Biology subjects

Constant, S.

Publications and source records attributed to Constant, S..

14 recordsLinked to original sources

bioRxiv2026

Third Harmonic Generation Microscopy Reveals Structure and Mucus Dynamics in Human Airway Epithelium Models

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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/717621v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@62e8acorg.highwire.dtl.DTLVardef@199a8b7org.highwire.dtl.DTLVardef@113bb84org.highwire.dtl.DTLVardef@7be3f8_HPS_FORMAT_FIGEXP M_FIG For Table of Contents Only C_FIG

biophysics↗

bioRxiv2026

Human airway organoids as a high-throughput screening platform for antiviral natural products discovery

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.

microbiology↗

bioRxiv2026

Bovine-derived influenza A virus (H5N1) shows efficient replication in well-differentiated human nasal epithelial cells without requiring genetic adaptation

Highly pathogenic avian influenza (H5N1) viruses of clade 2.3.4.4b have caused significant losses among bird populations worldwide and have repeatedly crossed the species barrier, infecting mammals, including humans. However, efficient human-to-human transmission has not yet been observed. Here, we demonstrate that an H5N1 virus isolated from bovine milk in Texas in 2024 (H5N1Tex/24) replicates in differentiated human nasal epithelial cells as efficiently as a 2009 pandemic H1N1 virus strain (H1N1HH4/09) at both 37 {degrees}C and 33 {degrees}C. The adaptive mutations PB2-M631L and PA-K497R do not affect H5N1Tex/24 replication at 37 {degrees}C but promote replication at 33 {degrees}C. Conversely, H5N1BE/22, a virus from the same clade isolated from a pelican in 2022 that lacks these mutations, replicates in human nasal epithelial cells at 37 {degrees}C as efficiently as H5N1Tex/24 but exhibits limited replication at 33 {degrees}C. Introducing the two mutations PB2-M631L and PA-K497R did not overcome this limitation. Furthermore, nasal epithelial cells express receptors for both human and avian influenza viruses. Accordingly, no mutations were detected in HA which are known to switch receptor preference. Finally, we demonstrate that H5N1Tex/24 remains sensitive to the antiviral effects of interferon-{lambda} (IFN-{lambda}), however, infected nasal epithelial cells secrete only small amounts of this cytokine. Overall, our results suggest that H5N1Tex/24 possesses intrinsic traits enabling efficient replication in the human upper airways.

microbiology↗

bioRxiv2025

Local tolerance and innate immune activation of primary human respiratory cells exposed to flagellin

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.

cell biology↗

bioRxiv2025

Early-Life Commensal Lung Bacteria Shape Immune Responses and RSV Susceptibility in Neonatal Mice

The lung mucosa during the neonatal period and early infancy is especially susceptible 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 contribution of early lung-colonizing bacterial strains to the establishment of the lung microbiota and susceptibility to RSV infection in neonatal mice remains poorly characterized. 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 {beta}-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. Author summaryRespiratory syncytial virus (RSV) is a major cause of severe respiratory infections in newborns and infants, a period during which the lungs and immune system are still maturing. Simultaneously, the respiratory tract undergoes its first microbial colonization by so-called primo-colonizing bacteria -- the first bacterial species to establish themselves on neonatal lower respiratory mucosal surfaces. These early microbial pioneers may play an unsuspected role in shaping how the immature immune system responds to viral threats. In this study, we investigated whether these early lung-colonizing strains influence RSV susceptibility. Using neonatal mouse models, lung explants, and alveolar macrophages, we found that distinct bacterial strains trigger markedly different innate immune responses. Prior exposure to one strain in particular, strain 17, was associated with enhanced type I interferon responses, reduced viral replication, and protection against harmful inflammation upon RSV reinfection later in life. In a human airway epithelium model, pre-exposure to strain 17 also restricted RSV spread without tissue damage, potentially through increased {beta}-defensin 2 production. These findings provide proof-of-concept that the bacterial strains establishing in the neonatal lung can influence future immune reactivity and antiviral responses, and suggest that early-life respiratory microbiota modulation could represent a promising strategy to prevent severe RSV disease.

immunology↗

bioRxiv2025

Flagellin-mediated TLR5 activation enhances innate immune responses in healthy and diseased human airway epithelium

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.

immunology↗

bioRxiv2024

The triple combination of Remdesivir (GS-441524), Molnupiravir and Ribavirin is highly efficient in inhibiting coronavirus replication in human nasal airway epithelial cell cultures and in a hamster infection model

The use of fixed dose-combinations of antivirals with different mechanisms of action has proven a key in the successful treatment of infections with HIV and HCV. For the treatment of infections with SARS-CoV-2 and possible future epi-/pandemic coronaviruses, it will be important to explore the efficacy of combinations of different drugs, in particular to avoid resistance development, such as in patients with immunodeficiencies. As a first effort, we studied the antiviral potency of combinations of antivirals. To that end, we made use of primary human airway epithelial cell (HAEC) cultures grown at the air-liquid interface that were infected with the beta coronavirus OC43. We found that the triple combination of GS-441524 (parent nucleoside of remdesivir), molnupiravir, and ribavirin resulted in a more pronounced antiviral efficacy than what could be expected from a purely additive antiviral effect. The potency of this triple combination was next tested in SARS-CoV-2 infected hamsters. To that end, for each of the drugs, intentionally suboptimal or even ineffective doses were selected. Yet, in the lungs of all hamsters that received triple prophylactic therapy with suboptimal/inactive doses of GS-441524, molnupiravir, and ribavirin, no infectious virus was detectable. Our finding indicate that co-administration of approved drugs for the treatment of coronavirus infections should be further explored but also against other families of viruses with epidemic and pandemic potential for which no effective antiviral treatment is available.

microbiology↗

bioRxiv2024

Development of Broad-spectrum β-cyclodextrins-Based Nanomaterials Against Influenza Viruses

In recent decades, epidemics and pandemics have multiplied throughout the world, with viruses generally being the primary agents responsible. Among these, influenza viruses play a key role, as they cause severe respiratory distress, representing a major threat to public health. To enhance the response to viral disease outbreaks, there is a need for ready-to-use broad-spectrum antivirals. We have engineered macromolecules (named CD-SA) consisting of a {beta}-cyclodextrin (CD) scaffold modified with hydrophobic linkers in the primary face, onto which unitary sialic acid (SA) epitopes are covalently grafted, this to mimic influenza virus host receptors. In this study, we demonstrated that CD-SA, with a unitary SA, without extensive polysaccharides or specific connectivity, acts as a potent virucidal antiviral against several variants of human influenza type A and type B viruses. We also assessed the genetic barrier to resistance of CD-SA in vitro and successfully delayed emergence of resistance by combining CD-SA with interferon-{lambda}1 (IFN {lambda}1). Finally, we completed the characterization of the antiviral activity by conducting both ex vivo and in vivo studies, demonstrating a potent antiviral effect in human airway epithelia and in a mouse model of infection, higher than that of Oseltamivir, a currently approved anti-influenza antiviral.

microbiology↗

bioRxiv2022

Molnupiravir combined with different repurposed drugs further inhibits SARS-CoV-2 infection in human nasal epithelium in vitro

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in late 2019, has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available, and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show high clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new virus variants, and generally short duration of immunity, the development of safe and effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing and approved drugs for accelerated clinical testing and potential emergency use authorizations. However, drug-repurposing using high throughput screenings in cellular assays, often identify hits that later prove ineffective in clinical studies. Our approach was to evaluate the activity of compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Secondly, we evaluated drug combinations using sub-maximal doses of each active single compound. Here, we report the antiviral effects of 95 single compounds and 30 combinations. The data show that selected drug combinations including 10 M of molnupiravir, a viral RNA-dependent RNA polymerase (RdRp) inhibitor, effectively inhibit SARS-CoV-2 replication. This indicates that such combinations are worthy of further evaluation as potential treatment strategies against coronavirus disease 2019 (COVID-19).

microbiology↗

bioRxiv2021

A novel anti-influenza combined therapy assessed by single cell RNA-sequencing

Influenza makes millions of people ill every year, placing a large burden on the healthcare system and the economy. To develop a novel treatment against influenza, we combined virucidal sialylated cyclodextrins with interferon lambda and demonstrated, in human airway epithelia, that the two compounds inhibit the replication of a clinical H1N1 strain more efficiently when administered together rather than alone. We investigated the mechanism of action of the combined treatment by single cell RNA sequencing analysis and found that both the single and combined treatments impair viral replication to different extents across distinct epithelial cell types. We also showed that each cell type comprises multiple sub-types, whose proportions are altered by H1N1 infection, and assess the ability of the treatments to restore them. To the best of our knowledge this is the first study investigating the effectiveness of an antiviral therapy by transcriptomic studies at the single cell level.

genomics↗

bioRxiv2021

Drug-free nasal spray as a barrier against SARS-CoV-2 infection: safety and efficacy in human nasal airway epithelia

BackgroundFor SARS-CoV-2 and other respiratory viruses, the nasal epithelium is a key portal for infection. Therefore, the nose is an important target of prophylactic and therapeutic interventions against these viruses. We developed a nasal spray (AM-301, a medical device marketed as Bentrio) to protect against infection by SARS-CoV-2 and potentially other viruses. Aims of the studyTo test the safety and efficacy of AM-301 against SARS-CoV-2 infection. MethodsAM-301 was tested on an in vitro 3D model of primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in prophylaxis and infection mitigation assays. ResultsAM-301 did not have any detrimental effect on the nasal epithelium. Prophylactic treatment with AM-301 reduced viral titer significantly vs. controls over 4 days, reaching a maximum reduction of 99%. When treatment with AM-301 was started 24 or 30 h after infection, epithelia that received the formulation had a 12- or 14-fold lower titer than controls. ConclusionAM-301 was found to be safe in vitro, and it significantly decelerated viral titer growth in experimental models of prophylaxis and mitigation. Its physical (non-pharmaceutical) mechanism of action, safety and efficacy pave the way for further investigation of its possible use against a broad spectrum of viruses, allergens and pollutants.

pharmacology and toxicology↗

bioRxiv2021

Pyrimidine biosynthesis inhibitors synergize with nucleoside analogs to block SARS-CoV-2 infection

The ongoing COVID-19 pandemic has highlighted the dearth of approved drugs to treat viral infections, with only [~]90 FDA approved drugs against human viral pathogens. To identify drugs that can block SARS-CoV-2 replication, extensive drug screening to repurpose approved drugs is underway. Here, we screened [~]18,000 drugs for antiviral activity using live virus infection in human respiratory cells. Dose-response studies validate 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Amongst these drug candidates are 16 nucleoside analogs, the largest category of clinically used antivirals. This included the antiviral Remdesivir approved for use in COVID-19, and the nucleoside Molnupirivir, which is undergoing clinical trials. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral, and we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogs synergistically inhibits SARS-CoV-2 infection in vitro and in vivo suggesting a clinical path forward.

microbiology↗

bioRxiv2020

Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia

In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the lack of biologically relevant pre-clinical experimental models of SARS-CoV-2 infection as a complement of classic cell lines represents a major barrier for scientific and medical progress. Here, we advantageously used human reconstituted airway epithelial models of nasal or bronchial origin to characterize viral infection kinetics, tissue-level remodeling of the cellular ultrastructure and transcriptional immune signatures induced by SARS-CoV-2. Our results underline the relevance of this model for the preclinical evaluation of antiviral candidates. Foremost, we provide evidence on the antiviral efficacy of remdesivir and the therapeutic potential of the remdesivir-diltiazem combination as a rapidly available option to respond to the current unmet medical need imposed by COVID-19. One Sentence SummaryNew insights on SARS-CoV-2 biology and drug combination therapies against COVID-19.

microbiology↗

bioRxiv2020

Non-Toxic Virucidal Macromolecules Show High Efficacy Against Influenza Virus Ex Vivo and In Vivo

Influenza is one of the most widespread viral infections worldwide and represents a major public health problem. The risk that one of the next pandemics is caused by an influenza strain is very high. It is very important to develop broad-spectrum influenza antivirals to be ready for any possible vaccine shortcomings. Anti-influenza drugs are available but they are far from ideal. Arguably, an ideal antiviral should target conserved viral domains and be virucidal, i.e. irreversibly inhibit viral infectivity. Here, we describe a new class of broad-spectrum anti-influenza macromolecules that meets these criteria and displays exceedingly low toxicity. These compounds are based on a cyclodextrin core modified on its primary face with long hydrophobic linkers terminated in 6sialyl-N-acetyllactosamine (6SLN) or 3SLN. SLN enables nanomolar inhibition of the viruses while the hydrophobic linkers confer irreversibility to the inhibition. The combination of these two properties allows for efficacy in vitro against several human or avian influenza strains, as well as against a 2009 pandemic influenza strain ex vivo. Importantly, we show that, in mice, the compounds provide therapeutic efficacy when administered 24h post-infection allowing 90% survival as opposed to no survival for the placebo and oseltamivir..

microbiology↗