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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication. HIGHLIGHTS: Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection; The versatile model cell line A549-AT is suitable for rapid and sensitive high-throughput assays; Additional gene specific expression cassettes allow the screening for compounds and cellular factors limiting SARS-CoV-2 replication.
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections. We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R. We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.51-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab. In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which, however, might be circumvented by combination therapy with casirivimab together.