key: cord-330121-eadu2ba3 authors: Gudmundsdottir, Ágústa; Scheving, Reynir; Lindberg, Fredrik; Stefansson, Bjarki title: Inactivation of SARS‐CoV‐2 and HCoV‐229E in vitro by ColdZyme® a medical device mouth spray against common cold date: 2020-09-25 journal: J Med Virol DOI: 10.1002/jmv.26554 sha: doc_id: 330121 cord_uid: eadu2ba3 BACKGROUND: The COVID‐19 pandemic calls for effective and safe treatments. SARS‐CoV‐2 causing COVID‐19 actively replicates in the throat, unlike SARS‐CoV, and shows high pharyngeal viral shedding even in patients with mild symptoms of the disease. HCoV‐229E is one of four coronaviruses causing the common cold. In this study, the efficacy of ColdZyme® (CZ‐MD), a medical device mouth spray, was tested against SARS‐CoV‐2 and HCoV‐229E in vitro. The CZ‐MD provides a protective glycerol barrier containing cod trypsin as an ancillary component. Combined, these ingredients can inactivate common cold viruses in the throat and mouth. The CZ‐MD is believed to act on the viral surface proteins that would perturb their entry pathway into cells. The efficacy and safety of the CZ‐MD has been demonstrated in clinical trials on the common cold. METHOD OF STUDY: The ability of the CZ‐MD to inactivate SARS‐CoV‐2 and HCoV‐229E was tested using an in vitro virucidal suspension test (ASTM E1052). RESULTS: CZ‐MD inactivated SARS‐CoV‐2 by 98.3% and HCoV‐229E by 99.9%. CONCLUSION: CZ‐MD mouth spray can inactivate the respiratory coronaviruses SARS‐CoV‐2 and HCoV‐229E in vitro. Although the in vitro results presented cannot be directly translated into clinical efficacy, the study indicates that CZ‐MD might offer a protective barrier against SARS‐CoV‐2 and a decreased risk of COVID‐19 transmission. This article is protected by copyright. All rights reserved. The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global health crisis 1 . COVID-19 starts as an infection of the respiratory tract and active viral replication of SARS-CoV-2 in the throat has recently been confirmed 2 . Coronaviruses are divided into 4 subgroups where SARS-CoV-2 as well as SARS-CoV and MERS-CoV are beta coronaviruses 3 . The alpha coronaviruses HCoV-229E and HCoV-NL63 and the beta coronaviruses HCoV-OC43 and HCoV-HKU1 are believed to cause about one-third of the common cold cases 4 . Treatments and vaccines against human coronavirus infections are lacking 3 . This article is protected by copyright. All rights reserved. ColdZyme ® (CZ-MD) is a commercially available CE marked Class III medical device mouth spray against the common cold. It contains glycerol and minor amounts of purified cold-adapted trypsin 5 The entry of coronaviruses into host cells is mediated by the spike (S) glycoprotein that forms homo-trimers protruding from the virus surface 11 . The S protein is frequently cleaved at the boundary between two functional subunits termed S1 and S2 3 . The S1 subunit comprises the receptor-binding domain (RBD) and contributes to stabilization of the pre-fusion state and the S2 subunit contains the fusion machinery 3 . SARS-CoV-2 and SARS-CoV use the ACE2 human cell receptor for cell entry 12 . The S protein is cleaved by host cell proteases such as furin, cathepsin and transmembrane serine protease TMPRSS2 3 . Cleavage at the S2′ site is located upstream of the fusion peptide. This supposedly activates the S protein for membrane fusion involving irreversible conformational changes 3 . Therefore, entry of coronavirus into susceptible cells is a complex process that requires the concerted action of the S protein, receptor-binding and proteolytic processing by host cell proteases to promote virus-cell fusion 3 . In contrast to SARS-CoV, active SARS-CoV-2 virus replication in the upper respiratory tract has been demonstrated 2 . Based on the article, SARS-CoV-2 virus shedding in the throat was shown to be high during the first week of symptoms. The presence of a polybasic furin-type cleavage site at the S1-S2 junction in the SARS-CoV-2 spike protein, not present in SARS-CoV, could explain the extension of tissue tropism of SARS-CoV-2 to the throat 2 . This article is protected by copyright. All rights reserved. Here we present research demonstrating the in vitro efficacy of a medical device mouth spray (CZ-MD) against SARS-CoV-2 and HCoV-229E. The results indicate that the CZ-MD may be active against a variety of coronaviruses in vivo. CZ-MD solution contained glycerol, water, cod trypsin, ethanol, calcium chloride, tris and menthol. Two lots were evaluated. Testing according to ASTM International E1052-11 method, "Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension" was carried out by an independent testing laboratory under GLP conditions; Microbac Laboratories, Inc., 105 Carpenter Drive, Sterling, VA 20164, USA. The controls included were as previously described 8 . The buffers described in the viral inactivation test were used in the controls where buffer was used, HCoV-229E test: 1xPB without sodium chloride, pH 7.5 and SARS-CoV-2 test: 20 mM Tris, 1 mM CaCl2, pH 8.2. The controls were done at the same time as the test samples. The virus inactivating ability of the CZ-MD solution against SARS-CoV-2 and HCoV-229E was determined as described under Material and methods (Table I) . The 50% Tissue Culture Infectious Dose (TCID50) endpoint assay was used to titrate samples from each incubation using the appropriate host cell system for each virus, see Materials and methods. The tests were done in duplicate for each CZ-MD lot and in duplicate for the viral recovery control where the mean is reported for the results ( Table I ). The results show that CZ-MD inactivated both viruses with a log10 reduction of 1.76 or 98.3% inactivation of SARS-CoV-2 and with a log10 reduction of 2.88 or 99.9% inactivation of HCoV-229E (Table I) . All the controls met the criteria for a valid test. There was no cytotoxicity detected at any dilution or cell line tested. In the cell viability control wells, virus was not detected, the cells remained viable and the media was sterile. Enough virus was recovered for the virus recovery control and the appropriate titer was used in the experiment based on the viral stock titer control (6.30 log10TCID50/mL for SARS-CoV-2 and 6.20 log10TCID50/mL for HCoV-229E) for each assay. Viral-induced CPE was distinguishable from uninfected cells. For the reference product control 2000 ppm NaOCl was used as test substance which showed a log reduction of ≥4.45 for SARS-CoV-2 and ≥4.24 for HCoV-229E (Table I) . For the neutralizer effectiveness/viral interference control, virus was detected in all wells. Based on the results presented in this study, CZ-MD was found to inactivate SARS-CoV-2 (98.3%) and HCoV-229E (99.9%) in vitro (Table I) There is a lack of treatment options against coronaviruses infecting humans such as SARS-CoV-2, HCoV-229E and other common cold coronaviruses. Coronaviruses cause about one-third of the common cold cases 4 Entry of coronaviruses into susceptible cells requires receptor-binding of the S protein and it´s proteolytic processing by host cell proteases that occurs in a concerted action to promote virus-cell fusion 3 . Externally added trypsin is sometimes used in in vitro studies as a surrogate for more biologically relevant host cell proteases as these This article is protected by copyright. All rights reserved. proteases have trypsin-like substrate specificity 14, 15 . However, the in vitro study presented here clearly demonstrates that the CZ-MD (containing cod trypsin) inactivates SARS-CoV-2 and HCoV-229E. In addition, the in vivo topical application of CZ-MD as a mouth spray limits the activity of cod trypsin to the surface of the oral and throat mucous layer. The oral and throat mucus protects epithelial surfaces by trapping pathogens and foreign particles 16 . The activity of endogenous proteases and external proteases, such as those found in food, are under strict control to prevent unintended proteolysis at the cellular level 16, 17 . Mucus membranes play a vital role in this anti-proteolytic process by preventing penetration of negatively charged proteins, such as cod trypsin, through its layers of glycosylated proteins containing protease inhibitors 16 . Although the in vitro results presented cannot be directly translated into clinical efficacy, the study indicates that CZ-MD might offer a protective barrier against coronaviruses such as SARS-CoV-2 and a decreased risk of COVID-19 transmission. The data that support the findings of this study are available from the corresponding author upon reasonable request. Stefansson and Ágústa Gudmundsdottir wrote, reviewed, edited and approved the manuscript. Reynir Scheving and Fredrik Lindberg reviewed, edited and approved the manuscript. Reynir Scheving and Bjarki Stefansson are employed at Zymetech. Ágústa Gudmundsdottir is Professor emeritus at the University of Iceland and partially employed at Zymetech. Fredrik Lindberg was employed at Enzymatica AB. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Virological assessment of hospitalized patients with COVID-2019 Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein The human coronavirus HCoV-229E Sprotein structure and receptor binding Biochemical characterization of a native group III trypsin ZT from Atlantic cod (Gadus morhua) Is the oral cavity relevant in SARS-CoV-2 pandemic? Hosts and Sources of Endemic Human Coronaviruses A medical device forming a protective barrier that deactivates four major common cold viruses A Randomized, Double-Blind, Placebo-Controlled Pilot Clinical Study on ColdZyme® Mouth Spray against Rhinovirus-Induced Common Cold Coldzyme® Mouth Spray reduces duration of upper respiratory tract infection symptoms in endurance athletes under free living conditions Structural insights into coronavirus entry Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2 Cell entry mechanisms of SARS-CoV-2 Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis HCoV-229E spike protein fusion activation by trypsin-like serine proteases is mediated by Accepted Article proteolytic processing in the S2' region The Interaction between Respiratory Pathogens and Mucus Proteases: Pivot Points in Functional Proteomics Table Table I