PEPTIDES HAVING PROTEASE ACTIVITY FOR USE IN THE TREATMENT OR PREVENTION OF CORONAVIRUS INFECTION

Abstract

The present invention provides a polypeptide having protease activity for use in the treatment or prevention of coronavirus infection in a mammal. In particular, the invention relates to treatment or prevention of a coronavirus infection in a human, using trypsins.

Claims

1. A method of treating or preventing coronavirus infection in a subject comprising administering to said subject a polypeptide having protease activity.

2. The method according to claim 1 wherein the coronavirus infection is an infection of the respiratory tract and/or of the gastrointestinal tract.

3. The method according to claim 1 wherein the coronavirus infection is selected from the group consisting of common cold, pneumonia, pneumonitis, bronchitis, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), sinusitis, otitis or pharyngitis.

4. The method according to claim 3 wherein the coronavirus infection is the common cold.

5. The method according to claim 1 wherein the coronavirus is selected from the group consisting of: (a) alphacoronavirus; (b) betacoronavirus; (c) gammacoronavirus; and (d) deltacoronavirus.

6. The method according to claim 1 wherein the coronavirus is a human coronavirus.

7. The method according to claim 6 wherein the human coronavirus is selected from the group consisting of: (a) human coronavirus 229E; (b) human coronavirus OC43; (c) Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) (d) human Coronavirus NL63 (HCoV-NL63, New Haven coronavirus); (e) human coronavirus HKU1; and (f) Middle East respiratory syndrome coronavirus (MERS-CoV).

8. The method according to claim 1 wherein the subject is a mammal, for example a human.

9. The method according to claim 1 wherein the polypeptide having protease activity is selected from the group consisting of serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases.

10. The method according to claim 9 wherein the protease is a serine protease.

11. The method according to claim 10 wherein the protease is a trypsin or chymotrypsin, or a component of a mixture thereof.

12. A polypeptide for use according to any one of the preceding claims wherein the polypeptide having protease activity is cold-adapted.

13. The method according to claim 1 wherein the polypeptide is naturally occurring.

14. The method according to claim 13 wherein the polypeptide is a marine serine protease.

15. The method according to claim 14 wherein the marine serine protease is obtained or obtainable from cod, pollock, salmon or krill.

16. The method according to claim 15 wherein the marine serine protease is obtained or obtainable from Atlantic cod.

17. The method according to claim 14 wherein the marine serine protease is a trypsin, for example trypsin I.

18. The method according to claim 1 wherein the polypeptide is trypsin I, trypsin X or trypsin ZT from Atlantic cod.

19. The method according to claim 1 wherein the activity of the trypsin ranges from 1 U/mg to 1 U/g of the polypeptide, for example between 50 U/mg and 500 U/mg of the polypeptide.

20. The method according to claim 1 wherein the polypeptide is non-naturally occurring.

21. The method according to claim 1 wherein the polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 12, or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the protease activity of said amino acid sequence.

22. The method according to claim 21 wherein the polypeptide comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 1 to 12.

23. The method according to claim 1 wherein the polypeptide comprises or consists of a fragment of the amino acid sequence according to SEQ ID NOs: 1 to 12.

24. A polypeptide for use according to claim 23 wherein the fragment comprises or consists of at least 15 contiguous amino acid of any one of SEQ ID NOs: 1 to 12, for example at least 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 contiguous amino acids of any one of SEQ ID NOs: 1 to 12.

25. The method according to any one of claims 1 to 21 wherein the polypeptide comprises or consists of a variant of the amino acid sequence according to any one of SEQ ID NOs: 1 to 12.

26. The method according to claim 25 wherein the variant is a non-naturally occurring variant.

27. The method according to claim 25 wherein the variant has an amino acid sequence which has at least 50% identity with the amino acid sequence according to any one of SEQ ID NOs: 1 to 12, or a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

28. The method according to claim 1 wherein the polypeptide is between 150 and 250 amino acids in length, for example between 200 and 250, between 210 and 240, between 220 and 230, or between 220 and 225 amino acids in length.

29. The method according to claim 1 wherein the polypeptide is a recombinant polypeptide.

30. The method according to claim 1 wherein the polypeptide is provided in an osmotically active solution.

31. The method according to claim 1 wherein the polypeptide is administered in combination with glycerol and a buffer.

32. The method according to claim 1 wherein the polypeptide is provided in a form suitable for delivery to the oropharynx.

33. The method according to claim 1 wherein the polypeptide is provided in a mouth spray, lozenge, pastille, tablet, syrup or chewing gum.

34. The method according to claim 1 wherein the polypeptide is for use in combination with one or more additional active agents.

35. The method according to claim 34 wherein the additional active agents are selected from the group consisting of antimicrobial agents (including antibiotics, antiviral agents and anti-fungal agents), anti-inflammatory agents (including steroids and non-steroidal anti-inflammatory agents) and antiseptic agents.

36. The method according claim 35 wherein the one or more antimicrobial agents are antibiotics selected from the group consisting of penicillins, cephalosporins, fluoroquinolones, aminoglycosides, monobactams, carbapenems and macrolides.

37. A method the preparation of a medicament comprising formulating a polypeptide according to claim 1 in a pharmaceutical composition.

38. The method according to claim 37 wherein the polypeptide is a trypsin or chymotrypsin, or a component of a mixture thereof.

39. The method according to claim 37 wherein the polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOS: 1 to 12, or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the trypsin activity of said amino acid sequence.

40. The method according to claim 39 wherein the polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOS: 1 to 121.

41. The method according to claim 37 wherein the coronavirus infection is selected from the group consisting of common cold, pneumonia, pneumonitis, bronchitis, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), sinusitis, otitis or pharyngitis.

42-49. (canceled)

Description

[0194] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

[0195] FIG. 1. Deactivation of coronavirus (CoV-229E-luc) by cod trypsin. This figure shows that cod trypsin lowers the ability of coronavirus in infecting human liver cells (Huh7). Y-bar shows the level of infection by the coronavirus on a log scale based on results from a Renilla luciferase assay (see Methods). X-bar shows the amount of cod trypsin used in U/mL.

[0196] FIG. 2. Deactivation of coronavirus (CoV-229E) by cod trypsin. This figure shows that cod trypsin lowers the ability of coronavirus in infecting human fetal lung fibroblast-like cells (MRC5). Y-bar shows the reduction of infection by the coronavirus on a log scale. The concentration of cod trypsin used was 1.22 U/mL (panel A) and 2.44 U/mL (panel B). Each concentration and time point was done in duplicate.

[0197] FIG. 3. Western blot analysis of coronavirus proteins in a coronavirus sample incubated with different concentrations of cod trypsin. This figure shows that coronavirus proteins are degraded in samples containing cod trypsin treated coronavirus. Proteins within cod trypsin treated coronavirus samples were resolved by SDS-PAGE and subjected to Western blot analyses using an antibody directed against coronaviral proteins (see Methods). The concentration of cod trypsin used is shown above each lane in the gel. The lane labelled coronavirus is a sample that did not contain cod trypsin.

[0198] FIG. 4. Western blot analysis of recombinant CoV-229E spike protein incubated with different concentrations of cod trypsin. This figure shows that recombinant coronavirus spike protein is degraded by cod trypsin. Proteins within samples containing recombinant coronavirus spike protein incubated with different concentrations of cod trypsin were resolved by SDS-PAGE and subjected to Western blot analyses. Migration of size standards is shown by bars and numbers (kDa) on the right side. As controls, a sample containing cod trypsin and a sample containing S1 spike protein were loaded on the gel. The amount of cod trypsin incubated with recombinant coronavirus spike protein is shown above each lane in the gel.

[0199] FIG. 5. SDS-PAGE analysis of recombinant SARS-CoV spike protein incubated with trypsin ZT or trypsin I for 10 min at 33 C. This figure shows that recombinant SARS-CoV spike protein is degraded by trypsin ZT and trypsin I. Proteins within samples containing recombinant SARS-CoV spike protein incubated with different concentrations of cod trypsin were resolved by SDS-PAGE. The gel was dyed with Coomassie blue and imaged on an Odyssey infrared imaging system. Migration of size standards is shown by bars and numbers (kDa) on the right side. As a control, a sample containing SARS-CoV spike protein was loaded on the gel. The amount of trypsin ZT and trypsin I incubated is shown above each lane in the gel.

EXAMPLES

Materials & Methods

Cells, Viruses and Enzymes

[0200] Huh7 cells and CoV-229E-luc (van den Worm, Eriksson et al. 2012) were obtained from Prof. Dr. Volker Thiel at the Institute of Virology and Immunology, University of Bern, Switzerland. Benzamidine purified cod trypsin was obtained from Zymetech (Reykjavik, Iceland). Trypsin ZT (unpublished data in a manuscript submitted for publication: Sandholt G B, Stefansson B, Gudmundsdottir A) and trypsin I were purified as previously described (Stefansson, Helgadottir et al. 2010). Activity of cod trypsin was measured using the substrate CBZ-GPR-pNA (Stefansson, Helgadottir et al. 2010).

Deactivation of CoV-229E-Luc by Cod Trypsin and Renilla Luciferase Assay

[0201] Cod trypsin diluted in Dulbecco's Modified Eagle's medium (DMEM) without Fetal Bovine Serum (FBS), was incubated with CoV-229E-luc (ORF 4 is replaced by Renilla luciferase) per mL at 33 C. for 3 hours. Previously, about 15.000 Huh7 cells were seeded per well into a 96-well plate and incubated at 37 C. and 5% CO.sub.2 in DMEM with 5% FBS. Twenty-four hours later, the cells were washed with PBS, infected with a multiplicity of infection (MOI) of 0.1 with cod trypsin treated CoV-229E-luc. The virus was allowed to adsorb to cells for 2 hours, cells were washed with PBS and cell media. The cells were incubated in DMEM with 5% FBS. Cell viability was assessed by using 10% (v/v) AlamarBlue assay (Fisher Scientific, Inc.) 22 hours post infection. AlamarBlue was incubated at 33 C. and 5% CO.sub.2 for further 24 hours before evaluation in a luminometer at 595 nm. The level of virus replication was determined using the Renilla luciferase Assay kit (Promega, USA) according to the manufactures instructions, using half the recommended substrate concentration, and a luminometer. Logarithmic scale was constructed by calculating log of the luminescence measured.

Deactivation of CoV-229E by Cod Trypsin

[0202] Cod trypsin was evaluated against a challenge virus (human coronavirus, strain 229E, ATCC VR-740) in suspension at two timepoints, 10 and 60 min in a virucidal efficacy suspension test. The test followed the ASTM International test method designated E1052-11 Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension. For each run, two separate dilutions were made. One dilution with 1 part trypsin and 1 part diluent (1 Phosphate buffered saline (PBS)) and the other dilution 1 part trypsin and 3 parts diluent. Each dilution (2.7 ml) was mixed with 0.3 mL of the challenge virus suspension (contained 0% serum) and mixed by vortexing. The reaction mixtures were incubated at 35-37 C. for 10 and 60 min (contact time). After incubation, an aliquot of the reaction mixture was immediately mixed with an equal volume of neutralizer. The neutralizer was minimum essential medium (MEM)+10% FBS+1% polysorbate 80. Sephacryl columns were used to separate the virus from cod trypsin for the test substance and the virus recovery control (see below) after the neutralization by the neutralizer. The quenched sample from the column was serially diluted with medium in tenfold increments and inoculated onto host cells (MRC-5 cells, ATCC CCL-171) to assay for infectious virus. Inoculated plates were incubated at 332 C. in 51% CO.sub.2 for 5-7 days. After incubation, the cultures were scored for viral infection by determining viral-induced cytopathic effect (CPE). The titer of the virus (log.sub.10 TCID.sub.50/mL) was calculated using the Spearman-Karber formula (Krber 1931). The viral load (log 10 TCID50) was calculated by adding the viral titer (log 10 TCID50/mL) to the log 10 (the volume of reaction mixture in mL times the volume correction). The volume correction accounted for the neutralization of the sample post contact time. The log 10 reduction factor was calculated by subtracting the output viral load (log 10) from the input viral load (log 10). The input load was 5.92 but it represents the virus units (log 10 TCID50) recovered after incubating the virus in medium before inoculation (virus recovery control, see below). The output load represents the virus unit (log 10 TCID50) recovered after mixing and incubating the virus with cod trypsin.

[0203] Controls included a virus recovery control, neutralizer effectiveness/viral interference control, a cytotoxicity control, cell viability control, a virus stock titer control, and a reference product control. The neutralizer effectiveness/viral interference control was performed to determine if residual active ingredients were present after neutralization and if the neutralized test substance interfered with virus infectivity. A mixture of 1.35 mL of cod trypsin and 1.35 mL of 1PBS was mixed thoroughly with 0.3 mL of medium (in lieu of the challenge virus), held for 60 min, neutralized and run through a sephacryl column. The quenched sample was divided into 2 portions, one for neutralizer effectiveness/viral interference control, and the other for cytotoxicity control, and each portion serially tenfold diluted. For the neutralizer effectiveness/viral interference control, 0.1 mL of a low titered virus was added to 4.5 mL of each dilution and held for a period equivalent or greater than 60 min. After incubation, the virally spiked dilutions were inoculated onto host cells. For the cytotoxicity control, the sample obtained from the neutralizer effectiveness/viral interference control run was serially diluted and inoculated onto host cells. The condition of the host cells was recorded at the end of the incubation period. For the virus recovery control, 2.7 mL of dilution medium (MEM+5% FBS) was mixed with 0.3 mL of the challenge virus suspension. The mix was held for 60 min, neutralized and run through a sephacryl column as for the test product runs. The quenched sample was serially diluted with dilution medium in tenfold increments and selected dilutions were inoculated onto host cells to assay for infectious virus. For the cell viability control, at least 4 wells were inoculated with media in each assay to demonstrate that cells remained viable and media was sterile throughout the assay. For the virus stock titer control, an aliquot of the virus was serially diluted and inoculated directly onto host cells. This was to demonstrate that the titer of the stock virus was appropriate for use and that the viral infectivity assay was performed appropriately. For the reference product control, a 2.7 mL aliquot of an approximately 1000 ppm NaOCl containing bleach solution was mixed with 0.3 mL of the challenge virus suspension. The mix was held for the two contact times and then neutralized. The quenched sample was serially diluted with dilution medium in tenfold increments and selected dilutions were inoculated onto host cells to assay for infectious virus. The viral stock titer control for each assay confirmed that the appropriate titer was used in the experiment and sufficient amount of virus was recovered for the virus recovery control. No virus was detected in the cell viability control wells, the cells remained viable and the media was sterile. Virus was detected in all the neutralizer effectiveness/viral interference control wells. Cytotoxicity was not detected at any dilution or cell line tested. Viral-induced CPE was distinguishable from uninfected cells. Thus, all the controls met the criteria for a valid test. The reference test substance, 1000 ppm NaOCl, had a log reduction of a 4.31 for all viruses tested.

[0204] The virucidal efficacy suspension test was carried out by an independent testing laboratory; Microbac Laboratories, Inc., 105 Carpenter Drive, Sterling, Va. 20164, USA.

Viral Protein Analysis of Cod Trypsin Treated CoV-229E-Luc

[0205] Cod trypsin diluted in DMEM (without FBS) was mixed with CoV-229E-luc virus. The mixture was incubated at 33 C. for 2 hours. After incubation, Laemmli buffer (4) was added and the mixture incubated for 5 min at 95 C. The sample was loaded on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (Bachofen, Bollinger et al. 2013) and Western blot analysis was conducted, see below.

Treatment of Recombinant CoV-229E S1 Spike Protein with Cod Trypsin

[0206] Cod trypsin diluted in serum free DMEM, was used to treat recombinant CoV-229E S1 spike protein at a final concentration of 86 g/mL. The mixture was incubated at 33 C. for 2 hours. After incubation, Laemmi buffer (4) was added and incubated for 5 min at 95 C. to inactivate the enzyme. The sample was loaded on a 10% SDS-PAGE gel (Bachofen, Bollinger et al. 2013) and Western blot analysis was conducted, see below. Recombinant CoV-229E S1 spike protein was obtained from Prof. Dr. Volker Thiel at the Institute of Virology and Immunology, University of Bern, Switzerland.

Western Blot Analysis

[0207] For Western blot analysis, SDS-PAGE (10%) was performed and the proteins were transferred onto a nitrocellulose membrane. After transfer, the membrane was blocked with PBS containing 5% milk and 0.5% Tween-20 for 60 min at room temperature (RT). Primary antibody (anti-coronavirus polyclonal goat antibodies produced from NP-40-dispersed CoV-229E (Wentworth and Holmes 2001)) was diluted 1:1000 in PBS-Tween (PBS-T) with 0.5% milk and incubated overnight with gentle agitation at 4 C. The membrane was washed with PBS followed by incubation with secondary antibody (anti-goat IgG conjugated to horse radish peroxidase, 1:1000 in 0.5% milk) for 60 min at RT. The immunoblot was developed using a charge-coupled device (CCD) camera.

Treatment of Recombinant SARS-CoV Spike Protein with Trypsin ZT or Trypsin I

[0208] A recombinant Spike Protein (Beijing02) (SARS-CoV) (eEnzyme, catalog number: SS-001-005P) (0.1 mg/mL) was incubated with trypsin ZT or trypsin I at 33 C. for 10 min in 17 mM Tris, 12.5 mM CaCl.sub.2, 2.5 mM ethanolamine at pH 8.5. The samples were incubated for 10 min at 95 C. in 4LDS sample buffer. As a positive control, spike protein was incubated at 33 C. for 10 min without trypsin isoenzymes. The mixture was run on a 12% SDS-PAGE gel (NuPAGE Bis-Tris, Novex by life technologies) using MOPS-SDS running buffer. The gel was stained with Coomassie blue (PageBlue Protein Staining Solution, Thermo Fisher Scientific) and imaged on an Odyssey infrared imaging system.

Results

Deactivation of Coronavirus by Cod Trypsin

[0209] To study the ability of cod trypsin in reducing the infectivity of coronavirus, an assay using CoV-229E-luc utilizing Renilla luciferase was used (van den Worm, Eriksson et al. 2012). Coronavirus was incubated (180 min) with or without cod trypsin and placed on human liver cells (Huh7). After the viral adsorption period, cells were incubated for two days and the level of coronavirus infection measured (see Methods). The level of infection was measured using the Renilla luciferase assay (FIG. 1).

[0210] Treatment with 1.4 and 2.8 U/mL cod trypsin lowered the level of coronaviral infection by about 3 log units compared to control (untreated coronavirus) (FIG. 1).

[0211] To further test the ability of cod trypsin to deactivate coronavirus, an experiment was performed using a standardized virucidal efficacy suspension test with coronavirus 229E. The coronavirus was incubated for 10 or 60 min with cod trypsin at a concentration of 1.22 U/mL or 2.44 U/mL, in duplicate. Samples from each incubation were titrated with the 50% Tissue Culture Infectious Dose (TCID.sub.50) endpoint assay (see Methods). The log.sub.10 reduction factor was calculated and the mean reported (FIG. 2).

[0212] Cod trypsin treatment of coronavirus resulted in greater than 4 log reduction in virus infectivity after incubation for 60 min at both concentrations. With incubation for 10 min, 2.5 and 3 log reduction was observed at 1.22 U/mL and 2.44 U/mL, respectively.

Degradation of Viral Proteins in Cod Trypsin Treated Coronavirus Samples

[0213] The ability of cod trypsin to degrade coronavirus proteins in samples containing infectious coronavirus was tested. A stock solution of coronavirus was treated with cod trypsin at a concentration of 1.4-5.6 U/mL for 3 hours at 33 C. and the samples subjected to Western blot analysis (FIG. 3). As can be seen in FIG. 3, cod trypsin is able to degrade coronavirus proteins at a concentration of 1.4-5.6 U/mL in 3 hours. The size of the protein recognized by the antibody (50 kDa) matches the size of CoV-229E nucleocapsid protein (Lo, Lin et al. 2013).

[0214] To further study the ability of cod trypsin to degrade proteins from coronavirus, recombinant CoV-229E spike protein was incubated with different concentrations of cod trypsin at 33 C. for 2 hours and subjected to Western blot analysis (FIG. 4).

[0215] As can be seen in FIG. 4, cod trypsin degrades the recombinant coronavirus spike protein at all concentrations tested.

[0216] It was of interest to test the ability of the cod trypsin isoenzymes trypsin ZT and trypsin I to degrade recombinant SARS-CoV spike protein. Both isoenzymes are found in the cod trypsin isolate used in the study. In FIG. 5 the SARS-CoV spike protein is seen around 180 kDa. The spike protein is degraded at both concentrations tested (0.35 and 0.7 U/mL) by trypsin ZT and trypsin I in 10 min at 33 C.

Discussion

[0217] In this study, it was demonstrated that cod trypsin reduced the level of infection of coronavirus by more than 4 log units in 60 min and up to 3 log units in only 10 min (FIG. 2). The ability of cod trypsin to deactivate coronavirus was also established using a coronavirus (CoV-229E-luc) where the viral titer was monitored with a Renilla luciferase assay (FIG. 1). The deactivating efficacy of cod trypsin against coronavirus is thought to be based on its capacity to degrade coronavirus spike proteins. This would diminish the ability of the coronavirus to bind to human cell receptors important for infection (Lim, Ng et al. 2016, Park, Li et al. 2016). In support of this mode of action, degradation of coronavirus proteins by cod trypsin was analyzed. CoV-229E-luc virus treated with cod trypsin was subjected to Western blot analysis using a polyclonal antibody raised against CoV-229E (FIG. 3). The results clearly demonstrate degradation of a protein in the size range of 50 kDa that correlates well with that of nucleocapsid protein (Lo, Lin et al. 2013). Furthermore, recombinant CoV-229E spike protein in a purified form was degraded by cod trypsin as seen in FIG. 4. The data obtained on coronavirus protein degradation are based on incubation of the virus with cod trypsin for 2 hours (FIG. 3). It is suspected that the spike proteins are most susceptible to cod trypsin degradation as they are located on the surface of the coronavirus. Based on our findings, incubation of coronavirus with cod trypsin starts with degradation of spike proteins (FIG. 4 and FIG. 5). However, after an extensive incubation period the nucleocapsid proteins are degraded (FIG. 3).

[0218] The benzamidine purified cod trypsin fraction used in the study contains different cod trypsin isoenzymes such as trypsin I, trypsin ZT and trypsin X (see above). All these trypsin isoenzymes cleave at arginine and lysine but trypsin I and trypsin ZT have been shown to have different subsite specificity (unpublished data; Sandholt G B, Stefansson B, Gudmundsdottir ). Therefore, it was of interest to test if these isoenzymes can cleave spike proteins from coronaviruses. For this purpose, recombinant spike protein from SARS-CoV was selected. This is of high importance as MERS and SARS have high mortality rates and are on the top of WHO's list of disease priorities needing urgent research due to the high likelihood of causing a major epidemic (Gralinski and Baric 2015, Mackay and Arden 2015).

[0219] Based on SDS-PAGE analysis, recombinant SARS-CoV spike protein was effectively degraded by both cod trypsin I and trypsin ZT at low concentrations in only 10 min (FIG. 5). The findings indicate that SARS coronavirus is likely to be susceptible to deactivation by cod trypsin at low concentrations. This suggests that the different coronaviruses that cause infection in humans could be deactivated by cod trypsin. The fact that the trypsin isoenzymes were effective in cleaving SARS-CoV spike protein in 10 min (FIG. 5) aligns well with the results in FIG. 2 where cod trypsin lowered the level of infection of coronavirus in 10 min.

[0220] The results presented in this study on the ability of cod trypsin to deactivate coronaviruses extend the findings of other studies that show efficacy of cod trypsin against different respiratory viruses (Gudmundsdottir, Hilmarsson et al. 2013). Interestingly, lysine and arginine rich amino acid sequences are frequently found in viral proteins (Suzuki, Orba et al. 2010, Jiang, Cun et al. 2012, Gallaher and Garry 2015). These positively charged basic amino acid residues are mainly exposed to the protein surface and are important for protein stability by forming electrostatic interactions (Sokalingam, Raghunathan et al. 2012). The fact that arginine and lysine residues are common in viral proteins can explain the ability of cod trypsin to efficiently cleave the coronavirus spike proteins tested in this study (FIG. 4 and FIG. 5).

[0221] In conclusion, the results identify that the ability of cod trypsin to deactivate coronaviruses via degradation of surface spike proteins that prevent adhesion of coronaviruses to cells.

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