RESUMEN
Primary differentiated human epithelial cell cultures have been widely used by researchers to study viral fitness and virus-host interactions, especially during the COVID19 pandemic. These cultures recapitulate important characteristics of the respiratory epithelium such as diverse cell type composition, polarization, and innate immune responses. However, standardization and validation of these cultures remains an open issue. In this study, two different expansion medias were evaluated and the impact on the resulting differentiated culture was determined. Use of both Airway and Ex Plus media types resulted in high quality, consistent cultures that were able to be used for these studies. Upon histological evaluation, Airway-grown cultures were more organized and had a higher proportion of basal progenitor cells while Ex Plus- grown cultures had a higher proportion terminally differentiated cell types. In addition to having different cell type proportions and organization, the two different growth medias led to cultures with altered susceptibility to infection with SARS-CoV-2 but not Influenza A virus. RNAseq comparing cultures grown in different growth medias prior to differentiation uncovered a high degree of differentially expressed genes in cultures from the same donor. RNAseq on differentiated cultures showed less variation between growth medias but alterations in pathways that control the expression of human transmembrane proteases including TMPRSS11 and TMPRSS2 were documented. Enhanced susceptibility to SARS-CoV-2 cannot be explained by altered cell type proportions alone, rather serine protease cofactor expression also contributes to the enhanced replication of SARS-CoV-2 as inhibition with camostat affected replication of an early SARS-CoV-2 variant and a Delta, but not Omicron, variant showed difference in replication efficiency between culture types. Therefore, it is important for the research community to standardize cell culture protocols particularly when characterizing novel viruses.
RESUMEN
Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an un-derstudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insights into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affects SCV2, but not IAV, viral replicative fitness and that SCV2 infected cultures are slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changes the basal transcriptomic landscape of epithelial cells, but that it also impacts the response to infection. The induction of interferon and other innate immune responses were not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remains consistent, there may be metabolic or signaling changes that affect how well the cultures are able to adapt to new pressures such as infection. Finally, we show that hNECs respond differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.
RESUMEN
Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an understudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insight into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affected SCV2, but not IAV, viral replicative fitness and that SCV2-infected cultures were slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changed the basal transcriptomic landscape of epithelial cells, but that it also impacted the response to infection. The induction of interferon and other innate immune responses was not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remained consistent, there may be metabolic or signaling changes that affect how well the cultures were able to adapt to new pressures, such as infection. Finally, we show that hNECs responded differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.
RESUMEN
From 2013 to 2016, the H1N1 component of live, attenuated influenza vaccine (LAIV) performed very poorly in contrast to the inactivated influenza vaccine. We utilized a primary, differentiated human nasal epithelial cell (hNEC) culture system to assess the replication differences between isogenic LAIVs containing the HA segment from either A/Bolivia/559/2013 (rBol), which showed poor vaccine efficacy, and A/Slovenia/2903/2015 (rSlov), which had reasonable vaccine efficacy. There were minimal differences in infectious virus production in Madin-Darby Canine Kidney (MDCK) cells, but the rSlov LAIV showed markedly improved replication in hNEC cultures at both 32 °C and 37 °C, demonstrating that the HA segment alone could impact LAIV replication in physiologically relevant systems. The rSlov-infected hNEC cultures showed stronger production of interferon and proinflammatory chemokines which might also be contributing to the increased overall vaccine effectiveness through enhanced recruitment and activation of immune cells. An M2-S86A mutation had no positive effects on H1 LAIV replication in hNEC cultures, in contrast to the increased infectious virus production seen in an H3 LAIV. No obvious defects in viral RNA packaging were detected, suggesting that HA function, rather than defective particle production, may be driving the differential infectious virus production in hNEC cultures. Overall, we have shown that not all H1 HA segments can be successfully used in LAIV, and this phenotype cannot be fully explained by segment incompatibilities. Physiologically relevant temperatures and primary cell cultures should be used to demonstrate that candidate LAIVs can replicate efficiently, which is a necessary property for effective vaccines.
