A discovery by Stanford Medicine researchers and colleagues may pave the way for a “morning after” or prophylactic nasal spray to prevent infection.
In a study published in Cell, Peter Jackson, PhD, a Stanford Medicine professor of pathology and of microbiology and immunology, and his colleagues brought that possibility closer to reality by pinpointing the routes that SARS-CoV-2, the COVID-19 virus, takes to enter and exit cells in our nasal cavity. “Our upper airways are the launchpad not only for infection of our lungs, but for transmission to others,” Jackson said.
The nose and airway are lined with epithelial tissue consisting mainly of three cell types: basal cells, goblet cells and multiciliated cells, which make up about 80% of all cells in the nasal epithelium. Multiciliated cells form a protective barrier to keep viruses from entering the airway. Jackson and his colleagues zoomed in on two structures found on multiciliated epithelial cells: cilia and microvilli. Although both are well known, neither structure has previously been implicated in how the virus enters or exits the cells lining the airway.
To see what happens during a nascent viral infection, Jackson and his associates used a sophisticated tissue culture method to generate what they call airway epithelial organoids, which mimic normal airways. While lacking blood vessels and immune cells, these organoids otherwise fully recapitulate the architecture of the nasal epithelium, including an intact mucus-mucin layer and well-developed multiciliated cells.
The scientists inoculated the cultures by incubating them in the same dish with SARS-CoV-2. With light and electron microscopy and immunochemical staining, they monitored the epithelial cells for viral entry, replication and exit.
Only ciliated cells became infected. Electron microscopy showed that the virus initially attaches only to cilia. Six hours after organoids were incubated with SARS-CoV-2, many virus particles were dotting the cilia’s sides from the tips down. Even 24 hours after inoculation, the virus was replicating only in a few cells. It took 48 hours for massive replication to occur.
SARS-CoV-2 needs a full day or two to start replicating full-tilt in real life, too. Depleting the cilia, by knocking down levels of a protein critical to cilia formation in nasal epithelial cells, severely slowed down SARS-CoV-2 infection.
“It’s clear that human ciliated nasal epithelial cells are the primary entry site for SARS-CoV-2 in nasal epithelial tissue,” Jackson said.
Suspecting that the delay in infection is due to the airway mucus-mucin barrier the virus has to cross, the researchers treated the airway organoids with a mucin-selective enzyme that breaks down the mucin-network mesh. It sped up virus entry at 24 hours from “barely detectable to easily detectable,” said Jackson, who concludes that eliminating mucin from this mesh prevented the mesh from blocking SARS-CoV-2 infection of the organoids.
At 48 hours, SARS-CoV-2 was infecting far fewer cells overall — it could infect only the immediately surrounding cells — suggesting that once SARS-CoV-2 has started replicating within infected cells, the virus relies on adequate mucus flow to help it spread throughout the upper airways.
The findings identify new targets for a nasally applied drug that, by impeding ciliary motion or microvilli gigantism, could prevent even unknown respiratory viruses. Jackson said substances used in these experiments could perhaps be optimized for use in, say, nasal sprays soon after a respiratory viral exposure, or as prophylactics.
