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The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models., Competing Interests: Competing interests statement:The authors declare no competing interest.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has now been continuing for more than two years. The infection causes COVID-19, a disease of the respiratory and cardiovascular system of variable severity. Here, the humoral immune response of 80 COVID-19 patients from the University Hospital Frankfurt/Main, Germany, was characterized longitudinally. The SARS-CoV-2 neutralization activity of serum waned over time. The neutralizing potential of serum directed towards the human alpha-coronavirus NL-63 (NL63) also waned, indicating that no cross-priming against alpha-coronaviruses occurred. A subset of the recovered patients ( n = 13) was additionally vaccinated with the mRNA vaccine Comirnaty. Vaccination increased neutralization activity against SARS-CoV-2 wild-type (WT), Delta, and Omicron, although Omicron-specific neutralization was not detectable prior to vaccination. In addition, the vaccination induced neutralizing antibodies against the more distantly related SARS-CoV-1 but not against NL63. The results indicate that although SARS-CoV-2 humoral immune responses induced by infection wane, vaccination induces a broad neutralizing activity against multiple SARS-CoVs, but not to the common cold alpha-coronavirus NL63.

Convalescent plasma is plasma collected from individuals after resolution of an infection and the development of antibodies. Passive antibody administration by transfusion of convalescent plasma is currently in clinical evaluations to treat COVID-19 patients. The level of neutralizing antibodies vary among convalescent patients and fast and simple methods to identify suitable plasma donations are needed. We compared three methods to determine the SARS-CoV-2 neutralizing activity of human convalescent plasma: life virus neutralization by plaque reduction assay, a lentiviral vector based pseudotype neutralization assay and a competition ELISA-based surrogate virus neutralization assay (sVNT). Neutralization activity correlated among the different assays; however the sVNT assay was overvaluing the low neutralizing plasma. On the other hand, the sVNT assay required the lowest biosafety level, is fast and is sufficient to identify highly neutralizing plasma samples. Though weakly neutralizing samples were more reliable detected by the more challenging lentiviral vector based assays or virus neutralization assays. Spike receptor binding competition assays are suitable to identify highly neutralizing plasma samples under low biosafety requirements. Detailed analysis of in vitro neutralization activity requires more sophisticated methods that have to be performed under higher biosafety levels., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused a pandemic with tens of millions of cases and hundreds of thousands of deaths. The infection causes coronavirus disease 2019 (COVID-19), a disease of the respiratory system of divergent severity. In the current study, humoral immune responses were characterized in a cohort of 143 patients with COVID-19 from the University Hospital Frankfurt am Main, Germany., Methods: SARS-CoV-2-specific-antibodies were detected by enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 and human coronavirus NL63 neutralization activity was analyzed with pseudotyped lentiviral vectors., Results: The severity of COVID-19 increased with age, and male patients encountered more serious symptoms than female patients. Disease severity was correlated with the amount of SARS-CoV-2-specific immunoglobulin (Ig) G and IgA and the neutralization activity of the antibodies. The amount of SARS-CoV-2-specific IgG antibodies decreased with time after polymerase chain reaction conformation of the infection, and antibodies directed against the nucleoprotein waned faster than spike protein-directed antibodies. In contrast, for the common flu coronavirus NL63, COVID-19 disease severity seemed to be correlated with low NL63-neutralizing activities, suggesting the possibility of cross-reactive protection., Conclusion: The results describe the humoral immune responses against SARS-CoV-2 and might aid the identification of correlates of protection needed for vaccine development., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread worldwide, with millions of cases and more than 1 million deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8 + and CD4 + T cell responses with S protein-specific killing activity were detected. Upon challenge using a mouse-adapted SARS-CoV-2, virus loads in vaccinated mice were significantly lower, while vaccinated Syrian hamsters revealed protection in a harsh challenge setup using an early-passage human patient isolate. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)