlight the potential of early-life microbiota modulation as a promising intervention for preventing RSV disease and its long-term respiratory consequences. ### Competing Interest Statement The authors have declared no competing interest. Air Liquide (France), https://ror.org/0431b2v07 Vaincre la Mucoviscidose, https://ror.org/00gp9bw49 Association Gregory Lemarchal, Région Ile de France - DIM Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-13-BSV3-0016, ANR-24-NEOMIS

Bacterial pneumonia poses a significant challenge to public health, often leading to antibiotic treatment failure. Enhancing innate immunity represents a promising adjunctive strategy to conventional antibiotic therapy. Bacterial flagellin, a Toll-like receptor 5 (TLR5) agonist, has been shown to stimulate innate immune defenses when delivered via the respiratory route, demonstrating efficacy in both preventing and treating bacterial pneumonia in murine models. This protective effect is primarily mediated through TLR5-driven activation of airway epithelial cells. This study aimed to characterize the immunomodulatory effects of flagellin on human primary respiratory epithelium. Using the MucilAir™ air-liquid interface model and RNA sequencing, we demonstrated that apical administration of flagellin induced robust immune responses in airway epithelium derived from healthy individuals, as well as patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). TLR5-mediated epithelial signaling triggered key immune-related pathways, including cytokine production, leukocyte chemotaxis, neutrophil recruitment, and antimicrobial defense, with strong commonalities across healthy and diseased airway epithelia. Furthermore, we demonstrated that flagellin effectively activated epithelial immune responses even in the presence of the bacteria Pseudomonas aeruginosa or Streptococcus pneumoniae . However, epithelial activation alone was insufficient to directly limit bacterial colonization or replication, highlighting the potential role of epithelial-immune cell interactions in achieving effective bacterial clearance. These findings support TLR5 activation as a promising therapeutic strategy to enhance host defense mechanisms and improve treatment outcomes for bacterial pneumonia in both healthy individuals and patients with COPD or CF. ### Competing Interest Statement JCS and NHV are the inventors of the patents WO2009156405, WO2011161491, and WO2015011254 that describes the use of FLAMOD as biologic against infectious diseases and the patent WO2023275292 on the formulation of FLAMOD. Authors declare no other competing interests. Vaincre la Mucoviscidose, https://ror.org/00gp9bw49, RF20160501644, RF20170502036 Agence Nationale de la Recherche, ANR-19-CE18-0030-01 European Union, 847786

Influenza viruses pose a significant threat due to annual epidemics and pandemic potential. Resistance to current antivirals underscores the need for new drugs and strategies to prevent its emergence. We previously developed two novel HA-targeting compounds (CD-6'SLN and CD-SA) with demonstrated efficacy against influenza A and B strains. Here, we compared their resistance barrier to that of FDA-approved oseltamivir (OS) and baloxavir marboxil (BXM). We established a resistance testing assay in human airway epithelia (HAE) and in mice. We also evaluated the impact of combination therapies on resistance emergence. In HAE, highly reduced inhibition (HRI) by CD-6'SLN and CD-SA occurred within 2 and 4 weeks respectively without fitness loss, while reduced inhibition (RI) by baloxavir acid (BXA) emerged within 4 weeks. No reduction of susceptibility to OS was observed in the same time frame. Of note, emergence of RI by CD-SA was not delayed in BXA/CD-SA co-treatment, and slightly reduced upon OS/CD-SA co-treatment. In mice, RI by CD-SA was observed after 8 passages in one of three mice treated with OS/CD-SA, but not in mice with single therapies. This study demonstrates that (1) HAE represents a relevant model to detect emergence of resistance and (2) HA-targeting compounds are prone to induce resistance followed by BXA and OS. Importantly, combination of clinically available antivirals and HA-targeting compounds did not prevent the emergence of variants with HA substitutions. Additional research is needed to develop anti-influenza antivirals with high resistance barrier and compounds should be tested in HAE before moving to animal experimentation.