key: cord-1023174-sqtk8dpm
authors: Rajan, Anubama; Weaver, Ashley Morgan; Aloisio, Gina Marie; Jelinski, Joseph; Johnson, Hannah L.; Venable, Susan F.; McBride, Trevor; Aideyan, Letisha; Piedra, Felipe-Andrés; Ye, Xunyan; Melicoff-Portillo, Ernestina; Yerramilli, Malli Rama Kanthi; Zeng, Xi-Lei; Mancini, Michael A; Stossi, Fabio; Maresso, Anthony W.; Kotkar, Shalaka A.; Estes, Mary K.; Blutt, Sarah; Avadhanula, Vasanthi; Piedra, Pedro A.
title: The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics
date: 2021-07-28
journal: bioRxiv
DOI: 10.1101/2021.07.28.453844
sha: 9cad7482955344ddfa4724be46e9b4d0c819cb80
doc_id: 1023174
cord_uid: sqtk8dpm

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

There is an unmet need for pre-clinical models to understand the pathogenesis of human 24 respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve 25 as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on 26 invasive techniques to obtain patient samples. Here, we report a non-invasive technique to 27 generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made 28 air liquid interface (ALI) cultures from HNOs and assessed infection with two major human 29 respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome 30 coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-31

CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus 32 hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system 33 to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the 34 that represent the fetal or developmental stages of the lung [2-6] and the Clevers group recently 48

reported an advanced method for long-term culturing of human lung tissue-derived AOs [7] . 49 Brewington et al [8] and Gamage et al [9] also modeled nasal epithelial cells using nasal biopsy 50 samples and nasal brushings. However, all the above methods utilize invasive techniques and 51 typically require physicians to obtain lung tissue or bronchoalveolar lavage or nasal brushings 52 from patients, which limit their application to the general researchers and for therapeutic 53 screening. Therefore, a critical need remains for the development of a non-invasive method for 54 generating AOs that can be readily applied to both pediatric and adult populations. Here we report 55 a novel, expandable, ex-vivo human nose organoid (HNO) model that capitalizes on non-invasive 56

techniques and yet retains the architecture of the respiratory epithelium. Additionally, we have 57 effectively modeled these nasal wash and swab derived HNOs to study the pathogenies of the 58 major pediatric respiratory viral pathogen, respiratory syncytial virus (RSV), and the foremost 59 global respiratory viral pathogen, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-60 2). 61

Globally, RSV infection in children <5 years results in 33.1 million cases, 3.2 million 62 hospitalizations, and up to 200,000 deaths, annually [10, 11] . RSV is the major cause of acute 63 lower respiratory tract illness (ALRTI) in children accounting for approximately 20% of all ALRTI 64 [12] . RSV infects almost all children by two years of age, and causes repeated reinfections 65 throughout life [13] . RSV is also a significant cause of respiratory disease morbidity and mortality 66 in older adults, immunocompromised adults, and those with chronic pulmonary disease [14] . 67

Unlike RSV, SARS-CoV-2 pandemic has resulted in a record-breaking global disaster. The rapidly 68 spreading SARS-CoV-2 has caused over 194 million cases and 4.1 million deaths as of July 26, 69 2021 [15] . Therefore, it is important to develop human model systems to study viral pathology and 70 to test therapeutics against them. In this study, we modeled an HNO-derived air liquid interface 71 (ALI) culture system to grow and study RSV, and SARS-CoV-2 infections. The HNO-ALI cultures 72 were readily infected by contemporaneous RSV strains (RSV/A/Ontario (ON) and RSV/B/Buenos 73 Aires (BA)), and SARS-CoV-2 (WA-1) resulting in epithelial damage reminiscent of human 74 pathology. Cytokine analysis of RSV and SARS-CoV-2 infected HNO epithelium demonstrated 75 cell polarity specific response thus highlighting the importance of using polarized cells to 76 understand the host immune response. Furthermore, this HNO respiratory virus model functions 77

as an ex-vivo human challenge model where we tested the efficacy of palivizumab, a monoclonal 78 antibody used for the prevention of RSV. 79

We established six lines of HNOs using stem cells isolated from nasal wash and mid-82 turbinate swab samples collected from human volunteers adopted from a recently published 83 protocol [7] ( Figure 1A ). The 3D HNOs formed between 2-3 weeks after initial adaptation of stem 84 cells to specialized growth media conditions ( Figure 1B-C) . Microscopically, the differentiated 85 HNO-ALI culture system was composed of polarized, pseudostratified airway epithelium 86 containing basal cells (keratin 5 positive, KRT5), secretory club cells SCGB1A1 (CC10 positive), 87 goblet cells (mucin 5AC positive, Muc-5AC), and ciliated cells (acetylated tubulin positive, Ace-88 tub) (Fig 1D-G) . Beating cilia of HNO-ALI were also visible under light microscopy (Movie V1). 89

We performed RNA sequencing of undifferentiated 3D HNOS, and early (21 day) and late 90 differentiated (31 day) HNO-ALI to analyze airway cell specific gene expression pattern. Our data reveals that the HNO-ALI transcriptome was highly enriched for several of airway epithelial cell 92 specific-markers including keratins, dynein, and secretoglobins (Appendix 1); and also showed 93 hallmark of ciliary function as shown by gene set enrichment analysis (GSEA) (Supplemental 94 Figure 1 ). In summary, our HNOs retained the in-vivo characteristics of human airway epithelium 95 viral infected cells, ciliary damage, and epithelial thinning ( Figure 3A -3D). Early infection (6hrs 116 and 1 dpi) was comparable between RSV/A/ON, RSV/B/BA, and SARS-CoV-2, but SARS-CoV-117 2 infection caused more damage at later time points (6-dpi). Also, RSV infection appeared 118

confined to the apical cell layer. By contrast, SARS-CoV-2 spike protein antigen was detected 119 deeper into the basal cell layer. Using fluorescence threshold analysis to quantify cilia expression 120 (acetylated tubulin or Ace-Tub), we found that SARS-CoV-2 caused significantly higher ciliary 121 damage in comparison to RSV ( Figure 3E ). The thickness of the epithelium also was significantly 122 lower in SARS-CoV-2 infected cells in comparison to RSV ( Figure 3F ). The H&E and PAS-AB 123 staining of HNO-ALI further revealed severe extrusion, epithelial thinning and rounding of apical 124 cells in SARS-CoV-2 infected samples (Supplemental Figure 2 ). In contrast, RSV but not SARS-125

CoV-2 showed hypersecretion of mucus as quantified by expression of MUC5AC marker ( Figure  126 3C, 3D, 3G and Supplemental Figure 3) . 127

inducible chemokine response in HNO-ALI cultures. 129

Characterizing host immune response and identification of biomarkers is crucial towards 130 modeling of RSV and SARS-CoV-2 pathogenesis in these advanced HNO-ALI culture systems. 131

To do this, we performed Luminex cytokine analysis of 29 cytokines/chemokines in HNO2-ALI 132 (Appendix 3). Bronchial and nasal epithelial cells are known to secrete inflammatory cytokines in 133 response to viral infections. We analyzed the levels of inflammatory cytokines induced by ALI cultures in response to (i) both RSV/A/ON and RSV/B/BA infection at 1-dpi, 2-dpi, 5-dpi, and 135 10-dpi (Appendix 3) and (ii) SARS-CoV-2 infection at 6hr, 3-dpi, and 6-dpi. RSV infection induced 136 a strong IFN-λ1/IL-29 response in HNO-ALI cultures at 5-dpi and 10-dpi. In striking contrast, 137

HNO2-ALI showed no changes in the levels of IFN-λ1 in response to SARS-CoV-2 infection 138 ( Figure 4A and 4G). Notably, for RSV strong inductions were observed for chemokine (C-X-C 139 motif) ligand 10 (IP-10), CXCL9, CXCL11/IP-9 and regulated on activation, normal T cell expressed and secreted (RANTES) from both the apical and basolateral side of the transwells. 141 ( Figure 4B -4E ). The C-X-C chemokine ligands are generally upregulated by IFN-gamma (γ) 142 produced from activated T cells and natural killer cells, none of which were present in the HNO-143 ALI cultures. Next, interleukin-8 (IL-8), a classical biomarker of RSV infection was also detected 144 at 5-dpi and 10-dpi at both the apical and basolateral side of HNO-ALI cultures (Supplemental 145 Table 3 ) [16] . RSV induced high levels of vascular endothelial growth factor A (VEGF-α) 146 specifically on the basolateral side of HNO-ALI. SARS-CoV-2 infections showed similar trends in 147 the levels of the same cytokines, but the concentration of these cytokines was much lower than 148 RSV ( Figure 4I -4L). In contrast, increase in CXCL10 (IP-10) by ~100-fold was measured on the 149 basolateral side of SARS-CoV-2 infected samples at 6-dpi, which has been reported as a 150 biomarker of COVID-19 disease severity ( Figure 4H ) [17] . 151

Increase in matrix metalloproteinase 9 (MMP-9) was reported in COVID-19 patients with 152 respiratory failure [18] and during RSV infection [19] . However, we did not detect significant 153 changes in the levels of MMP-9 or MMP-7 to either RSV or SARS-CoV-2 (Appendix 3). Perhaps, 154 this could be due to the inhibitory effect of TIMP-1 on MMPs. Additionally, for both RSV and 155 SARS-CoV-2, we did not detect any changes in levels of some pulmonary fibrosis biomarkers 156 such as transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), granulocyte 157 colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-158 CSF) [20] . On the other hand, VEGF was elevated and has been associated with disease severity 159 in idiopathic pulmonary fibrosis [20] . Though measured in low amounts (less than 100 pg/ml), we 160 noticed an increase in levels of proinflammatory immune mediators such as IL-6, and IL-1α for 161 both RSV and SARS-CoV-2 as seen in clinical disease (Appendix 3). In contrast, only for RSV 162 and at the basolateral side, monocyte chemoattractant protein 1 (MCP-1), MIP-1β, and tumor 163 necrosis factor-α (TNF-α) levels increased at 5-dpi. Eotaxin, IL-1β, and MCP-3 were both 164 increased at apical and basolateral side at low levels in response to RSV infection (Appendix 3).

Although multiple pre-clinical animal models are available to recapitulate some 167 pathognomonic aspects of RSV and SARS-CoV-2 infection, they do not faithfully represent the 168 physiology of human airway epithelium [21] [22] [23] [24] [25] [26] . Additionally, there are advanced iPS and lung 169 tissue derived AO models where the disease can be modeled but they require access to clinical 170 samples to establish these culture systems [7, 27, 28] . Thus, there is an unmet and urgent need 171 for physiologically relevant, yet easily accessible pre-clinical human airway models for respiratory 172 viral diseases to test the efficacy of therapeutics. To address this, we tested the ability of the 173 HNO-ALI system to act as an ex-vivo human challenge respiratory virus model to test the efficacy 174 of a known therapeutic monoclonal antibody (mAb) to RSV infection. Palivizumab (Synagis®), is 175 a neutralizing mAb targeted against the F-glycoprotein of RSV that prevents RSV-cell fusion and 176 hence reduces RSV replication [29] . We introduced the palivizumab mAb in the basolateral 177 compartment and monitored its neutralizing capacity on the apical lumen mimicking the 178 neutralizing effects of mAb in circulation on the virus exposed airway epithelium (Supplemental 179 Figure 3B ). The palivizumab concentrations used were in the biological range shortly after 180 intravenous injection (640µg/ml) or prior to the next administration dose (80µg/ml) [30] . 181 HNO2-ALI cultures pre-incubated with palivizumab at 640µg/ml suppressed 182 RSV/A/Tracy replication up until 2-dpi. Thereafter, RSV replication resumed at 4-dpi although at 183 reduced levels as compared to the no palivizumab control, and replication reached a peak at 6-184 dpi, and finally plateaued at 8-dpi ( Figure 5A ). This demonstrated both the efficacy of palivizumab 185 to reduce infection in the apical ciliated cells and also suggested a decline in bioavailability of 186 palivizumab to persistently prevent RSV replication at later time points. In a subsequent 187 experiment, HNO-ALI cultures were pre-incubated with palivizumab prior to the virus inoculation 188 and given a second dose at 4-dpi. In this experimental setup, RSV/A/Tracy failed to replicate in 189 HNO-ALI culture at both 640µg/ml and 80µg/ml of palivizumab ( Figure 5B ). Nonetheless, palivizumab resistant strain-RSV/Tracy P-Mab R [31] readily replicated in palivizumab pre-treated 191 HNO-ALI cultures demonstrating the specificity of palivizumab in preventing RSV infection 192 ( Figure 5C ). Furthermore, we analyzed the cytokines expressed by palivizumab pre- incubated-193 HNOs that were inoculated with RSV. Palivizumab pre-treatment with either the 80 or 640 µg/ml 194 dose, followed with second dose not only prevented RSV replication ( Figure 5B and 5E) but also 195 abolished the virus-induced inflammatory cytokines that would have been released into the apical 196 lumen and basolateral compartment ( Figure 5G -5K) . 197

Airway organoids are three-dimensional airway culture systems that were first developed 199 in 1993 with self-organizing 3D structures [32] . To date, there are only a handful of AO models 200 that are produced using invasive lung biopsy or bronchoalveolar lavage (BAL) patient-donor 201 materials [4, 6, 7, [33] [34] [35] [36] [37] [38] [39] . In this study for the first time, we describe a non-invasive, reproducible 202 and a reliable approach to establish human nose organoids (HNOs) that allows for long-term 203 expansion. Earlier studies used invasive nasal brushing samples or biopsy samples that are 204 traditionally obtained from human subjects undergoing bronchoscopy procedures. In our protocol, 205 we used human nasal wash and mid-turbinate swab samples from healthy volunteers to generate 206 nasal organoids. The ease in obtaining the nasal wash/mid-turbinate swab samples facilitates our 207 non-invasive approach into the general adult population as well as the vulnerable pediatric 208 population. These nasal wash and mid-turbinate samples can either be self-collected or through 209 a trained laboratory technician. A notable difference between our HNO-ALI culture and other ALI 210 In our HNO-ALI model, the apical side of the epithelium is both accessible and air exposed, thus 230 making it both physiologically relevant and highly suitable for studying respiratory viral infections. 231

Here, we report that the HNO-ALI system readily supports the replication and growth of RSV and 232 SARS-CoV-2 (Figure 2A -2C) and the newly released virions from these cultures are infectious 233 ( Figure 2D, and 2E ). Using immunohistochemistry, we also showed that the apical ciliated an increase in mucus secretion ( Figure 3B, and 3D) . 244

We performed a thorough characterization of host immune response to RSV and SARS-245

CoV-2 infection in HNO-ALI system. Although RSV infection predominantly occurred at the apical 246 surface, we measured higher levels of cytokine response to viral infection on the basolateral side 247

suggesting translocation of infection signals from the apical side to the basolateral compartment 248 mimicking physiological relevance. We also observed a strong type-III interferon (IFN-λ1/IL-29) 249 response for RSV and not SARS-CoV-2 suggesting stark differences in the role of the epithelial 250 cells in initiating the early innate and antiviral immune signaling. It was interesting to observe that, 251 the increase in IFN-λ1/IL-29 responses to RSV at 5-dpi was associated with a reduced or stalled 252 viral replication beyond 5-dpi as noted by plateauing of viral copy number measured by RT-PCR 253 shown and supported the growth of RSV and SARS-CoV-2 only up to 4-dpi [7, 28, 40] . However, 267

in our system, we extended the viral infection for RSV beyond 8 days and for SARS-CoV-2 beyond 268 6 days. This allows for long-term study of viral infections. Thus, we hope that this HNO-derived 269 airway epithelial model will greatly enhance the understanding of RSV and SARS-CoV-2 270

Currently, there is an unmet need for an easily available preclinical human model to study 272 RSV and SARS-CoV-2 infections that recapitulates the human experience. While animal models 273 of infection have provided major insights, they have also misdirected our understanding of viral 274 pathogenesis and prevention [25] . Also, the cost, safety and ethical issues associated with human 275 challenge models limit their use and access [49] . Our ex-vivo HNO-ALI model is an alternative to 276 the human challenge model. In our study, HNO-ALI cultures were pre-treated with palivizumab to 277 model immunoprophylaxis treatment to prevent RSV infection. A single dose of palivizumab pre-278 treatment of HNO-ALI culture was only partially effective in reducing RSV replication (1-dpi at 279 80µg/ml; and up to 2-dpi at 640µg/ml). Nonetheless, when a second dose of palivizumab was 280 administered at 4-dpi, RSV was unable to replicate in HNO-ALI system and the HNOs did not 281 produce inflammatory cytokines in response to RSV infection demonstrating the 282 immunoprophylactic protection against RSV infection and disease. Indeed, our HNO-ALI system 283 more closely resembled the human experience where therapeutic mAb or polyclonal antibodies 284 are administered intramuscularly or intravenously, respectively, to get into the blood circulation 285 and provide protection of the airways against RSV infection [50] [51] [52] . Thus, these HNOs will 286

provide a more precise human milieu and can function as a pre-clinical human model to 287 investigate promising therapeutics while recapitulating the complex interactions between the 288 drug, the virus, and the airway cells.

We are of the opinion that, at the current stage, the HNO-ALI system remains at a highly 290 reductionist level and has the potential for improvements. This includes advancements of HNO-291 ALI system with 1) addition of immune cells, 2) endothelial cells to further mimic the complex 292 physiology of organs and 3) furthermore genetic knock-ins and knockouts can be made to study 293 the role of specific host components in microbial infections and human diseases. With these future 294 advancements, the HNOs can be optimized to develop next-generation in-vivo human airway 295 models and used as a valuable tool to evaluate pathogenesis, therapeutics, and vaccine 296 candidates for major global respiratory viral pathogens. In addition, the HNOs retain the genetic 297 background of the individual, thus allowing the possibility to screen drugs for cancer therapeutics, 298 genetic-disease modeling, and development of personalized medicine. 299

Obtaining nasal wash/ nasal swab samples: The samples were collected under the Institutional 301

Review Board (IRB) of the Baylor College of Medicine (BCM), Houston, Texas, USA with written 302 informed consent. Self-collected nasal wash samples were obtained by instilling 3 ml of saline 303 into each nostril and collecting the fluid into a sterile cup. A paired mid-turbinate nasal swab from 304 the same volunteer was also obtained using a flocked swab. The paired nasal wash and nasal 305 swab samples were mixed and stored on ice until further processed. 306

The generation of nasal wash and nasal swab derived HNOs was based on the published protocol 308

[7]. The nasal wash along with the flocked swab was spun at 80g for 5 minutes at 4⁰C. 309

Supernatant was carefully removed and sheared using a 29-gauge insulin syringe to break up 310 mucus (if any). Digestion medium [10ml AO medium + 10mg Collagenase (Sigma C9407) +100µl 311 Fungizone (Amphotericin B)] was added and the falcon tube was kept on a rocker for 30-60 312 minutes at 37⁰C. After digestion, the nasal swab was discarded, and fetal bovine serum (FBS) was added to inactivate collagenase. The above solution was sheared using a syringe, strained 314 through a 100µm strainer, and spun at 80g for 5 minutes at 4⁰C. The supernatant was removed, 315 and the cell pellet was washed twice with Wash Medium (96 ml Advanced DMEM/F12 + 1 ml 316

Glutamax 100x + 1 ml HEPES 1M + 1ml Pen/Strep + 1ml Fungizone) and spun at 80g for 5 317 minutes at 4⁰C. Finally, the Wash Medium was removed, and the cell pellet was suspended in 318

Matrigel® and plated onto a 24 well plate and incubated for 10 minutes at 37⁰C. Once Matrigel® 319 had solidified, 500µl of AO medium with Penicillin Streptomycin Amphotericin (PSA) was added 320

to each well and the plate was transferred into 37⁰C incubator. AO medium was replaced every 4 321 days and passaged every other week at 1:2 ratio (wells) for expansion. 322

The mature 3D HNOs were enzymatically and mechanically sheared to make ALI cultures using 324 our previous method adopted from enteroid monolayer technology and conditions utilized for 325 growing human bronchial epithelial cells [33, [53] [54] [55] [56] . Clear transwells (Corning Costar, Catalog # 326 3470) were pre-coated with 100µl of Bovine Type I collagen at 30µg/ml (Gibco, Catalog # 327 A1064401) and placed in an incubator for 1.5 hour at 37⁰C. HNO cultured in AO medium for 7 328 days were dissociated using 0.5mM EDTA and spun at 300g for 5 minutes at 4⁰C. Single cells 329

were obtained by adding 0.05% Trypsin/0.5mM EDTA (Invitrogen, Catalog # 25300054) for 4 330 minutes at 37⁰C. Trypsin was inactivated by addition of AO medium containing 10% FBS. The 331

HNOs were dissociated vigorously using pipette tips, passed through 40µm strainer (Falcon, 332

Catalog # 352340) and pelleted at 400g at room temperature for 5 minutes to generate single 333 cells. The pellet was resuspended in AO medium containing 10µM Y-27632 + Epidermal Growth 334

Factor (EGF) (Peprotech-AF-100-15). The collagen coating from transwells was removed, 335 washed with phosphate buffered saline (PBS) and the single cells were added at a seeding 336 density of 3 x 10 5 cells/well. 750µl of AO medium + EGF containing 10µM Y-27632 (Sigma, 337

Catalog # Y-0503) was added into the lower compartment of the transwells. After 4 days, confluent monolayers are cultured in air-exposed conditions using differentiation medium (PneumaCult-ALI 339 medium from STEMCELL Technologies) in lower compartments for Transwell until 21 days. 340 Viral infection, PCR, and plaque assays. SARS-CoV-2 as previously described [57, 58] . RSV titer was measured using semi-quantitative 358 plaque assay as previous described [59] . 

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emission). Z stacks (0.25µm) of the whole section (~10µm) were acquired before applying a 388 conservative restorative algorithm for quantitative image deconvolution using SoftWorx v7.0. Max 389 intensity projections were used for image analysis and processed using ImageJ/Fiji. Between 6 390 and 10 slides were imaged per treatment/biological replicate and analyzed for IF studies. Every 391 experiment was performed in two technical transwell replicates and repeated for minimum of two 392 biological replicates. For visualization, RSV/SARS-CoV-2 spots were enhanced by histogram 393 stretching across treatments, post image acquisition in Fiji. The average area of ciliated epithelium 394 was quantified by cell counts with acetylated-tubulin in Fiji at the beginning of each infection (day 395 1 for RSV, 6hr for SARS-CoV-2), the midpoint (day 5 for RSV, 3 days for SARS-CoV-2), and the 396 endpoint of infection assays (day 10 for RSV and day 6 for SARS-CoV-2). Quantification of 397 amount of epithelial damage by RSV-A, and SARS-CoV-2 was measured by calculating the 398 average thickness of the epithelium in µm. Percentage of goblet cells (MUC5AC labeled area) 399 relative to DAPI after infection with RSV-A, RSV-B and SARS-CoV-2 was measured using the 400 formula "μm 2 MUC5AC label / μm 2 of epithelium (DAPI)" at the beginning of each infection (day 1 401 for RSV, 6hr for SARS-CoV-2), the midpoint (day 5 for RSV, 3 days for SARS-CoV-2), and the 402 endpoint of infection assays (day 10 for RSV and day 6 for SARS-CoV-2). GraphPad Prism 9.0 403 was used to construct graphs and perform statistical tests.