key: cord-0822450-jcekiymg authors: Ritzmann, F.; Chitirala, P.; Yao, Y.; Krueger, N.; Hoffmann, M.; Zuo, W.; Lammert, F.; Smola, S.; Seiwert, N.; Tov, N.; Alagem, N.; Mozafari, B.; Guenther, K.; Seibert, M.; Hoersch, S.; Volk, T.; Lepper, P. M.; Danziger, G.; Poehlmann, S.; Beisswenger, C.; Herr, C.; Bals, R. title: Therapeutic application of alpha-1-antitrypsin in COVID-19 date: 2021-04-06 journal: nan DOI: 10.1101/2021.04.02.21252580 sha: aae6c0218a51a95253cae81e28c6ede315a94f06 doc_id: 822450 cord_uid: jcekiymg Rationale: The treatment options for COVID-19 patients are sparse and do not show sufficient efficacy. Alpha-1-antitrypsin (AAT) is a multi-functional host-defense protein with anti-proteolytic and anti-inflammatory activities. Objectives: The aim of the present study was to evaluate whether AAT is a suitable candidate for treatment of COVID-19. Methods: AAT and inflammatory markers were measured in the serum of COVID-19 patients. Human cell cultures were employed to determine the cell-based anti-protease activity of AAT and to test whether AAT inhibits the host cell entry of vesicular stomatitis virus (VSV) particles bearing the spike (S) protein of SARS-CoV-2 and the replication of authentic SARS-CoV-2. Inhaled and / or intravenous AAT was applied to nine patients with mild-to-moderate COVID-19. Measurements and Main Results: The serum AAT concentration in COVID-19 patients was increased as compared to control patients. The relative AAT concentrations were decreased in severe COVID-19 or in non-survivors in ratio to inflammatory blood biomarkers. AAT inhibited serine protease activity in human cell cultures, the uptake of VSV-S into airway cell lines and the replication of SARS-CoV-2 in human lung organoids. All patients, who received AAT, survived and showed decreasing respiratory distress, inflammatory markers, and viral load. Conclusion: AAT has anti-SARS-CoV-2 activity in human cell models, is well tolerated in patients with COVID-19 and together with its anti-inflammatory properties might be a good candidate for treatment of COVID-19. Coronavirus disease 2019 (COVID-19) is a novel illness and is a rapidly evolving pandemic with unprecedented impact on global health in the last 100 years. COVID-19 is caused by "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), a member of the Coronaviridae family that is regarded a spill-over of an animal coronavirus to humans (1) . At the time of submission of this work, SARS-CoV-2 has caused 80 million diagnosed infections and 1·8 million known deaths. The trajectories of the pandemic are still difficult to predict. While vaccines are currently developed and enter public health prevention measures, the treatment options for COVID-19 patients are sparse and do not show sufficient efficacy. Remdesivir (2) and dexamethasone (3) are used in hospitalized patients and use of tocilizumab has also shown beneficial outcomes for this patient group (4) . The options for patients with mild COVID-19 or asymptomatic infection are scant. At the present time, there are no efficacious drugs available that can be applied non-invasively and decrease the likelihood of disease progression towards organ failure (5) . SARS-CoV-2 uses several host factors to infect target cell including the transmembrane protease serine 2 (TMPRSS2) for activation of the viral spike (S) protein, and angiotensin-converting enzyme 2 (ACE2) for entry (6) (7) (8) . The disease pathomechanisms comprise the direct consequences of cell and tissue damage and a systemic inflammatory syndrome that includes innate and adaptive immune activation, hypercoagulation and other features (9, 10) . Alpha-1-antitrypsin (AAT) is a multi-functional host-defense and acute phase protein and member of the serpin superfamily (11) . AAT is mainly produced in the liver and secreted into the blood stream. In addition, AAT is synthesized in macrophages and All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.02.21252580 doi: medRxiv preprint lung epithelial cells. Genetic deficiency results in increased susceptibility for early chronic obstructive lung disease (COPD) or chronic liver injury (11) . AAT has a broad anti-protease activity and has been implicated in several biological processes such as the regulation of inflammation (12, 13) . Intravenous or inhaled AAT has been used in multiple clinical trials for COPD or cystic fibrosis and has a good safety profile (14, 15) . In the context of hepatitis C virus infection, it has been shown that AAT inhibits TMPRSS2 (16), the same host serine protease that is necessary for the activation of the S-protein of SARS-CoV-2 (6). Based on potential inhibition of virus entry by AAT and its broad anti-inflammatory activity, the aim of this study was to investigate whether AAT can be considered as candidate for the treatment of COVID-19. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Patients for observational measurements of AAT serum levels were recruited within the COVID-19 cohort study CORSAAR (n = 35) and its control cohort PULMOHOM. Both studies have been approved by the ethics committee of the Landesärztekammer Saarland, and all patients or their legal representatives gave their informed consent. Serum C-reactive protein (CRP), AAT, and interleukin-6 (IL-6) levels were measured by multiplex arrays. Moderate disease severity was defined as hospitalized patients without need for intensive care treatment, severe disease was defined as patients with need for intensive care treatment. AAT (A6150), aprotinin (trasylol) (A3428), and camostat mesylate (SML0057) were purchased from Merck (Germany). AAT was additionally obtained from Kamada (Rehovot, Israel). Camostat mesylate (Tocris, cat. no. 3193) was reconstituted in dimethyl sulfoxide (DMSO), yielding a stock concentration of 10 mM. HEK-293T (human embryonic kidney; DSMZ no. ACC 635) cells were cultivated in Dulbecco's modified Eagle medium containing 10 % fetal bovine serum (FCS, Biochrom), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech). Calu-3 cells (human lung adenocarcinoma; kindly provided by Stephan Ludwig, Institute of Virology, University of Münster, Germany) were cultivated in minimum essential All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.02.21252580 doi: medRxiv preprint medium supplemented with 10 % FCS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (from 100x stock, PAA) and 1 mM sodium pyruvate (Thermo Fisher Scientific). 16-HBE cells, a SV40immortalized human bronchial epithelial cell line, were grown in Opti-MEM with 10 % FCS, penicillin, and streptomycin. All cell lines were incubated at 37 °C in a humidified atmosphere with 5 % CO2. HEK-293T were transfected by calcium-phosphate precipitation. Airway basal cells were isolated and cultured as described before (17) . Briefly, basal cells from the donors were cultured using Airway Epithelial Cell Growth Medium (C-21060; PromoCell) supplemented with 1 µM of A83-01 (TGF inhibitor) (2939; Biotechne), 0.2 µM of DMH-1 (BMP4 inhibitor) (4126; Biotechne) and 5 µM of Y27632 (ROCK inhibitor) (1254; Biotechne) to preserve prolonged basal cell phenotype with high proliferative capacity. Human lung organoids were cultured as described before (17) . Human bronchial epithelial cells (HBECs) were isolated from fresh brush biopsies from patients who underwent bronchoscopy. Cells were washed from the brushes with AEC media (PromoCell, Germany) and expanded in conventional 2D co-culture on NIH-3T3 feeder cells and presence of 10 µM RHO/ROCK Inhibitor (Y-27632, StemCell) to avoid loss of differentiation capacity. For organoid culture, cells were harvested and transferred in differentiation media (BEGM, Lonza, with added supplements except for retinoic acid and T3, mixed 1:1 with DMEM-F12). A 96-well plate was precoated with 40 µl differentiation media containing 25% Matrigel. 80 µl of a cell suspension with 3 x 10 4 cells/ml in differentiation media and 5% Matrigel was plated in each well. 120 µl differentiation media with 5% Matrigel was added on top on day 2 and changed every 4 days. The organoids were used for the experiments after 21 days of maturation. For this purpose, 120 µl of medium was removed and the Matrigel was dissolved by All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. The effect of AAT on epithelial cell trypsin-like protease activity was assessed using the synthetic peptide analogue (trypsin substrate), Boc-Gln-Ala-Arg-AMC (3135-V; PeptaNova GmbH, Germany). 16-HBE cell or organoids in 96 well plates were incubated with trasylol (100 or 200 µg / ml), AAT (1or 2 mg / ml), or camostat (250 µM). Finally, 2 µl of 100 µM peptide substrate was added to cells and fluorescence was measured immediately at 360 / 465 mm of excitation/emission wavelength every 2 minutes for 6 hours at 37°C. Trypsin-EDTA was used as positive control. All the compounds were dissolved in Opti-MEM. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. Expression plasmids for severe acute respiratory syndrome coronavirus 2 spike glycoprotein (SARS-2-S) and vesicular stomatitis virus glycoprotein (VSV-G) have been described elsewhere (6) . In addition, we used empty pCG1 expression plasmid (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Based on its anti-viral and anti-inflammatory activities together with large experience from therapeutic applications of AAT and the low number of side effects of treatment, we decided to offer patients with moderate COVID-19 inhaled or intravenous AAT as individual medication. As a consequence, the focus of this approach was the care for the individual patient and not a controlled clinical trial. This approach was reported to the ethics committee of the Ärztekammer des Saarlandes. As inhaled drug we used nebulized Prolastin, which is a medicinal product nationally licensed for treatment of AAT-deficiency dependent lung disease. Prolastin has been used for inhaled treatment in several trials earlier (20) . In addition, we used AAT (Kamada) for intravenous and All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Continuous data are presented as mean +/-SD (standard deviation) when normally distributed, if not otherwise indicated. Categorical data are presented as percentages. Statistical differences between continuous variables were determined using Student's t-test or chi-squared test for categorical variables. Differences between more than two groups were analyzed with ANOVA with Tukey post-hoc testing. Data were analyzed with Microsoft Excel (Microsoft, Redmond, USA), SPSS 28 (IBM, Ehningen, Germany) and graphed using GraphPad Prism 8 Software (San Diego, USA). The P level for significance was 0.05. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.02.21252580 doi: medRxiv preprint AAT is an acute phase protein that has anti-protease and anti-inflammatory properties. Earlier results from COVID-19 patients indicated a relative deficiency of AAT in relation to inflammatory markers (21) . We determined the AAT serum concentration and found that patients with COVID-19 have increased levels in the blood as compared to patients undergoing elective surgery for extra-pulmonary disease (Fig. 1A) . Within the COVID-19 group, there were no differences of the AAT serum concentration between severe cases vs. moderate cases or survivors vs. non-survivors (data not shown). In the present study, the ratio IL-6 / AAT was significantly increased in non-survivors (Fig. 1B) , The ratio CRP / AAT was significantly increased in patients with more severe disease (Fig. 1C) . These data indicate that in COVID-19 there is a relative AAT deficiency with inadequate production of AAT in response to systemic inflammation. 3D organoid cultures of airway epithelium were developed for high-throughput screening to identify possible TMPRSS2 inhibitors. To investigate whether TMPRSS2 and ACE2 (6) are expressed in epithelial cells of the organoids, immunostaining was performed and showed expression of both TMPRSS2 and ACE2 ( Fig. 2A) . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.02.21252580 doi: medRxiv preprint Next, we analyzed the effect of protease inhibitors on the activity of trypsin-like proteases, including TMPRSS2, in in 2D and 3D cultures of airway epithelial cells using a fluorescence-based assay. The protease activity was measured by incubating cell cultures with the synthetic peptide analogue Boc-Gln-Ala-Arg-7-Amino-4methylcoumarin, a substrate for trypsin-like proteases, including TMPRSS2. We found that AAT inhibits the protease activity in a concentration between 1 and 5 mg/ml in submersed, undifferentiated epithelial cells (Fig. 2B) or organoids (Fig. 2C) . Camostat and trasylol were more active in cell lines (Fig. 2B) . Next, we investigated whether AAT inhibits SARS-CoV-2 spike protein (SARS-2-S)driven entry into the human lung cell line Calu-3, which expresses endogenous ACE2 and TMPRSS2. For this, we employed previously described vesicular stomatitis virus (VSV)-based vectors bearing either SARS-2-S or the glycoprotein of VSV (VSV-G) (6) . The protease inhibitor camostat, which is known to block TMPRSS2, was included as positive control in these experiments. Camostat reduced SARS-2-S but not VSV-Gdriven entry efficiently and in a dose-dependent manner, as expected (6) . Inhibition by AAT was also observed but was modest and only detected at high concentration ( Fig. 3A ). At 20 mg/ml a slight, unspecific inhibition of VSV-G-driven entry was detected. We next analyzed whether these results translate to human lung organoids and authentic SARS-CoV-2. Camostat efficiently inhibited SARS-CoV-2 infection of lung organoids, while an approximately 50% reduction of SARS-CoV-2 infection was observed at 10 mg/ml of AAT (Fig. 3B) . These results suggest that AAT exerts moderate antiviral effects in human lung cells. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Based on the anti-viral and anti-inflammatory activities of AAT, we offered inhaled or combined inhaled / intravenous application to patients with mild to moderate COVID- 19 . Four patients were treated with inhaled AAT (100 mg/day for 7 days) and five patients were treated with inhaled (100 mg/day for 7 days) and intravenous treatment (60 mg/kg body weight, day 1, 3, 5) . For further analysis we matched two COVID-19 patients from the CORSAAR cohort based on age and disease severity (patient characteristic in Table S1 ). All patients treated with AAT survived and were discharged from the hospital in good functional status. To monitor disease activity, we recorded clinical measurements, inflammatory blood parameters and viral load. The respiratory status eventually improved in all patients; three patients experienced an initial deterioration (Fig. 4A) . Serum CRP levels decreased over the days after initiation of the AAT application with individual variations (Fig. 4B) . Virus load was monitored at irregular intervals and turned negative in all patients (Fig. 4C) . We did not observe any drug-associated side effects such as allergic reactions. In the group of matched patients, three patients died, the decrease of CRP was delayed as compared to the AAT treatment group (Fig. 4B ). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.02.21252580 doi: medRxiv preprint The main finding of this work is that AAT is a candidate for treatment of COVID-19. Evidence from this work and from the literature shows that AAT has anti-viral and antiinflammatory activity. Administration of AAT to patients with mild to moderate COVID-19 was associated with clinical improvement with decreasing inflammation and good functional reconstitution. AAT is an acute phase protein and its production in the liver is induced by inflammatory mediators (22) . Clinical data showed that in severe COVID-19 the AAT serum concentration relative to the amount of systemic inflammation is decreased. These data confirm similar findings from the McElvaney group (21) and suggest that reduced activity of AAT could represent a host factor in the modulation of the course of COVID-19 due to the anti-inflammatory properties of AAT (12, 13) . In addition to its anti-inflammatory activities, AAT inhibits SARS-CoV-2 replication in a cell-based human model of COVID-19 in airway organoids, although with modest efficiency. Antiviral activity is likely due to blockade of TMPRSS2 as shown by the decreased broad serine-protease activity of cell supernatants. TMPRSS2 has been shown earlier to activate SARS-CoV-2 and several other viral respiratory pathogens (23) . Several other studies have suggested a potential role of AAT in COVID-19. Two studies showed AAT-dependent inhibition of entry of pseudotyped VSV or SARS-CoV-2 into airway cells (24) (25) (26) . Another study could not detect a significant effect of AAT in cell-based SARS-CoV-2 infection models (27) . Differences within the cell models likely contribute to this variation. We treated 9 COVID-19 patients with AAT, and all patients showed clinical improvement within several days associated with full functional recovery, decreasing All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Other studies showed that between 21.1 and 24.5 % of the patients hospitalized deteriorated and died (28) (29) (30) . AAT might represent a good candidate for rapid development for clinical use in COVID- This study has limitations that need to be addressed, first of all the lack of data from a controlled trial and the missing deep mechanistic characterization of the mode of action of AAT. These items are beyond this report, which shows that AAT has anti-SARS-CoV-2 activity, is well tolerated in patients with COVID-19 and together with its antiinflammatory properties can be considered a promising candidate for treatment of COVID-19 that should be further evaluated in a randomized controlled trial for inhaled and / or intravenous treatment. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The data collected for the study, including individual participant data and a data dictionary defining each field in the set, will be made available to others upon reasonable request. The data that will be made available include anonymized clinical data and data dictionaries. Other related documents are available, including the study protocol, statistical analysis plan, informed consent. The data will be available with publication through the email of the corresponding author. Data will be shared after approval by the author group. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. t-test, data are shown as the mean ± SD with ** = p < 0.01 and *** = p < 0.001. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. with Tukey post-hoc test. Data are shown as the mean ± SD with **p < 0.01 and ***p < 0.001. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. indicate SD with **p < 0.01 and ***p < 0.001. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.02.21252580 doi: medRxiv preprint C) The viral load in oro-or nasopharyngeal swabs decreased over time. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 6, 2021. ; https://doi.org/10.1101/2021.04.02.21252580 doi: medRxiv preprint Coronaviridae Study Group of the International Committee on Taxonomy of V. 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No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.