The Human Nose Organoid Respiratory Virus Model: an Ex Vivo Human Challenge Model To Study Respiratory Syncytial Virus (RSV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Pathogenesis and Evaluate Therapeutics

Anubama Rajan, Ashley Morgan Weaver, Gina Marie Aloisio, Joseph Jelinski, Hannah L Johnson, Susan F Venable, Trevor McBride, Letisha Aideyan, Felipe-Andrés Piedra, Xunyan Ye, Ernestina Melicoff-Portillo, Malli Rama Kanthi Yerramilli, Xi-Lei Zeng, Michael A Mancini, Fabio Stossi, Anthony W Maresso, Shalaka A Kotkar, Mary K Estes, Sarah Blutt, Vasanthi Avadhanula, Pedro A Piedra, Anubama Rajan, Ashley Morgan Weaver, Gina Marie Aloisio, Joseph Jelinski, Hannah L Johnson, Susan F Venable, Trevor McBride, Letisha Aideyan, Felipe-Andrés Piedra, Xunyan Ye, Ernestina Melicoff-Portillo, Malli Rama Kanthi Yerramilli, Xi-Lei Zeng, Michael A Mancini, Fabio Stossi, Anthony W Maresso, Shalaka A Kotkar, Mary K Estes, Sarah Blutt, Vasanthi Avadhanula, Pedro A Piedra

Abstract

There is an unmet need for preclinical 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 noninvasive technique to generate human nose organoids (HNOs) as an alternative 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 hypersecretion. 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 alternative to lung organoids to study respiratory viruses and test therapeutics. IMPORTANCE Preclinical models that recapitulate aspects of human airway disease are essential for the advancement of novel therapeutics and vaccines. Here, we report a versatile airway organoid model, the human nose organoid (HNO), that recapitulates the complex interactions between the host and virus. HNOs are obtained using noninvasive procedures and show divergent responses to SARS-CoV-2 and RSV infection. SARS-CoV-2 induces severe damage to cilia and the epithelium, no interferon-λ response, and minimal mucus secretion. In striking contrast, RSV induces hypersecretion of mucus and a profound interferon-λ response with ciliary damage. We also demonstrated the usefulness of our ex vivo HNO model of RSV infection to test the efficacy of palivizumab, an FDA-approved monoclonal antibody to prevent severe RSV disease in high-risk infants. Our study reports a breakthrough in both the development of a novel nose organoid model and in our understanding of the host cellular response to RSV and SARS-CoV-2 infection.

Keywords: ALI cultures; RSV; SARS-CoV-2; air-liquid interface (ALI) culture; airway; airway organoids; cilia; cytokines; epithelium; immunoprophylaxis; mucus; nose organoids; palivizumab; respiratory syncytial virus (RSV); severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therapeutics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Derivation and characterization of human nose organoids (HNOs). (A) Schematic representation of the workflow for making of HNOs. (B) Bright-field image of 3D HNOs in culture. Scale bar equals 100 μm. (C) Hematoxylin and eosin (H&E) staining of 3D HNOs. (D) H&E staining of pseudostratified airway epithelium of HNO-ALI culture. (E to G). Immunofluorescence of HNOs. Basal cells are labeled by keratin 5 (KRT5), in red. DAPI stains each nucleus. (E) Goblet cells are shown in green, labeled by mucin 5AC (MUC5AC). (F) Ciliated epithelium is shown in green, labeled by acetylated alpha tubulin (Ace-tubulin) and (G) club cells in green, labeled by CC10. Scale bar equals 10 μm.
FIG 2
FIG 2
Infection of human nose organoids (HNOs) with respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (A to C) HNO air-liquid interface (ALI) cells were apically infected with RSV/A/Ontario (ON) and RSVB/Buenos Aires (BA) at a multiplicity of infection (MOI) of 0.01. Apical and basolateral samples were collected at 1, 2, 5, and 10 days postinoculation (dpi) in two distinct HNO cell lines (HNO2 and HNO204). RNA was isolated from media, and copy numbers of RSV N gene RNA were determined using quantitative real-time PCR (qRT-PCR). Levels of RSV N genes (copies/mL) from (A) RSV/A/ON at different time points and (B) RSV/A/BA at different time points. (C) HNO-ALI cells were apically infected with SARS-CoV-2 at an MOI of 0.01. Apical and basolateral samples were collected at the time of infection and 3 and 6 dpi in three distinct HNO cell lines (HN02, HNO204, and HNO918). RNA was isolated from media, and SARS-CoV-2 copy numbers of N gene RNA were determined using qRT-PCR. (D and E) Infectious viral titers reported as PFU per mL for RSV/A/ON and RSV/B/BA using a quantitative plaque assay. Data shown were from two individual experiments with two technical replicates per group in each experiment and are represented as the mean ± standard deviation (SD).
FIG 3
FIG 3
Immunofluorescence and morphological analysis of respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human nose organoid air-liquid interface (HNO-ALI) system. (A to D) Representative deconvoluted epifluorescence images of HNO cells showing nuclei (DAPI, blue) and basal cells (keratin 5 [KRT5], red). In panel A cilia (acetylated alpha tubulin [ace-tubulin], green) and RSV (F-protein, yellow) are shown; (B) cilia (ace-tubulin, green) and SARS-CoV-2 (spike protein, yellow); (C) goblet cells (mucin 5AC [MUC5AC], green) and RSV (fusion protein, yellow); (D) goblet cells (MUC5AC, green) and SARS-CoV-2 (spike protein, yellow). (E to G) Quantification of (E) ciliary damage, (F) epithelial damage, and (G) MUC5AC expression of HNO-ALI infected by RSV/A/ON, RSV/B/BA, and SARS-CoV-2. Data were collected from 10 representative images per group in each experiment and are represented as the mean ± SD. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Scale bar equals 10 μm.
FIG 4
FIG 4
Immune cytokine/chemokine profile of human nose organoids (HNOs) infected with respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). HNO air-liquid interface (HNO-ALI) cultures were infected apically with RSV/A/Ontario (ON), RSVB/Buenos Aires (BA), and SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. The cultured supernatants from both the apical and basolateral compartments were harvested from mock-, RSV-, and SARS-CoV-2-infected HNO-ALI cultures. Profiles of extracellular cytokines and chemokines released in the apical and basolateral compartments were determined by the multiplex-Luminex cytokine assay. (A to F) Levels of cytokines and chemokines released from HNO2 cells infected with contemporaneous RSV strains RSV/A/ON and RSV/B/BA. (G and L) Levels of cytokines and chemokines released from HNO2 cells infected with SARS-CoV-2 (WA-1 strain). The data shown were from two technical replicates per group, and the Luminex assay was run with each sample in duplicate and assessed for cytokines.
FIG 5
FIG 5
Immunoprophylaxis treatment for respiratory syncytial virus (RSV) infection in human nose organoids (HNOs). (A to C) Viral copy numbers of RSV/A/Tracy after treatment with palivizumab of 0 μg/mL, 80 μg/mL, and 640 μg/mL at 1, 2, 4, 6, and 8 dpi were measured. Panel A demonstrates only an initial but not sustained decrease in RSV copy number with a short exposure of palivizumab pretreatment. Panel B shows a significant reduction in RSV replication during the long exposure of palivizumab across the infection period in a dose-dependent manner with the addition of a second dose of palivizumab at day 4. Panel C exhibits specificity of palivizumab pretreatment, as strain RSV/A/TracyP-Mab-R is resistant to the antibody and hence showed no decrease in infection. (D to F) Quantification of infective viral particles using a plaque assay for the experiments described above. The data shown were gathered from two technical replicates per group in each experiment and are represented as the mean ± SD. (G to K) The levels of inflammatory cytokines released by RSV-infected HNOs under palivizumab treatment conditions.

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Source: PubMed

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