Study indicates that SARS-CoV-2 has evolved to gain increased replicative fitness and become well-adapted in epithelial cells of human airways

By Bhavana KunkalikarApr 20 2023Reviewed by Lily Ramsey, LLM

In a recent study published in the Proceedings of the National Academy of Sciences Journal, researchers explored increasing replicative fitness of emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in human nasal and airway organoids.

Study: Human airway and nasal organoids reveal escalating replicative fitness of SARS-CoV-2 emerging variants. Image Credit: MIA Studio/Shutterstock.com

Background

SARS-CoV-2 variants have evolved efficiently since late 2021. The rising number of SARS-CoV-2 Omicron BA.5 sublineage cases worldwide directly manifested its improved transmissibility over related subvariants and past variants.

Studies have reported the replicative efficacy of emerging variants in inducible pluripotent stem cells (iPSC)-derived respiratory organoids with increased viral gene copy numbers for BA.5 cases compared to BA.2 cases.

As opposed to the widespread documentation related to immune escape, little is known regarding SARS-CoV-2 intrinsic fitness within human respiratory cells, which are the main target of SARS-CoV-2.

About the study

In the present study, researchers examined the replicative fitness of SARS-CoV-2 Omicron BA.5 subvariant and prior variants in respiratory organoids.

The team first evaluated the replication kinetics related to a BA.5 clinical isolate detected in airway organoids compared to a B.1.1.529 isolate and a wildtype HKU-001a strain (WT).

Only the monolayers of the nasal and airway organoids which were placed on transwell inserts were employed since these monolayers could sustain stronger SARS-CoV-2 growth as compared to their 3D counterparts.

The team harvested cell-free culture media obtained from infected airway organoids after inoculation with 0.1 multiplicity of infection (MOI), and estimated viral growth by examining viral gene copy numbers along with viral titration.

SARS-CoV-2 viruses were routinely propagated in VeroE6-transmembrane serine protease 2 (TMPRSS2) cells before titrating them in the cells. The team investigated viral replication kinetics after 0.1 MOI. The replication kinetics of B.1.1.529, BA.5, and WT in alveolar organoids that recapitulated cellular contents, functionality, and morphological characteristics of the native alveolar epithelium were also explored.

Results

The study showed that B.1.1.529 replication achieved a considerably higher titer than WT. The TCID59 assay revealed that BA.5 had the highest replicative capacity, almost two to three log units more than B.1.1.529 and over four log units more than WT.

Also, BA.5 displayed a peak viral titer of more than seven log units/mL after 24 hours post-infection (h.p.i.). The team found virions generated by BA.5- and B.1.1.529-inoculated airway organoids, creating typical plaques within VeroE6-TMPRSS2 monolayers.

Yet, none of the plaque formations were discernible after the culture media was inoculated from WT-infected airway organoids. Furthermore, the plaque assay showed robust BA.5 replication, with the highest viral titer of 2.2 log units more than that of B.1.1.529.

BA.5 viral titer was the highest at 24 h.p.i., which declined at 72 h.p.i., indicating BA.5 could cause a productive infection even with a lower MOI. Therefore, the team infected airway organoids with three viral strains at both 0.01 and 0.001 MOI, which showed that B.1.1.529 and WT negligibly replicated within airway organoids after inoculations of low MOI. On the other hand, BA.5 robustly replicated.

During confocal imaging, the team noted apparent syncytium formation within BA.5-infected airway and nasal organoids. Syncytial bodies which were positive for pneumocyte markers were detected in the lung tissue autopsy of deceased SARS-CoV-2-infected patients, which was an in vivo characterization of the fusogenic ability of the SARS-CoV-2 spike protein.

Viral nucleoprotein (NP+) cells can group in the organoid monolayers infected with WT- and B.1.1.529, especially in a more active infection in nasal organoids.

Yet, individual infected cells within the cluster displayed a clear boundary marked by cellular F-actin filaments. Additionally, NP+ multinucleated syncytia characterized by typical morphology were easily detectable in BA.5-infected airways and nasal organoid monolayers.

Conclusion

The study findings showed that BA.5 revealed a significantly improved fitness compared to a WT strain and the SARS-CoV-2 Omicron B.1.1.529 subvariant in the human nasal and airway organoids.

The researchers believe that the main reason for enhanced fitness is the ability of BA.5 spike protein to elicit syncytium formation in the nasal and airway organoids.

Considering this, highly effective intranasal vaccination to trigger a potent mucosal immune response, production of antivirals against SARS-CoV-2, and therapeutic methods could provide effective techniques to curb SARS-CoV-2 transmission.