Since the cellular serine protease TMPRSS2 causes S-protein cleavage required for efficient entry and infection by SARS-CoV-2, it is possible that the presence of specific serpins in the HBEC ALI cultures of group low inhibits S-protein cleavage and thereby prevents infection. Among the upregulated genes in group low, four individual serpins were found (SERPINA1, SERPINE1, SERPINE2, and SERPINF1), although SERPINA1 had a slightly lower significance (
P = 0.088) than the original cutoff (
P = 0.05). First, we addressed if the reduction in infection in group low could be explained by the presence of these serpins. HEK-293T cells were made susceptible to SARS-CoV-2 infection by transfection with plasmids expressing ACE2 and TMPRSS2. These cells were also cotransfected with plasmids expressing individual serpins, followed by infection with SARS-CoV-2. We focused our attention on SERPINA1 and SERPINE1, as SERPINF1 lacks serine protease inhibitory activity and SERPINE1 and -E2 are paralogs. Additionally, SERPINC1, known as antithrombin III, is an FDA-approved anticoagulant and was included here due to its therapeutic potential against COVID-19 (ClinicalTrials.gov registration no. NCT04745442). To investigate the role of serpins during the first round of infection, a 6-h time frame was chosen, allowing replication, but not release, of progeny viruses and reinfection. All three serpins efficiently reduced infection of SARS-CoV-2 in HEK-293T cells as determined by quantification of viral RNA in cells 6 h postinfection (
Fig. 4A). Next, the possible inhibition of SARS-CoV-2 entry by the serpins was analyzed. SARS-CoV-2 was added to HEK-293T cells coexpressing ACE2 and TMPRSS2 with or without individual serpins for 2 h followed by trypsination of the cells to remove bound but not internalized virus particles. A strong reduction in SARS-CoV-2 entry as measured by viral RNA could be observed with all three serpins (
Fig. 4B). After viral membrane fusion, the viral genome is translated and negative-strand RNA synthesis is initiated. As TMPRSS2 cleavage of the spike is important for membrane fusion, we also specifically measured the synthesis of negative-stranded RNA in the presence and absence of the different serpins. A strong reduction of the negative-strand RNA to the level of ACE2-transfected cells was detected when coexpressing the serpins (
Fig. 4C). This indicated that the antiviral effect of serpins occurs during the initial steps of the SARS-CoV-2 life cycle, preventing viral membrane fusion probably by inhibiting TMPRSS2-mediated cleavage of the viral S-protein. To investigate this further, we first performed surface plasmon resonance, allowing us to study the ability of these serpins to specifically interact with TMPRSS2, a prerequisite for inhibition. As a positive control, the natural target tissue plasminogen activator (tPA) was analyzed against SERPINE1. All three serpins showed a strong interaction with TMPRSS2 with nanomolar (nM) affinity (
Fig. 4D). To ensure that this interaction leads to serpin-specific inhibition of TMPRSS2-mediated cleavage of the viral S-protein, an
in vitro cleavage assay was performed. Recombinant TMPRSS2 and viral S-protein were incubated with or without recombinant SERPINA1, SERPINE1, and SERPINC1 protein or the positive control, nafamostat mesylate (
20), followed by detection of SARS-CoV-2 S-protein and its cleavage products by Western blotting. In the presence of TMPRSS2, several cleavage products of the S-protein were detected, and these were reduced when adding the protease inhibitor nafamostat mesylate (
Fig. 4E and
F). All three serpins reduced the amount of cleavage product with a similar efficiency as the positive control, indicating that the serpins specifically inhibit TMPRSS2-mediated S-protein cleavage. Next, we wanted to see if the recombinant serpins could reduce SARS-CoV-2 infection of an HBEC ALI culture from group high. HBEC ALI cultures were pretreated apically with individual recombinant SERPINA1, SERPINE1, and SERPINC1 proteins, and the infection was monitored over time. All serpins tested reduced SARS-CoV-2 infection at 48 h and 72 h postinfection (
Fig. 4G). Finally, as the expression of serpins in our HBEC ALI-cultures was based on transcriptional data (
Fig. 3A,
Table S4), we wanted to validate the presence of serpins in the apical secretions from these cultures. Apical samples were collected from uninfected HBEC ALI cultures of group high and group low, and the concentrations of SERPINA1 and SERPINE1 proteins were determined by enzyme-linked immunosorbent assay (ELISA). Although SERPINA1 was present in group high, the concentration of SERPINA1 was 4-fold higher in group low (
Fig. 4H). The concentration of SERPINE1 was generally low, and no difference was observed between the two groups. These results indicate a larger antiviral contribution by SERPINA1 and support the antiviral effect of the apical secretions in Calu3 cells (
Fig. 3F). The low levels of SERPINE1 in the apical secretions were rather unexpected, as the transcriptional difference between the two groups was high. From these data, we conclude that SERPINA1, -E1, and -C1 can prevent SARS-CoV-2 infection by specifically binding to and inhibiting TMPRSS2-mediated S-protein cleavage and subsequent entry and fusion of SARS-CoV-2 into target cells. However, the cellular distribution of the individual serpins and their relative antiviral effect against SARS-CoV-2 remain unclear.