Sex and disease regulate major histocompatibility complex class I expression in human lung epithelial cells.

Major histocompatibility complex class I (MHC I) molecules present peptides to CD8+ T-cells for immunosurveillance of infection and cancer. Recent studies indicate lineage-specific heterogeneity in MHC I expression. While respiratory diseases rank among the leading causes of mortality, studies in mice have shown that lung epithelial cells (LECs) express the lowest levels of MHC I in the lung. This study aims to answer three questions: (i) Do human LECs express low levels of MHC I? (ii) Is LEC MHC I expression modulated in chronic respiratory diseases? (iii) Which factors regulate MHC I levels in human LECs? We analyzed human LECs from parenchymal explants using single-cell RNA sequencing and immunostaining. We confirmed low constitutive MHC I expression in human LECs, with significant upregulation in chronic respiratory diseases. We observed a sexual dimorphism, with males having higher MHC I levels under steady-state conditions, likely due to differential redox balance. Our study unveils the complex interplay between MHC I expression, sex, and respiratory disease. Since MHC I upregulation contributes to the development of immunopathologies in other models, we propose that it may have a similar impact on chronic lung disease.

Hypoxia Promotes Invadosome Formation by Lung Fibroblasts.

Lung parenchymal hypoxia has emerged as a cardinal feature of idiopathic pulmonary fibrosis (IPF). Hypoxia promotes cancer cell invasion and metastasis through signaling that is dependent upon the lysophosphatidic acid (LPA) receptor, LPA1 (LPAR1). Abundant data indicate that LPA1-dependent signaling also enhances lung fibrogenesis in IPF. We recently reported that fibroblasts isolated from the lungs of individuals with IPF have an increased capacity to form subcellular matrix-degradative structures known as invadosomes, an event that correlates with the degree of lung fibrosis. We therefore hypothesized that hypoxia promotes invadosome formation in lung fibroblasts through LPA1-dependent signaling. Here, it is demonstrated that invadosome formation by fibroblasts from the lungs of individuals with advanced IPF is inhibited by both the tyrosine receptor kinase inhibitor nintedanib and inhibition of LPA1. In addition, exposure of normal human lung fibroblasts to either hypoxia or LPA increased their ability to form invadosomes. Mechanistically, the hypoxia-induced invadosome formation by lung fibroblasts was found to involve LPA1 and PDGFR-Akt signaling. We concluded that hypoxia increases the formation of invadosomes in lung fibroblasts through the LPA1 and PDGFR-Akt signaling axis, which represents a potential target for suppressing lung fibrosis.

Evaluation of interleukin-1 and interleukin-6 receptor antagonists in a murine model of acute lung injury.

The acute exudative phase of acute respiratory distress syndrome (ARDS), a severe form of respiratory failure, is characterized by alveolar damage, pulmonary oedema, and an exacerbated inflammatory response. There is no effective treatment for this condition, but based on the major contribution of inflammation, anti-inflammatory strategies have been evaluated in animal models and clinical trials, with conflicting results. In COVID-19 ARDS patients, interleukin (IL)-1 and IL-6 receptor antagonists (IL-1Ra and IL-6Ra, kineret and tocilizumab, respectively) have shown some efficacy. Moreover, we have previously developed novel peptides modulating IL-1R and IL-6R activity (rytvela and HSJ633, respectively) while preserving immune vigilance and cytoprotective pathways. We aimed to assess the efficacy of these novel IL-1Ra and IL-6Ra, compared to commercially available drugs (kineret, tocilizumab) during the exudative phase (day 7) of bleomycin-induced acute lung injury (ALI) in mice. Our results first showed that none of the IL-1Ra and IL-6Ra compounds attenuated bleomycin-induced weight loss and venous P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ increase. Histological analyses and lung water content measurements also showed that these drugs did not improve lung injury scores or pulmonary oedema, after the bleomycin challenge. Finally, IL-1Ra and IL-6Ra failed to alleviate the inflammatory status of the mice, as indicated by cytokine levels and alveolar neutrophil infiltration. Altogether, these results indicate a lack of beneficial effects of IL-1R and IL-6R antagonists on key parameters of ALI in the bleomycin mouse model.

Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K+) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

Rationale: Pseudomonas aeruginosa is the major bacterial pathogen colonizing the airways of adult patients with cystic fibrosis (CF) and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evade the immune system and antibiotic therapy. Although the ability of P. aeruginosa to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. Objectives: To detect and characterize intracellular P. aeruginosa in CF airway epithelium from human lung explant tissues. Methods: We sampled lung explant tissues from patients with CF undergoing lung transplantation and non-CF lung donor control tissue. We analyzed lung tissue sections for the presence of intracellular P. aeruginosa using quantitative culture and microscopy, in parallel to histopathology and airway morphometry. Measurements and Main Results: P. aeruginosa was isolated from the lungs of seven patients with CF undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P. aeruginosa within airway epithelial cells in three of the seven patients analyzed at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. Conclusions: This is the first study describing the presence of intracellular P. aeruginosa in CF lung tissues. Although intracellular P. aeruginosa in airway epithelial cells is likely relatively rare, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

Utilization of Transcriptomic Profiling to Identify Molecular Markers Predicting Successful Recovery Following Endoscopic Sinus Surgery for Chronic Rhinosinusitis.

OBJECTIVES: Successful recovery from chronic rhinosinusitis (CRS) following endoscopic sinus surgery (ESS) can be characterized by minimal presence of symptoms and absence of disease on endoscopy. However, molecular markers of surgical success remain to be characterized. These could allow for better tailoring of perioperative therapy. This study aims to identify novel molecular markers associated with surgery responsive patient. STUDY DESIGN: Prospective cohort study. SETTING: Single academic hospital center. METHOD: One hundred eighteen consecutive patients with CRS at high risk of recurrence after surgery were followed prospectively following ESS in an academic medical center. Symptomatic and endoscopic outcomes were assessed at 4 months, with success rigorously defined subjectively as minimal or no symptoms (no symptom greater than 1 on an ordinal scale of 0-3) and objectively by the absence of nasal polyposis on sinus cavity endoscopy and Lund-Kennedy endoscopic edema score no greater than 1. Samples were obtained at the time of surgery and at 4-month postoperatively. Changes associated with surgery were determined by gene expression profiling using Affymetrix's Clariom S Human HT arrays. RESULTS: Successful ESS was characterized by a mild upregulation in Type 1 inflammation, upregulation of cell cycle progression, and epithelial barrier and proliferation-associated genes and pathways. ESS failure was associated to very high levels of Type 1 inflammation along with downregulation of epithelial barrier function and regeneration genes and pathways. CONCLUSION: Successful recovery from ESS involves restoration of epithelial function and regulated activation of Type 1 inflammation. Excessively elevated Type 1 inflammation is associated with epithelial barrier dysfunction.

Azithromycin Mechanisms of Action in CRS Include Epithelial Barrier Restoration and Type 1 Inflammation Reduction.

OBJECTIVE: Previous in vitro transcriptomic profiling suggests azithromycin exerts its effects in patients with chronic rhinosinusitis (CRS) via modulation of type 1 inflammation and restoration of epithelial barrier function. We wished to verify these postulated effects using in vitro models of epithelial repair and in vivo transcriptional profiling. STUDY DESIGN: Functional effects of azithromycin in CRS were verified using in vitro models of wounding. The mechanism of the effect of azithromycin was assessed in vivo using transcriptomic profiling. SETTING: Academic medical center. METHODS: Effects of azithromycin on the speed of epithelial repair were verified in a wounding model using primary nasal epithelial cells (pNEC) from CRS patients. Nasal brushings collected pre-and posttreatment during a placebo-controlled trial of azithromycin for CRS patients unresponsive to surgery underwent transcriptomic profiling to identify implicated pathways. RESULTS: Administration of azithromycin improved the wound healing rates in CRS pNECs and prevented the negative effect of Staphylococcus aureus on epithelial repair. In vivo, response to azithromycin was associated with downregulation in pathways of type 1 inflammation, and upregulation of pathways implicated in the restoration of the cell cycle. CONCLUSION: Restoration of healthy epithelial function may represent a major mode of action of azithromycin in CRS. In vitro models show enhanced epithelial repair, while in vivo transcriptomics shows downregulation of pathways type 1 inflammation accompanied by upregulation of DNA repair and cell-cycle pathways. The maximal effect in patients with high levels of type 1-enhanced inflammation suggests that azithromycin may represent a novel therapeutic option for surgery-unresponsive CRS patients.

Association of low-density neutrophils with lung function and disease progression in adult cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) neutrophils fail to eradicate infection despite their massive recruitment into the lung. While studies mostly focus on pathogen clearance by normal density neutrophils in CF, the contribution of low-density neutrophil (LDNs) subpopulations to disease pathogenesis remains unclear. METHODS: LDNs were isolated from whole blood donations of clinically stable adult CF patients and from healthy donors. LDN proportion and immunophenotype was assessed by flow cytometry. Associations of LDNs with clinical parameters were determined. RESULTS: LDN proportion was increased in CF patients' circulation compared with healthy donors. LDNs are a heterogeneous population of both mature and immature cells in CF and in healthy individuals. Moreover, a higher proportion of mature LDN correlates with a gradual decline in lung function and repeated pulmonary exacerbations in CF patients. CONCLUSIONS: Collectively, our observations suggest that low-density neutrophils are linked to CF pathogenesis and underscore the potential clinical relevance of neutrophil subpopulations in CF.

High-quality read-based phasing of cystic fibrosis cohort informs genetic understanding of disease modification.

Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. We sequenced 477 individuals with cystic fibrosis (CF) using linked-read sequencing, which display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, demonstrating its utility for understanding complex CF alleles. These are visualized in a Web app, CFTbaRcodes, that enables interactive exploration of CFTR haplotypes present in this cohort. We perform fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus, an intestinal obstruction at birth associated with more severe CF outcomes and pancreatic disease. A 20-kb deletion polymorphism and a PRSS2 missense variant p.Thr8Ile (rs62473563) are shown to independently contribute to meconium ileus risk (p = 0.0028, p = 0.011, respectively) and are PRSS2 pancreas eQTLs (p = 9.5 × 10-7 and p = 1.4 × 10-4, respectively), suggesting the mechanism by which these polymorphisms contribute to CF. The phase information from linked reads provides a putative causal explanation for variation at a CF-relevant locus, which also has implications for the genetic basis of non-CF pancreatitis, to which this locus has been reported to contribute.

Correlation between Neutrophil Extracellular Traps (NETs) Expression and Primary Graft Dysfunction Following Human Lung Transplantation.

Primary graft dysfunction (PGD) is characterized by alveolar epithelial and vascular endothelial damage and inflammation, lung edema and hypoxemia. Up to one-third of recipients develop the most severe form of PGD (Grade 3; PGD3). Animal studies suggest that neutrophils contribute to the inflammatory process through neutrophil extracellular traps (NETs) release (NETosis). NETs are composed of DNA filaments decorated with granular proteins contributing to vascular occlusion associated with PGD. The main objective was to correlate NETosis in PGD3 (n = 9) versus non-PGD3 (n = 27) recipients in an exploratory study. Clinical data and blood samples were collected from donors and recipients pre-, intra- and postoperatively (up to 72 h). Inflammatory inducers of NETs' release (IL-8, IL-6 and C-reactive protein [CRP]) and components (myeloperoxidase [MPO], MPO-DNA complexes and cell-free DNA [cfDNA]) were quantified by ELISA. When available, histology, immunohistochemistry and immunofluorescence techniques were performed on lung biopsies from donor grafts collected during the surgery to evaluate the presence of activated neutrophils and NETs. Lung biopsies from donor grafts collected during transplantation presented various degrees of vascular occlusion including neutrophils undergoing NETosis. Additionally, in recipients intra- and postoperatively, circulating inflammatory (IL-6, IL-8) and NETosis biomarkers (MPO-DNA, MPO, cfDNA) were up to 4-fold higher in PGD3 recipients compared to non-PGD3 (p = 0.041 to 0.001). In summary, perioperative elevation of NETosis biomarkers is associated with PGD3 following human lung transplantation and these biomarkers might serve to identify recipients at risk of PGD3 and initiate preventive therapies.

Human Lung Tissue Implanted on the Chick Chorioallantoic Membrane as a Novel In Vivo Model of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with no curative pharmacological treatment. Current preclinical models fail to accurately reproduce human pathophysiology and are therefore poor predictors of clinical outcomes. Here, we investigated whether the chick embryo chorioallantoic membrane (CAM) assay supports the implantation of xenografts derived from IPF lung tissue and primary IPF lung fibroblasts and can be used to evaluate the efficacy of antifibrotic drugs. We demonstrate that IPF xenografts maintain their integrity and are perfused with chick embryo blood. Size measurements indicate that the xenografts amplify on the CAM, and Ki67 and pro-collagen type I immunohistochemical staining highlight the presence of proliferative and functional cells in the xenografts. Moreover, the IPF phenotype and immune microenvironment of lung tissues are retained when cultivated on the CAM and the fibroblast xenografts mimic invasive IPF fibroblastic foci. Daily treatments of the xenografts with nintedanib and PBI-4050 significantly reduce their size, fibrosis-associated gene expression, and collagen deposition. Similar effects are found with GLPG1205 and fenofibric acid, two drugs that target the immune microenvironment. Our CAM-IPF model represents the first in vivo model of IPF that uses human lung tissue. This rapid and cost-effective assay could become a valuable tool for predicting the efficacy of antifibrotic drug candidates for IPF.

Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease.

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10-44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases.

Invadosome Formation by Lung Fibroblasts in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by abnormal fibroblast accumulation in the lung leading to extracellular matrix deposition and remodeling that compromise lung function. However, the mechanisms of interstitial invasion and remodeling by lung fibroblasts remain poorly understood. The invadosomes, initially described in cancer cells, consist of actin-based adhesive structures that coordinate with numerous other proteins to form a membrane protrusion capable of degrading the extracellular matrix to promote their invasive phenotype. In this regard, we hypothesized that invadosome formation may be increased in lung fibroblasts from patients with IPF. Public RNAseq datasets from control and IPF lung tissues were used to identify differentially expressed genes associated with invadosomes. Lung fibroblasts isolated from bleomycin-exposed mice and IPF patients were seeded with and without the two approved drugs for treating IPF, nintedanib or pirfenidone on fluorescent gelatin-coated coverslips for invadosome assays. Several matrix and invadosome-associated genes were increased in IPF tissues and in IPF fibroblastic foci. Invadosome formation was significantly increased in lung fibroblasts isolated from bleomycin-exposed mice and IPF patients. The degree of lung fibrosis found in IPF tissues correlated strongly with invadosome production by neighboring cells. Nintedanib suppressed IPF and PDGF-activated lung fibroblast invadosome formation, an event associated with inhibition of the PDGFR/PI3K/Akt pathway and TKS5 expression. Fibroblasts derived from IPF lung tissues express a pro-invadosomal phenotype, which correlates with the severity of fibrosis and is responsive to antifibrotic treatment.

Impact of KvLQT1 potassium channel modulation on alveolar fluid homeostasis in an animal model of thiourea-induced lung edema.

Alveolar ion and fluid absorption is essential for lung homeostasis in healthy conditions as well as for the resorption of lung edema, a key feature of acute respiratory distress syndrome. Liquid absorption is driven by active transepithelial sodium transport, through apical ENaC Na+ channels and basolateral Na+/K+-ATPase. Our previous work unveiled that KvLQT1 K+ channels also participate in the control of Na+/liquid absorption in alveolar epithelial cells. Our aim was to further investigate the function of KvLQT1 channels and their interplay with other channels/transporters involved in ion/liquid transport in vivo using adult wild-type (WT) and KvLQT1 knock-out (KO) mice under physiological conditions and after thiourea-induced lung edema. A slight but significant increase in water lung content (WLC) was observed in naïve KvLQT1-KO mice, relative to WT littermates, whereas lung function was generally preserved and histological structure unaltered. Following thiourea-induced lung edema, KvLQT1-KO did not worsen WLC or lung function. Similarly, lung edema was not aggravated by the administration of a KvLQT1 inhibitor (chromanol). However, KvLQT1 activation (R-L3) significantly reduced WLC in thiourea-challenged WT mice. The benefits of R-L3 were prevented in KO or chromanol-treated WT mice. Furthermore, R-L3 treatment had no effect on thiourea-induced endothelial barrier alteration but restored or enhanced the levels of epithelial alveolar AQP5, Na+/K+-ATPase, and ENaC expressions. Altogether, the results indicate the benefits of KvLQT1 activation in the resolution of lung edema, probably through the observed up-regulation of epithelial alveolar channels/transporters involved in ion/water transport.

Impact of temperature on the affinity of SARS-CoV-2 Spike glycoprotein for host ACE2.

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2.

The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Cystic fibrosis-related diabetes onset can be predicted using biomarkers measured at birth.

PURPOSE: Cystic fibrosis (CF), caused by pathogenic variants in the CF transmembrane conductance regulator (CFTR), affects multiple organs including the exocrine pancreas, which is a causal contributor to cystic fibrosis-related diabetes (CFRD). Untreated CFRD causes increased CF-related mortality whereas early detection can improve outcomes. METHODS: Using genetic and easily accessible clinical measures available at birth, we constructed a CFRD prediction model using the Canadian CF Gene Modifier Study (CGS; n = 1,958) and validated it in the French CF Gene Modifier Study (FGMS; n = 1,003). We investigated genetic variants shown to associate with CF disease severity across multiple organs in genome-wide association studies. RESULTS: The strongest predictors included sex, CFTR severity score, and several genetic variants including one annotated to PRSS1, which encodes cationic trypsinogen. The final model defined in the CGS shows excellent agreement when validated on the FGMS, and the risk classifier shows slightly better performance at predicting CFRD risk later in life in both studies. CONCLUSION: We demonstrated clinical utility by comparing CFRD prevalence rates between the top 10% of individuals with the highest risk and the bottom 10% with the lowest risk. A web-based application was developed to provide practitioners with patient-specific CFRD risk to guide CFRD monitoring and treatment.

SARS-CoV-2 Spike Expression at the Surface of Infected Primary Human Airway Epithelial Cells.

Different serological assays were rapidly generated to study humoral responses against the SARS-CoV-2 Spike glycoprotein. Due to the intrinsic difficulty of working with SARS-CoV-2 authentic virus, most serological assays use recombinant forms of the Spike glycoprotein or its receptor binding domain (RBD). Cell-based assays expressing different forms of the Spike, as well as pseudoviral assays, are also widely used. To evaluate whether these assays recapitulate findings generated when the Spike is expressed in its physiological context (at the surface of the infected primary cells), we developed an intracellular staining against the SARS-CoV-2 nucleocapsid (N) to distinguish infected from uninfected cells. Human airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. We observed robust cell-surface expression of the SARS-CoV-2 Spike at the surface of the infected pAECs using the conformational-independent anti-S2 CV3-25 antibody. The infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of the Spike present at the surface of the infected primary cells is maintained in serological assays involving expression of the native full-length Spike.