Development of a new platform for testing antiviral drugs using coronavirus-infected human nasal mucosa organoids

doi:10.12122/j.issn.1673-4254.2024.11.20

Abstract

Abstract:

Objective To establish a coronavirus (CoV) infection model using human nasal mucosa organoids for testing antiviral drugs and evaluate the feasibility of using human nasal mucosa organoids with viral infection as platforms for viral research and antiviral drug development. Methods Human nasal mucosa organoids were tested for susceptibility to SARS-CoV-2 and HCoV-OC43 pseudoviruses. In a P3 laboratory, nasal mucosa organoids were infected with the original strain of SARS-CoV-2 and 4 variant strains, and the infection conditions were optimized. The viral loads in the culture supernatants were measured at different time points using RT-qPCR, and immunofluorescence assay was employed to localize SARS-CoV-2 nucleocapsid protein to determine the type of the infected cells. In the optimized nasal mucosa viral infection model, the antiviral effects of camostat and bergamot extract (which were known to inhibit SARS-CoV-2) were tested and the underlying molecular mechanisms were explored. Results In the optimized nasal mucosa organoid models infected with SARS-CoV-2 and HCoV-OC43 pseudoviruses, the viral load in the culture supernatants increased significantly during the period of 2 to 24 h following the infection, which confirmed infection of the organoids by both of the pseudoviruses. The nasal mucosa organoids could be stably infected by the original SARS-CoV-2 strain and its 4 variant strains, validating successful establishment of the viral infection model, in which both camostat and bergamot extract exhibited dose-dependent antiviral effects. Conclusions Human nasal mucosa organoids with SARS-CoV-2 infection can serve as platforms for screening and testing antiviral drugs, particularly those intended for nasal administration.

Key words: organoids, nasal mucosa, virus infection, disease model

Yan YU, Junyuan CAO, Rong LIU, Minmin ZHOU, Jinyan WEI, Hairui ZHENG, Wei WANG, Gang LI. Development of a new platform for testing antiviral drugs using coronavirus-infected human nasal mucosa organoids[J]. Journal of Southern Medical University, 2024, 44(11): 2227-2234.

Figures/Tables 7

Tab.1 Nasal mucosa sample information

Sample number	Source	Passage number	Days	Cryopreservation date	Culture status
RNM-0126	Respiratory region	P1	4	2021-12-17	Success
RNM-0179	Respiratory region	P1	6	2021-12-28	Success
ONM-0325	Olfactoryactory region	P0	4	2022-01-08	Success
RNM-0160	Respiratory region	P2	10	2022-03-10	Success
RNM-0234	Respiratory region	P1	8	2022-03-10	Success
RNM-0370	Respiratory region	P0	0	2022-03-10	Failure
RNM-0328	Respiratory region	P1	6	2022-03-14	Success
RNM-0344	Respiratory region	P0	8	2022-03-16	Success
RNM-0350	Respiratory region	P0	7	2022-03-16	Success
RNM-0351	Respiratory region	P0	7	2022-03-16	Success
ONM-0375	Olfactoryactory region	P0	10	2022-03-18	Success
RNM-0359	Respiratory region	P1	5	2022-03-21	Failure
RNM-0408	Respiratory region	P0	8	2022-03-23	Success
ONM-0489	Olfactoryactory region	P0	8	2022-03-23	Success

Fig.1 Observation of growth of nasal mucosal organoids under inverted microscope at different time points and HE staining of the organoids on Day 5, P25 (Scale bar=100 μm).

Fig.2 SARS-CoV-2 (A) and HCoV-OC43 (B) pseudovirus infection of nasal mucosal organoids. ***P<0.001, ****P<0.0001.

Fig.3 Modeling of SARS-CoV-2 infection in nasal mucosal organoids. A: Virus content in the supernatant at different time points following infection ay MOI of 1 and 10. B: Detection of viral content in the supernatant following infection with different viruses. *P<0.05, ***P<0.001, ****P<0.0001.

Fig.4 Immunohistochemistry for detecting coronavirus receptors and immunofluorescence staining for detecting SARS-CoV-2-infected goblet cells (scale bar=50 μm). A: Immunohistochemistry analysis of ACE2 and TMPRSS2, the key proteins for coronavirus infection (scale bar: 50 μm). B: Immunofluorescence staining of goblet cells infected by SARS-CoV-2 showing DAPI staining of the cell nuclei, MUC5AC staining of goblet cells, and viral nucleoprotein.

Fig.5 Inhibitory effect of the drugs for testing on SARS-CoV-2 in human nasal mucosal organoids. A: Camostat dose-dependently inhibits SARS-CoV-2 infection in human nasal mucosal organoids. B: Bergamottin dose-dependently inhibits SARS-CoV-2 infection in human nasal mucosal organoids. **P<0.01, ***P<0.001, ****P<0.0001 vs DMSO.

Fig.6 Bergamottin and camostat interact with ACE2 to block coronavirus infection. A: Docking position of bergamottin with human ACE2. B: Docking position of camostat with human ACE2. C: Binding position of lisinopril with human ACE2. D: Binding details of lisinopril with human ACE2. E: Docking energy of bergamottin, camostat and lisinopril against human ACE2.

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