An endogenously activated antiviral state restricts SARS-CoV-2 infection in differentiated primary airway epithelial cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media.

The culture of differentiated human airway epithelial cells allows the study of pathogen-host interactions and innate immune responses in a physiologically relevant in vitro model. As the use of primary cell culture has gained popularity the availability of the reagents needed to generate these cultures has increased. In this study we assessed two different media, Promocell and PneumaCult, during the differentiation and maintenance of well-differentiated primary nasal epithelial cell cultures (WD-PNECs). We compared and contrasted the consequences of these media on WD-PNEC morphological and physiological characteristics and their responses to respiratory syncytial virus (RSV) infection. We found that cultures generated using PneumaCult resulted in greater total numbers of smaller, tightly packed, pseudostratified cells. However, cultures from both media resulted in similar proportions of ciliated and goblet cells. There were no differences in RSV growth kinetics, although more ciliated cells were infected in the PneumaCult cultures. There was also significantly more IL-29/IFNλ1 secreted from PneumaCult compared to Promocell cultures following infection. In conclusion, the type of medium used for the differentiation of primary human airway epithelial cells may impact experimental results.

The Secretome Profiling of a Pediatric Airway Epithelium Infected with hRSV Identified Aberrant Apical/Basolateral Trafficking and Novel Immune Modulating (CXCL6, CXCL16, CSF3) and Antiviral (CEACAM1) Proteins.

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

Comparative Therapeutic Potential of ALX-0171 and Palivizumab against Respiratory Syncytial Virus Clinical Isolate Infection of Well-Differentiated Primary Pediatric Bronchial Epithelial Cell Cultures.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections in young infants. There are no RSV-specific treatments available. Ablynx has been developing an anti-RSV F-specific nanobody, ALX-0171. To characterize the therapeutic potential of ALX-0171, we exploited our well-differentiated primary pediatric bronchial epithelial cell (WD-PBEC)/RSV infection model, which replicates several hallmarks of RSV disease in vivo Using 2 clinical isolates (BT2a and Memphis 37), we compared the therapeutic potential of ALX-0171 with that of palivizumab, which is currently prescribed for RSV prophylaxis in high-risk infants. ALX-0171 treatment (900 nM) at 24 h postinfection reduced apically released RSV titers to near or below the limit of detection within 24 h for both strains. Progressively lower doses resulted in concomitantly diminished RSV neutralization. ALX-0171 was approximately 3-fold more potent in this therapeutic RSV/WD-PBEC model than palivizumab (mean 50% inhibitory concentration [IC50] = 346.9 to 363.6 nM and 1,048 to 1,090 nM for ALX-0171 and palivizumab, respectively), irrespective of the clinical isolate. The number of viral genomic copies (GC) was determined by quantitative reverse transcription-PCR (RT-qPCR), and the therapeutic effect of ALX-0171 treatment at 300 and 900 nM was found to be considerably lower and the number of GCs reduced only moderately (0.62 to 1.28 log10 copies/ml). Similar findings were evident for palivizumab. Therefore, ALX-0171 was very potent at neutralizing RSV released from apical surfaces but had only a limited impact on virus replication. The data indicate a clear disparity between viable virus neutralization and GC viral load, the latter of which does not discriminate between viable and neutralized RSV. This report validates the RSV/WD-PBEC model for the preclinical evaluation of RSV antivirals.

Characterisation of morphological differences in well-differentiated nasal epithelial cell cultures from preterm and term infants at birth and one-year.

BACKGROUND: Innate immune responses of airway epithelium are important defences against respiratory pathogens and allergens. Newborn infants are at greater risk of severe respiratory infections compared to older infants, while premature infants are at greater risk than full term infants. However, very little is known regarding human neonatal airway epithelium immune responses and whether age-related morphological and/or innate immune changes contribute to the development of airway disease. METHODS: We collected nasal epithelial cells from 41 newborn infants (23 term, 18 preterm) within 5 days of birth. Repeat sampling was achieved for 24 infants (13 term, 11 preterm) at a median age of 12.5 months. Morphologically- and physiologically-authentic well-differentiated primary paediatric nasal epithelial cell (WD-PNEC) cultures were generated and characterised using light microscopy and immunofluorescence. RESULTS: WD-PNEC cultures were established for 15/23 (65%) term and 13/18 (72%) preterm samples at birth, and 9/13 (69%) term and 8/11 (73%) preterm samples at one-year. Newborn and infant WD-PNEC cultures demonstrated extensive cilia coverage, mucous production and tight junction integrity. Newborn WD-PNECs took significantly longer to reach full differentiation and were noted to have much greater proportions of goblet cells compared to one-year repeat WD-PNECs. No differences were evident in ciliated/goblet cell proportions between term- and preterm-derived WD-PNECs at birth or one-year old. CONCLUSION: We describe the successful generation of newborn-derived WD-PNEC cultures and their revival from frozen. We also compared the characteristics of WD-PNECs derived from infants born at term with those born prematurely at birth and at one-year-old. The development of WD-PNEC cultures from newborn infants provides a powerful and exciting opportunity to study the development of airway epithelium morphology, physiology, and innate immune responses to environmental or infectious insults from birth.

In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs).

The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis.

wichealth.org: impact of a stages of change-based Internet nutrition education program.

OBJECTIVE: To determine the usefulness and impact of on stage of change associated with 8 WIC client nutrition issues. DESIGN: Cross-sectional design. Data were collected through an online survey and via Web pages visited by clients for each module. SETTING: intervention and data collection are Internet-based. PARTICIPANTS: 39,541 WIC participants from 7 states completed a module and online survey. Subjects were likely between the ages of 18 and 34, residing in Michigan, Illinois, or Indiana, and accessing the Internet from home. INTERVENTIONS: Intervention included 5 online modules focusing on parent-child feeding behaviors. VARIABLES MEASURED: Impact variables included stage of change movement, user belief in ability to engage in behavior, and perception of site usefulness. ANALYSIS: Data were reported using frequency, ANOVA (analysis of variance) (P < . 01), and chi-square (P < .01) analyses. RESULTS: Movement in stage was greatest for the "picky eater" (PE) module. Contemplation as the beginning stage had the greatest stage movement. Participants responded well to all measures of site usefulness. User belief in ability to engage in behavior was associated with 7 of the 8 modules. IMPLICATIONS FOR RESEARCH AND PRACTICE: is a highly popular and viable method for impacting movement in stage of change with a number of parent-child feeding issues.