A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents

There are, besides Remdesivir (RDV), no approved antivirals for the treatment and/or prophylaxis of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI) and infected at the apical side with either one of two different SARS-CoV-2 isolates. The virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35°C than at 37°C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. GS-441524 (the parent nucleoside of Remdesivir), EIDD-1931 (the active metabolite of Molnupiravir) and IFN (β1 and λ1) all resulted in a dose-dependent inhibition of viral RNA and infectious virus titers at the apical side. However, AT-511 (a guanosine nucleotide previously reported to inhibit SARS-CoV-2) failed to inhibit viral replication. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.

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Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells

10.1101/637058 ◽

2019 ◽

Author(s):

Bianca Maria Rotoli ◽

Rossana Visigalli ◽

Amelia Barilli ◽

Francesca Ferrari ◽

Massimiliano G. Bianchi ◽

...

Keyword(s):

Epithelial Cells ◽

Pulmonary Delivery ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Apical Side ◽

Basolateral Side ◽

Subsequent Degradation ◽

Carnitine Transport ◽

Air Liquid Interface

ABSTRACTIn human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by β-oxidation. Aim of the present study is to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI were used as comparison. In EpiAirway™, ATB0,+ was highly expressed and functional on the apical side while OCTN2 transporter was active on the basolateral side. Calu-3 cells showed a different pattern of expression and activity for ATB0,+: indeed, L-carnitine uptake on apical side was evident in Calu-3 at 8 days of culture but not in fully differentiated 21d ALI culture. As both ATB0,+ and OCTN2, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the studies of drug inhalation and pulmonary delivery.

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