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Alpha-1 Antitrypsin Inhibits SARS-Cov-2 Infection

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Alpha-1 Antitrypsin Inhibits SARS-Cov-2 Infection bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.183764; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Alpha-1 antitrypsin inhibits SARS-CoV-2 infection Lukas Wettstein1, Carina Conzelmann1, Janis A. Müller1, Tatjana Weil1, Rüdiger Groß1, Maximilian Hirschenberger1, Alina Seidel1, Susanne Klute1, Fabian Zech1, Caterina Prelli Bozzo1, Nico Preising2, Giorgio Fois3, Robin Lochbaum3, Philip Knaff4,5, Volker Mailänder4,5, Ludger Ständker2, Dietmar Rudolf Thal6,7, Christian Schumann8, Steffen Stenger9, Alexander Kleger10, Günter Lochnit11, Konstantin Sparrer1, Frank Kirchhoff1, Manfred Frick3, & Jan Münch1,2* 1 Institute of Molecular Virology, Ulm University Medical Center, Ulm, 89081, Germany 2 Core Facility Functional Peptidomics, Ulm University Medical Center, Ulm, 89081, Germany 3 Institute of General Physiology, Ulm University, Ulm, 89081, Germany 4 Dermatology Clinic, University Medicine Mainz, Mainz, 55131, Germany 5 Max-Planck-Institute for Polymer Research, Mainz, 55129, Germany 6 Laboratory of Neuropathology, Department of Imaging and Pathology, KU-Leuven and Department of Pathology, UZ-Leuven, Leuven, Belgium 7 Laboratory of Pathology – Institute of Pathology, Ulm University, Ulm, 89081, Germany 8 Pneumology, Thoracic Oncology, Sleep and Respiratory Critical Care Medicine, Clinics Kempten- Allgäu, Kempten and Immenstadt, 87435, Germany 9 Institute for Microbiology and Hygiene, Ulm University Medical Center, Ulm, 89081, Germany 10 Department of Internal Medicine 1, Ulm University Hospital, Ulm, 89081, Germany 11 Institute of Biochemistry, Justus-Liebig University Giessen, Giessen, 35392, Germany Corresponding author: [email protected] Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). To identify factors of the respiratory tract that suppress SARS-CoV-2, we screened a peptide/protein library derived from bronchoalveolar lavage, and identified α1-antitrypsin (α1-AT) as specific inhibitor of SARS-CoV-2. α1-AT targets the viral spike protein and blocks SARS-CoV-2 infection of human airway epithelium at physiological concentrations. Our findings show that endogenous α1-AT restricts SARS-CoV-2 and repurposes α1-AT-based drugs for COVID-19 therapy. Main SARS-CoV-2 is mainly transmitted through inhalation of contaminated droplets and aerosols and consequently infects cells of the respiratory tract. In most cases, infection is limited to the upper airways resulting in no or only mild symptoms. Severe disease is caused by viral dissemination to the lungs ultimately resulting in acute respiratory distress syndrome, cytokine storm, multi-organ failure, septic shock, and death. The airway epithelium acts as a frontline defense against respiratory pathogens, not only as a physical barrier and through the mucociliary apparatus but also via its immunological functions1. The epithelial lining fluid is rich in innate immunity peptides and proteins with antibacterial and antiviral activity, such as lysozyme, lactoferrin or defensins2. Currently, our knowledge about innate immune defense mechanisms against SARS-CoV-2 in the respiratory tract is very limited. To identify endogenous antiviral peptides and proteins, we previously generated peptide/protein libraries from body fluids and tissues and screened the resulting fractions for antiviral factors3. This approach allowed to identify novel modulators of HIV-14,5, CMV6 and HSV-27 infection, with prospects 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.183764; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. for clinical development as antiviral drugs8. Here, we set out to identify factors of the respiratory tract that inhibit SARS-CoV-2. For this, we extracted polypeptides from 6.5 kg of homogenized human lung or 20 liters of pooled bronchoalveolar lavage (BAL), and separated them by chromatographic means. The corresponding fractions were added to Caco2 cells and infected with pseudoparticles carrying the SARS-CoV-2 spike protein9. None of the fractions of the lung library suppressed infection (Extended Data Fig. 1a). In contrast, fractions 42-45 of the BAL library prevented SARS-CoV-2 pseudoparticle infection almost entirely (Fig. 1a). The antiviral effect was comparable to that of 10 µM EK1, a spike- specific peptide fusion inhibitor used as control10 (Fig. 1a). Titration of BAL fractions 42 to 45 onto Caco2 cells confirmed that they prevent infection by SARS-CoV-2 spike pseudoparticles in a dose- dependent manner (Extended Data Fig. 2). To isolate the antiviral factor, the corresponding mother fraction 42 was further separated chromatographically and the resulting sub-fractions analyzed for antiviral activity. As shown in Fig. 1b, sub-fractions 42_4 to 42_9 and 42_57 reduced, and sub-fraction 42_55 almost completely prevented SARS-CoV-2 spike pseudoparticle infection. Analysis of these inhibitory fractions by gel electrophoresis showed distinct protein bands in sub-fractions 42_55 and 42_57, whereas no peptide/protein was detectable in sub-fractions 42_5 to 42_7 (Extended Data Fig. 3). The most active sub-fraction 42_55 contained a prominent band at ~ 52 kDa, which was also detectable in other active fractions but hardly present in those showing no antiviral activity (e.g. 42_49) (Extended Data Fig. 3). This band was excised from the gel and subjected to MALDI-TOF-MS revealing a 100 % sequence identity to α1-antitrypsin (SERPINA1) (Extended Data Fig. 4 and extended Table 1), a 52 kDa protease inhibitor11. The presence of α1-antitrypsin (α1-AT) in inhibitory fractions 42_55 and 42_57 was confirmed by Western Blots with an α1-AT-specific antibody (Extended Data Fig. 5). Fig. 1. Identification of α1-AT as SARS-CoV-2 inhibitor. a) Fractions 42-45 of the bronchoalveolar lavage library and EK1 prevent SARS-CoV-2 spike pseudoparticle infection of Caco2 cells. b) Fraction 55 of further fractionated mother fraction 42 prevents SARS-CoV-2 spike pseudoparticle infection. Subsequent analysis of the antiviral fraction identified α1-AT as major component. c) α1-AT and the control small molecule inhibitor Camostate mesylate (CM) inhibit infection of pseudoparticles carrying SARS-CoV-2 spike but not VSV-G. d) α1-AT inhibits SARS-CoV-2 spike pseudoparticle infection at an early stage. CaCo2 cells were treated for indicated hours with α1-AT and then infected with SARS-CoV-2 spike pseudoparticles. e) α1-AT and controls block SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells. Values shown in a and b are means from duplicate infections, values in c and d are means from two independent experiments performed in triplicates ± SEM, and values shown in e are means from triplicate infections ± SD. To test whether α1-AT inhibits SARS-CoV-2 infection, we used a commercially available α1-AT preparation from human blood (Prolastin®). Camostat mesylate (CM), a small molecule inhibitor of 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.183764; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. the spike priming protease TMPRSS2 was included as control9,12. α1-AT and CM both suppressed SARS-CoV-2 spike pseudoparticle infection of Caco2 cells, with half-maximal inhibitory concentrations (IC50) of ~ 2 mg/ml for α1-AT, and ~ 0.05 µM for CM, respectively (Fig. 1c). Cell viability assays showed that α1-AT displays no cytotoxic effects at concentrations of up to 8.2 mg/ml, whereas CM reduced cell viability at concentrations of 200 µM, due to DMSO in the stock (Extended Data Fig. 6). The antiviral activity of α1-AT and CM was specific for the corona virus spike because infection of pseudoparticles carrying the G-protein of VSV was not affected (Fig. 1c). These data suggest that α1-AT may inhibit an early step in the SARS-CoV-2 replication cycle. In fact, time of addition experiments showed that α1-AT blocks infection if added to cells 1 to 4 hours prior to or during SARS-CoV-2 pseudovirus infection (Fig. 1d) , but not if added 2 hours post infection (Extended Data Fig. 7). To determine whether α1-AT inhibits not only Spike-containing pseudoparticles but also wild-type SARS-CoV-2, we examined its activity against two SARS-CoV-2 isolates from France and the Netherlands. For this, we assessed survival rates of Vero cells infected in the absence or presence of EK1, CM or α1-AT. In the absence of drugs, infection by both SARS-CoV-2 isolates resulted in a massive virus-induced cytopathic effect (CPE) and reduced cell viability by about 80% (Extended Data Fig. 8). Microscopic evaluation revealed the absence of CPE in the presence of high concentrations of the compounds (not shown). MTS assays confirmed a concentration-dependent inhibition of cell killing and viral replication by EK1 and CM (Extended Data Fig. 8, Fig. 1e) with average IC50 values against both SARS-CoV-2 isolates of 2.81 ± 2.52 µM for EK1, and 3.62 ± 0.03 µM for CM, respectively (Fig. 1e). Strikingly, α1-AT suppressed the French SARS-CoV-2 isolate with an IC50 of 0.58 mg/ml, and the Dutch strain with an IC50 of 0.81 mg/ml (Fig. 1e). Almost complete rescue of cell viability was observed at α1-AT concentration of 2-4 mg/ml (Extended Data Fig. 8, Fig. 1e). Fig. 2. α1-AT inhibits SARS-CoV-2 infection of human airway epithelium cultures. The apical site of HAEC was exposed to buffer (mock), α1-AT (500 µg/ml) and remdesivir (5 µM) and then inoculated with SARS-CoV- 2. Tissues were fixed 2 days later, stained with DAPI (cell nuclei), a SARS-CoV-2 specific spike antibody and an α-tubulin-specific antibody.
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