BROMELAIN PROTEASE, BROMELAIN, JACALIN-LIKE LECTIN, EXTRACT FROM THE STEM AND/OR THE FRUIT OF A PINEAPPLE PLANT, COMBINATION PREPARATION, BROMELAIN PROTEASE INHIBITOR, PROTEIN/PROTEASE MIX, AND GLYCATED BROMELAIN PROTEIN FORMED BY EXOGENOUS NON-ENZYMATIC GLYCATION, FOR USE IN THE TREATMENT OR PROPHYLAXIS OF VIRUS INFECTIONS CAUSED BY CORONAVIRUSES IN A HUMAN OR ANIMAL

Abstract

The present invention addresses the problem of indicating active-ingredient classes which can treat virus diseases caused by coronaviruses in a human or animal. Said treatment includes the acute treatment of an already existing virus disease and the prophylaxis of same. The present invention relates to a bromelain protease, bromelain, jacalin-like lectin, extract from the stem and/or the fruit of a pineapple plant, combination preparation, bromelain protease inhibitor, protein/protease mix, and glycated bromelain protein formed by exogenous non-enzymatic glycation, for use in the treatment or prophylaxis of virus infections caused by coronaviruses in a human or animal.

Claims

1-40. (canceled)

41. A method of treating a human or animal infected by a coronavirus, or providing prophylaxis against a coronavirus infection, the method comprising administering to the human or animal an effective amount of a compound selected from the group consisting of bromelain protease, a jacalin-related lectin, a combination preparation of a bromelain protease and a jacalin-related lectin, an extract from the stem and/or from the fruit of a pineapple plant, a bromelain protease inhibitor comprising at least one peptide having an amino acid sequence that is at least 90% identical with one of the sequences of SEQ ID NO: 1-7, a glycated bromelain protein, and a bromelain protease mixture, which is a peptide mass, wherein the bromelain protease mixture contains less than 5.0% (w/w) bromelain protease inhibitors in the peptide mass.

42. The method of claim 41, wherein the bromelain protease is selected from the group consisting of stem bromelain (SBM) (EC 3.4.22.32), fruit bromelain (EC 3.4.22.33), ananain (EC 3.4.22.31), mixtures and combinations thereof.

43. The method of claim 41, wherein the jacalin-related lectin is selected from the group consisting of mannose-specific and glucose-specific lectins.

44. The method of claim 41, wherein the jacalin-related lectin is selected from the group consisting of pineapple lectin (jacalin-related lectin from Ananas comosus (AcmJRL)), jacalin, artocarpin lectin, MPA lectin, heltuba lectin agglutinin, griffithsin, and mixtures and combinations thereof.

45. The method in accordance with claim 41, wherein the pineapple plant is selected from the group consisting of Ananas comosus and Ananas sativus.

46. The method in accordance with claim 41, wherein the extract comprises a bromelain protease selected from the group consisting of stem bromelain (SBM) (EC 3.4.22.32), fruit bromelain (EC 3.4.22.33), ananain (EC 3.4.22.31), and mixtures and combinations thereof.

47. The method of claim 41, comprising administering to the human or animal an effective amount of a combination preparation containing at least one bromelain protease and at least one jacalin-related lectin.

48. The method according to claim 47, wherein the weight ratio of the totality of the at least one bromelain protease to the totality of the at least one jacalin-related lectin amounts to 50:50 to 0.1:99.9.

49. The method according to claim 47, wherein the total concentration of all the bromelain proteases combination preparation amounts to 0.01 to 50.0 wt % and/or the total concentration of all the jacalin-related lectin in the extract amounts to 0.01 to 60.0 wt %.

50. The method of claim 41, wherein the bromelain protease inhibitor comprises at least two peptides that have amino acid sequences that are at least 90% identical with one of the sequences of SEQ ID NO: 1-7.

51. The method of claim 41, wherein at least one peptide of the bromelain protease inhibitor has a post-translational modification characteristic for the pineapple plant or has no post-translational modification.

52. A glycated bromelain protein produced by exogenous non-enzymatic glycation, comprising at least one sugar unit covalently bound to a bromelain protein.

53. The glycated bromelain protein according to claim 52, wherein the glycated bromelain protein is selected from the group consisting of glycated jacalin-like lectin, glycated bromelain-protease, glycated bromelain-protease inhibitor, and mixtures thereof.

54. The glycated bromelain protein according to claim 52, wherein the at least one sugar unit covalently bound to the bromelain protein is a monomeric hexose or an oligomeric hexose.

55. The glycated bromelain protein according to claim 52, wherein the glycated bromelain protein has 1 to 10 sugar units that are covalently bound to the bromelain protein.

56. The glycated bromelain protein according to claim 52, wherein the glycated bromelain protein is prepared by mixing at least one bromelain protein with at least one reducing sugar and by carrying out a Maillard reaction.

57. The method according to claim 41, wherein the coronavirus is selected from the group consisting of alphacoronaviruses, betacoronaviruses, orthocoronaviruses, and leto viruses.

58. The method according to claim 57, wherein the alphacoronaviruses are selected from the group consisting of colacovirus, decacovirus, Duvinacovirus, luchacovirus, minacovirus, minunacovirus, myotacovirus, pedacovirus, Scotophilus bat coronavirus 512, Rhinacovirus, Setracovirus, and Tegacovirus, and/or the betacoronaviruses are selected from sarbecoviruses.

59. The method according to claim 41, wherein the coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) and synonymous or non-synonymous mutants thereof and variants thereof and those selected from the group consisting of B.1.1.7, B.1.351, B.1.617, P.1, B.1.525, P.2, B.1.427, B.1.429, L452R, Fin-796H, B.1.526, S:H66D, S:G142V, S:D215G, S:V483A, S:D614G, S:H655Y, S:G669S, S:Q949R, S:N1187D, ORF6:K23?, ORF6: V24?, ORF6:S25?, ORF6:I26?, ORF6:W27?, ORF6:N28?, ORF6:L29?, ORF6:D30?, ORF6: Y31?, S:Y144?, E484K, VOC 20I/484Q, B.1, R.1, A.2.5, C.36, B.1.1.318, B.1.621, B.1.623, SARS-COV-1, Embecovirus, bovine coronavirus (BCoV), equine coronavirus (ECoV-NC99), human coronavirus OC43 (HCoV-OC43), porcine hemagglutinating encephalomyelitis virus (HEV), Puffinosis coronavirus (PCOV), human enteric coronavirus (HECoV), China rattus coronavirus HKU24, human coronavirus HKU1 (HCoV-HKU1), Murine coronavirus, mouse hepatitis virus (MHV), rat coronavirus (RtCoV), hibecovirus; merbecovirus, MERSr-COV, hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus (MERS-COV), Pipistrellus bat coronavirus HKU5, Tylonycteris bat coronavirus HKU4, Nobecovirus, Rousettus bat coronavirus GCCDC1, Rousettus bat coronavirus HKU9, Sarbecovirus, Severe acute respiratory syndrome-related coronavirus (SARS-associated coronavirus), SARS-COV-1), severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), BatCoV RaTG13, Manis-COV SRR10168377, and SRR10168378; the gammacoronavirus is selected from the group consisting of cegacovirus and igacovirus; and the deltacoronavirus is selected from the group consisting of andecovirus, buldecovirus, herdecovirus, and moordecovirus.

60. The method of claim 41, wherein a symptom caused by an infection includes fever, coughing, pneumonia, lymphopenic community acquired pneumonia (L-CAP), pleuritis, shortage of breath, indisposition and/or fatigue, sputum, anosmia, ageusia, shortness of breath, muscular inflammation and/or inflammations of the throat, joint pain, chest pain, sore throat, headache, backache, ague, nausea, vomiting, colds, diarrhea, coughing blood, lymphopenia, skin rash on hands, feet, or in the mouth, nonsuppurative conjunctivitis at both sides, hypotonia, shock, hypercytokinemia, dysfunction of the cardiac muscle, inflammation of the pericardium and/or of the cardiac valve, blood coagulation dysfunctions, multisystem inflammatory syndromes in children (MIS-c), or welling of the conjunctiva, or a combination thereof.

61. The method of claim 41, wherein the weight ratio of the totality of the at least one bromelain protease to the totality of the at least one jacalin-related lectin in the combination preparation amounts to 1:99 to 99.9:0.1.

62. The method of claim 61, wherein the at least one bromelain protease is included in an amount of 0.1 to 80.0 wt. % or the at least one jacalin-related lectin is included in an extract in an amount of 0.01 to 50.0 wt. %.

63. The method of claim 41, wherein the bromelain protease, a jacalin-related lectin, an extract, a combination preparation, a bromelain protease inhibitor, a bromelain protease mixture, or a glycated bromelain protein is in the form of or as a component of a powder, a granulate, a tablet, a hard capsule, a soft capsule, an effervescent tablet, a solution, an emulsion, a suspension, a salve, a cream, a paste, a gel, a tincture, eye drops, an inhalation powder, a nose spray, a suppository, or a transdermal patch.

64. The method of claim 41, wherein the bromelain protease, jacalin-related lectin, bromelain protease inhibitor, bromelain protease mixture, or a glycated bromelain protein is administered orally, intravenously, subcutaneously, or intramuscularly, by infusion onto the mucous membranes of the nose, of the mouth or of the pharynx, by inhalation, by application onto the surface of the eyes, rectally and/or by a transdermal patch.

65. The method of claim 41, wherein the administration takes place from once a week up to once an hour.

Description

[0077] The subject matter in accordance with the invention will be explained in more detail with reference to the following Figures and to the following example without intending to restrict it to the specific embodiments shown here.

[0078] FIG. 1 shows the primary structure and molecular mass of previously known isoforms of bromelain protease inhibitors (according to Hatano et al., (2002) Biol. Chem., Vol. 383, 1151-1156);

[0079] FIG. 2 shows the elution profile of strong cation exchange chromatography (SEC) with BBP;

[0080] FIG. 3 shows the purification profile of size exclusion chromatography (SCX) with BBP;

[0081] FIG. 4 shows a schematic overview of the HCov229E test;

[0082] FIG. 5 shows lectin with anti-coronavirus activity from the HCoV229E reporter virus screening; shows bromelain and lectin with anti-coronavirus activity from the HCoV229E reporter virus screening; A) to F) The compounds (starting concentrations are specified) were preincubated with the HCoV229E virus in a ratio of 1:10 for 30 minutes (Table 2) followed by serial titration on the target cells Huh-7.5-FLuc and an incubation for 48 h. The activity of RLuc and FLuc was measured as a measure for the residual infectivity and cell viability. The data were standardized to the TBS control. The black arrows mark the 1:500 dilution or the 1:2500 dilution that were applied standardized in FIG. 6;

[0083] FIG. 6 shows lectin with anti-coronavirus activity from the HCoV229E reporter virus screening. The compounds were preincubated for 30 minutes with the HCoV229E virus at a ratio of 1:10 (Table 2) followed by a titration along the target cells. A) The values of the HCoV229E virus replication and of the cell viability were shown standardized for the 1:500 dilution (black arrows in FIG. 5) against the TBS control. B) Standardized representation of the 1:2,500 dilution;

[0084] FIG. 7 shows lectin with anti-coronavirus activity from the HCoV229E reporter virus screening. The compounds were preincubated for 30 minutes with the HCoV229E virus at a ratio of 1:2 (Table 2) followed by a titration along the target cells. Remdesivir was used as the positive control, DMSO as the negative control. A) Dosage action titration of lectin starting with a 1:500 dilution. The HCoV229E virus infectivity (absolute values) is shown on the left side and the corresponding cell viability (logarithmic values) of the Huh-7.5-FLuc cells is shown on the right side. B) The values of the HCoV229E virus replication and of the cell viability were shown standardized for the 1:500 dilution or the 1:2,500 dilution (red rectangles in A) against the BSA control;

[0085] FIG. 8 shows exemplary UV/VIS spectra of various manufactured anlec batches;

[0086] FIG. 9 shows a detailed evaluation of an exemplary anlec UV/VIS spectrum by forming the second derivation. The maximum of the UV/VIS spectrum is at 280.0 nm; significant subbands are shown in red; they correspond to the typical absorption layers of the aromatic amino acids Phe, Tyr, and Trp;

[0087] FIG. 10 shows an SDS-PAGE analysis of representative lectin batches. There are shown: lanes 1-3: (5.6/11.1/22.2) ?g HZI 2-09. Lane 4: 4.5 ?l SERVAChrom Protein Standard III. lanes 5-7: (8.9/17.8/35.6) ?g HZI 2-10. (T=14%, C=5%; samples were reduced before application with DTT and were heat denatured while adding SDS at 95? C. for 3 min.);

[0088] FIG. 11 shows an RP-HPLC UV-chromatograph of the anlec batch HZI 2-89. Detection of the absorption at ?=214 nm (blue) and ?=280 nm (red);

[0089] FIG. 12 shows surface plasmon resonance spectroscopy (SPR) binding kinetics and an affinity test for SARS-COV-2-spike protein and different lectin batches isolated from bromelain A) HZI 2-05, (B) HZI 2-06, C) HZI 2-09, D) HZI 2-10, and E) Acm-JRL 2). Increasing concentrations of the ligand (highest concentration: A) 100 ?M, B) 80 ?M, C+D) 50 M, E) 20 ?M, starting therefrom 5 1:1 dilutions) were injected for single cycle measurements. The sensorgraph shows a faster binding than dissociation of the lectins to the spike protein (on-off rate). The surface containing the spike protein was regenerated with 80% ethylene glycol between the individual batches;

[0090] FIG. 13 shows surface plasmon resonance spectroscopy (SPR) binding kinetics and an affinity test for ACE-2 receptor and lectin HZI 2-09. Increasing concentrations of the ligand (highest concentration: 50 ?M, starting therefrom 5 1:1 dilutions) were injected for single cycle measurements;

[0091] FIG. 14 shows deconvoluted mass spectograms of recombinant SARS-COV-2 spike (shown at the top) and recombinant SARS-COV-2 spike digested with PNGase F (shown at the bottom) to detect the glycosylation;

[0092] FIG. 15 shows an SDS-PAGE for detecting the proteolytic degradation of the recombinant SARS-COV-2 spike and of the inhibiting function of HZI 2-07. SARS-COV-2 spike with bromelain protease (HTI 2-08) and bromelain inhibitor (HZI 2-07) in the quantity of material ratio 1:1:1. 1) PageRulerPrestained. The rectangle marks the intact spike protein at approximately 150 kDa; the dashed rectangle shows the spike degradation product at approximately 24 kDa;

[0093] FIG. 16 shows an SDS-PAGE for detecting the proteolytic degradation of the recombinant SARS-COV-2 spike and of the inhibiting function of HZI 2-07 for the bromelain protease fraction HZI 2-08. SARS-COV-2 spike with bromelain protease function HZI 2-08 and bromelain inhibitor HZI 2-07 in the quantity of material ratio 1:1:1 and 1:1:10 1) PageRulerPrestained. The rectangle marks the intact spike protein at approximately 150 kDA;

[0094] FIG. 17 shows an SDS-PAGE for detecting the proteolytic degradation of the recombinant SARS-COV-2 spike and of the inhibiting function of HZI 2-07 for the bromelain protease fraction HZI 2-08. SARS-COV-2 spike with bromelain protease (HZI 2-08) in the quantity of material ratio 10:1 and 100:1 and SARS-COV-2 spike with bromelain protease HZI 2-08 and bromelain inhibitor HZI 2-07 in the quantity of material ratio 1:1:0.5. 1) PageRulerPrestained. The rectangle marks the intact spike protein at approximately 150 kDa; the dashed rectangle shows the spike degradation product at approximately 24 kDa;

[0095] FIG. 18 shows a total ion chromatograph and a deconvoluted mass spectrograph of recombinant SARS-COV-2 spike incubated with bromelain protease fraction HZI-2-08 in the quantity of material ratio 100:1, 10 min., 37? C. The arrow marks the intact spike protein at approximately 150 kDA;

[0096] FIG. 19 shows a total ion chromatograph and a deconvoluted mass spectrograph of recombinant SARS-COV-2 spike incubated with bromelain protease fraction HZI-2-08 in the quantity of material ratio 100:1, 2 h, 37? C. The right arrow marks the intact spike protein at approximately 150 kDA; the left arrow shows the spike degradation product of 24 kDa;

[0097] FIG. 20 shows a total ion chromatograph and a deconvoluted mass spectrograph of recombinant SARS-COV-2 spike incubated with bromelain protease fraction HZI-2-08 in the quantity of material ratio 1:1, 2 h, 37? C. The right arrow marks the intact spike protein at approximately 150 kDA; the left arrow shows the spike degradation product of 24 kDa;

[0098] FIG. 21 shows a total ion chromatograph and a deconvoluted mass spectrograph of recombinant SARS-COV-2 spike incubated with bromelain protease fraction HZI-2-08 and bromelain inhibitor HZI 2-07 in the quantity of material ratio 1:1:1, 2 h, 37? C. The arrow marks the intact spike protein at approximately 150 kDA;

[0099] FIG. 22 shows an SDS PAGE gel in which recombinant SARS-COV-2 spike protein incubated with bromelain (raw fraction) was applied; SARS-COV-2 spike protein not bromelain incubated is also shown for comparison. Details of SDS-PAGE analysis: Lane 1: 2 ?g lectin batch HZI 2-09. Lane 2: 2 ?g lectin batch HZI 2-10. Lane 4: 40 ?g bromelain (Ursapharm). Lanes 5-6: 1 ?g recombinant SARS-COV-2 spike and 0.4 ?g bromelain after 1 h incubation at 37? C. (quantity of material ratio protein:protease?1:1). Lanes 7/8: 1/1.5 ?g recombinant SARS-COV-2 spike. Lane 9: 2 ?g horseradish peroxidase Lanes 3/10: 3.5 ?l SERVA Triple Color Protein Standard III. (T=14%, C=5%; samples were reduced before application with DTT and were heat denatured and centrifuged while adding SDS at 95? C. for 3 min.).

[0100] FIG. 1 shows the primary structure (amino acid sequence) of the seven known bromelain protease inhibitors I, II, III, IV, V, VI, and VII in tabular form. The average and monoisotopic molecular mass in Dalton is furthermore respectively given for every isoform.

[0101] FIG. 2 shows the elution profile of BBP that was applied to a preparative SEC. The y axis stands for the absorption strength at 214 nm while the x axis reflects the elution volume. The first signal of the elution profile (1) is a component of BBP having a molecular mass of approximately 24 kDa that was identified as bromelain proteases. The second signal of the elution profile (2) is a component of BBP having a molecular mass of approximately 6 kDa that was identified as a mixture of bromelain protease inhibitors. The two signals at (3) are the result of first and second rechromatographies of the pooled fraction of the second signal (2).

[0102] FIG. 3(a) shows the profile of a purification of BPP over a strong cation exchange column (SCX). The y axis represents the absorption signal at 280 nm while the x axis characterizes the volume of elution buffer. (a) A strong signal occurs in the pass of the SCX column (signal I.), i.e. molecules evidently do not bind to the SX column. These molecules could be identified as bromelain protease inhibitors via SDS-PAGE and mass spectrometry. At a higher volume of elution buffer and a higher ion concentration (linear gradient) a further signal occurs (signal II). The assumption was able to be confirmed by SDS-PAGE and mass spectrometry that they are bromelain proteases here. (b) A rechromatography of the pooled fractions of the first signal (signal I.), that is of the pass of the first chromatography, using the same SCX column under the same conditions as in the first run, demonstrates that the second signal (peak II.) no longer occurs. The bromelain proteases that are responsible for the second signal could thus be separated from the bromelain protease inhibitors.

EXAMPLE 1

Examination of the Efficacy of Bromelain Over the Alphacoronavirus HCoV229E

1. Sample Preparation

[0103] Bromelain was dissolved in TRIS buffered saline solution (TBS) having a pH of 7.3 (10 mg/ml). Dry substances (HZI 2-01 to ?03) were dissolved in TBS buffer.

TABLE-US-00001 Inhibitor manufacture Note: digestion trials for spike protein (HZI 2-07) Starting material: Bromelain (Merck, Order #. 1.01651.0025, Batch # K38171251 719) Eluent A: Dist. water + 0.05% TFA Eluent B: 60% acetone nitrile for the preparative LC in dist. water + 0.05% TFA Sample solution: 50 mg/ml bromelain dissolved in dist. water and centrifuged at 9384 g for 15 minutes Separation column: Jupiter C4 5 ?m 300 A, 250*10 mm Flow: 5 ml/min Column volume: 19.6 ml Sample feed: 10? after one another per 5 ml of the sample solution Starting conditions: 6% acetonitrile Gradient: 20 minutes of 6% acetonitrile on 60% acetonitrile Performance: The sample feed took place manually on the column equalized with 6% acetonitrile and having a flow rate of 5 ml/min. The column was subsequently flushed with approximately 50-100 ml 6% acetonitrile until approximately the level of the absorption signal prior to the sample application has again been reached. The elution took place by means of a gradient elution of 6-60% acetonitrile over 20 minutes. The peaks occurring during the elution were collected in fractioned form. 10 identical passes with 1 ml sample application each were performed. Lyophilization: Pooled inhibitor fractions

[0104] The suspension was briefly centrifuged to pelletize the insoluble components. The samples HZI 2-01 and HZI 2-02 were not soluble and were not used for further tests. HZI 2-07 was suspended in 200 mM NaCl 20 mM TRIS pH 8.0.

TABLE-US-00002 Bromelain enzyme Note: digestion trials for spike protein (HZI 2-08) Starting material: Bromelain (Merck, Order #. 1.01651.0025, Batch # K38171251 719) Buffer A: 50 mM Tris + 150 mM NaCl + 100 mM D-mannose, pH 7.4 Sample solution: 40 mg/ml bromelain dissolved in buffer A and centrifuged at 9384 g for 15 minutes Separation column: Superdex 30 prep grade, GE Healthcare, self-packed, 2.5*47 cm Flow: 3 ml/min Column volume: approx. 230 ml packed Sample feed: 2 ? 5 ml of the sample solution Starting conditions: Buffer A: Elution: Isocratically with buffer A Performance: The sample feed took place manually on the column equalized with buffer A and having a flow rate of 3 ml/min. The pass was carried out isocratically with buffer A. The peaks occurring were collected in fractioned form. Two identical passes with 5 ml sample application each were performed. The collected fractions 2 + 3 of both passes were concentrated to a volume of approximately 5 ml over a 10 kD filtration membrane (Satorius Vivaspin 20) and were subsequently stored in the refrigerator. SEC: A rechromatography of the concentrated fractions 2 + 3 was carried out by means of SEC. Buffer A: 50 mM Tris + 150 mM NaCl, pH 7.2 Sample solution: Concentrated fractions 2 + 3 from both SEC passes (volume approximately 5 ml) Separation column: Superdex 30 prep grade, GE Healthcare, self-packed, 2.5*47 cm Flow: 3 ml/min Column volume: approx. 230 ml packed Sample feed: 5 ml of the sample solution (conc. Fr 2 + 3) Starting conditions: Buffer A Performance: The sample feed took place manually on the column equalized with buffer A and having a flow rate of 3 ml/min. The pass was carried out isocratically with buffer A. The peaks occurring were collected in fractioned form. The collected fractions 2-4 of the pass were pooled.

[0105] Table 1 almost all the sample used together.

TABLE-US-00003 TABLE 1 Sample Sample description Concentration Remarks Bromelain Bromelain stem solution in TBS 10 mg/ml Bromelain Acm-JRL 1 Anlec in TBS 2.2 mg/ml Lectin 143 ?M Acm-JRL 2 Anlec in TBS, Batch 2 2.04 mg/ml Lectin 133 ?M HZI 2- 03 Anlec lyophilizate ~10 mg/ml; contains Lectin (Bioaffinity chromatography, D- larger quantities of mannose, dialyzed against glycine, citrate sodium citrate, and citrate, pH 6.9) HZI 2- 04 Anlec in TBS 1.77 mg/ml Lectin (Bioaffinity chromatography, D- 115 ?M mannose, dialyzed against 2 l water, dialyzed against 50 mM TBS buffer, concentrated with 5 kDa membrane) HZI 2- 05 Anlec in TBS 4.22 mg/ml Lectin (Bioaffinity chromatography, D- 336 ?M mannose, dialyzed against 2 l water, dialyzed against 50 mM TBS buffer, concentrated with 5 kDa membrane) HZI 2- 06 Anlec in dist. water (bioaffinity 2.9 mg/ml Lectin chromatography, D-mannose, 188 ?M hydrophobic interaction chromatography C6, concentrated with 5 kDa membrane) HZI 2- 07 Bromelain inhibitor lyophilizate 1.05 mg/ml Inhibitor (RP-HPLC C4, freeze dried) HZI 2- 08 Protease mixture in mM TRIS + 150 mM 1.14 mg/ml Protease NaCL pH 7.2 (size exclusion chromatography) mixture HZI 2- 09 Anlec in TBS 2.2 mg/ml Lectin (Bioaffinity chromatography, D- 143 ?M mannose, dialyzed against 50 mM TBS buffer, concentrated with 5 kDa membrane) HZI 2- 10 Anlec in TBS 3.6 mg/ml Lectin (Protease inhibitor with MMTS, 234 ?M bioaffinity chromatography, D- mannose, dialyzed against 50 mM TBS buffer, concentrated with 5 kDa membrane) MMTS: Methyl methanethiosulfonate; RP-HPLC: reversed-phase high performance liquid chromatography; TBS: TRIS buffered saline solution (pH 7.3)

2. Performance of the Tests for Antiviral In Vitro Activity of Bromelain, Fractions, and Lectin Against HCoV229E

[0106] Bromelain and lectin were dissolved in TBS. The suspension was briefly centrifuged to pelletize the insoluble components. Firefly luciferase expressing Huh 7.5 FLuc cells were cultivated in DMEM medium (Gbico #41965-039)+10% fetal calf serum (FCS)+1% penicillin/streptomycin+1% L-glutamine+2% non-essential amino acids (=DMEM complete)+5 mg/ml blasticidin. 2?10.sup.4 cell/well (96 well plate) were completely sowed in 100 ?l DMEM 24 h before the experiment.

[0107] The alphacoronavirus HCoV229E that contains a renilla luciferase (RLuc) was preincubated with the compounds at RT in a ratio of 10:1 (90 ?l virus+10 ?l compound) for 30 min. The titer of the virus stock solution amounted to 3.41?10.sup.6 TCID50/ml (virus concentration at which 50% of the cells are infected per ml for all the experiments. Corresponds here to the number of infectious particles per ml.). The mixture was diluted in media and titrated on target cells in 1:5 steps after the preincubation. FIG. 4 shows a schematic representation of the test performance for the alpha-coronavirus CoV229E.

[0108] The preincubation was carried out in a ratio of 1:2 (10 ?l virus+10 ?l compound) for further experiments. Firefly luciferase expressing Huh 7.5 FLuc cells were infected with HCoV229E one day after the sowing in the presence of the specified concentrations of the compound [serial titration (1:5) of the cells]. The viral starting dilution amounted to 1:500, based on the starting concentration in the original sample and are shown in Table 2. The specifications for the bromelain sample having the stock concentration 10 mg/ml are analog to the sample HZI 2-03 and are not shown separately.

TABLE-US-00004 TABLE 2 Bromelain and lectin concentrations for the HCoV229E test Bromelain Acm-JRL 1 HZI 2- 03 HZI 2- 04 HZI 2- 05 2 mg/ml Concentration of 2.2 mg/ml 10 mg/ml 1.77 mg/ml 4.22 mg/ml 2 mg/ml stock solution 143 ?M 115 ?M 336 ?M 1:10 predilution 0.22 mg/ml 1 mg/ml 0.177 mg/ml 0.422 mg/ml 0.2 mg/ml 14.3 ?M 11.5 ?M 33.6 ?M 1:2 predilution 1.1 mg/ml 5 mg/ml 0.885 mg/ml 2.11 mg/ml 1 mg/ml 71.5 ?M 57.5 ?M 168 ?M Serial dilution 0.0044 mg/ml 0.02 mg/ml 0.0035 mg/ml 0.0084 mg/ml 0.004 mg/ml 1:500 0.286 ?M 0.23 ?M 0.672 ?M Stock solution 3.41 ? 10.sup.6 3.41 ? 10.sup.6 3.41 ? 10.sup.6 3.41 ? 10.sup.6 3.41 ? 10.sup.6 HCoV229E TCID50/ml TCID50/ml TCID50/ml TCID50/ml TCID50/ml 1:10 predilution 3.1 ? 10.sup.6 3.1 ? 10.sup.6 3.1 ? 10.sup.6 3.1 ? 10.sup.6 3.1 ? 10.sup.6 (90 ?l virus + TCID50/ml TCID50/ml TCID50/ml TCID50/ml TCID50/ml 10 ?l compound) .fwdarw. per ml: .fwdarw. per ml: .fwdarw. per ml: .fwdarw. per ml: .fwdarw. per ml: 3.1 ? 10.sup.6 3.1 ? 10.sup.6 3.1 ? 10.sup.6 3.1 ? 10.sup.6 3.1 ? 10.sup.6 virus particles on virus particles on virus particles on virus particles on virus particles on 0.22 mg compound 1 mg compound 0.177 mg compound 0.422 mg compound 0.2 mg compound 1:2 predilution 1.705 ? 10.sup.6 1.705 ? 10.sup.6 1.705 ? 10.sup.6 1.705 ? 10.sup.6 1.705 ? 10.sup.6 (10 ?l virus + TCID50/ml TCID50/ml TCID50/ml TCID50/ml TCID50/ml 10 ?l compound) .fwdarw. per ml: .fwdarw. per ml: .fwdarw. per ml: .fwdarw. per ml: .fwdarw. per ml: 1.75 ? 10.sup.6 1.75 ? 10.sup.6 1.75 ? 10.sup.6 1.75 ? 10.sup.6 1.75 ? 10.sup.6 virus particles on virus particles on virus particles on virus particles on virus particles on 1.1 mg compound 5 mg compound 0.885 mg compound 2.11 mg compound 1 mg compound TCID50/ml: Virus concentration at which 50% of the cells are infected per ml. Corresponds here to the number of infectious particles per ml.

[0109] 48 h after the inoculation and incubation of the cells at 33? C. and 5% CO.sub.2, the virus inoculum was removed and the cells were washed twice in phosphate buffered saline solution (PBS) and lyzed in 50 ?l PBS/0.5% Triton X-100. The lysis of the cells was further amplified by freezing the plates at ?20? C. 20 ?l of the lyzate were used for the measurement of the cell viability via the firefly luciferase signal and a respective 20 ?l of the lyzate were used for the analysis of the virus replication/infection efficiency via the renilla luciferase signal.

3. Results of the Tests for Antiviral In Vitro Activity of Bromelain, Fractions, and Lectin Against HCoV229E

[0110] Bromelain and lectin batches were first tested by a HCoV229E renilla luciferase reporter virus, an alpha coronavirus, using Huh-7.5-FLuc cells that are highly permeable for an HCoV229E infection. These cells are designed such that they express a firefly luciferase reporter gene, which makes the evaluation of the cell viability in a dual luciferase reporter assay possible. All the lectin samples consisted of different isolates of the lectin from bromelain. Samples HZI 2-01 and 2-02 were not used due to the insolubility. Bromelain was used as the control in concentrations of 2 mg/ml or 10 mg/ml.

[0111] It was able to be demonstrated that bromelain and lectin displayed a promising antiviral activity against the HCoV229E reporter virus (FIG. 5).

[0112] High concentrations of bromelain (FIG. 5E and FIG. 5F) resulted in a small cell viability. At 100% cell viability (indicated by the dashed line), an approximately 50% reduction (round dots) of the virus replication of the alphacoronavirus HCoV229E was able to be observed for bromelain. The black arrows mark the 1:500 dilution or the 1:2500 dilution that were applied standardized in FIG. 6; The 1:2,500 dilution shows a smaller cytoxicity per se due to the reduced compound concentration, whereby any occurring antiviral effectsand thereby also an improved cell viabilitycan be better demonstrated.

[0113] A virus replication reduced by approximately 25% can also be recognized at the 1:500 dilution on a standardization of the results with approximately 100% cell viability in the samples Acm-JR 1 and HZI 2-05. HZI 2-03 shows an approximately 20% reduction, whereas HZI 2-04 is inactive. Bromelain is accompanied at both concentrations (2 and 10 mg/ml) by a great reduction of the cell viability so that valid conclusions cannot be drawn on a reduction of the virus replication. The reduction of the cell viability is mainly caused by the protease activity of the bromelain, whereby a detaching of the cells from the well base takes place. The subsequent luciferase assay only includes the adherent cells and does not provide a differentiation of dead and vital cells floating in the culture medium, whereby a false result of the cell viability occurs.

[0114] A reduction of the virus replication of approximately 20-25% can be recognized in the 1:2,500 dilution in all lectins at approximately 100% cell viability. Bromelain even shows an approximately 45-50% reduction with a cell viability>100% at a stock concentration of 2 mg/ml. Only the highly concentrated bromelain having a stock concentration of 10 mg/ml shows a reduced cell viability so that valid conclusions cannot be drawn on a reduction of the virus replication. However, a drop in the virus replication can also be documented here.

[0115] The results should be confirmed by the lectins in a further trial. In addition, the antiviral action should be amplified with a higher substance to virus ratio in the preincubation (1:2 instead of 1:10). The starting concentrations of the titration 1:500 are furthermore set to correspond to the first pilot trials. The samples Acm-JRL 1, HZI 2-03, and HZI 2-05 were used for the experiment. Remdesivir (1 m in TBS), DMSO (1:40 in TBS), and BSA (5 mg/ml in TBS) were used as controls (FIG. 7A).

[0116] The positive control remdesivir brings about a complete reduction of the virus replication. At the 1:500 dilution, the lectins Acm-JRL 1 and HZI 2-05 displayed moderate antiviral effects on the HCoV229E reporter virus (FIG. 7B) while HZI 2-03 had no antiviral effect. The samples Acm-JRL 1 and HZI 2-05 displayed an approximately 25% reduction of the HCoV229E virus replication at >100% cell viability. If the 1:2,500 dilution is looked at, sample HZI 2-05 displayed a 50-60% reduction of the virus replication at >100% cell viability.

[0117] The results of the preceding trial were able to be confirmed by this trial. A further reduction of the virus replication in sample HZI 2-05 was able to be achieved by a higher concentration of the lectins [ratio 1:2 approximately 50-60% reduction vs. 1:10=approximately 20$ reduction HCoV229E replication (shown in FIG. 6B)]

[0118] It was able to be shown that an infection by the HCoV229E can be prevented in the in vitro trial.

EXAMPLE 2

Isolation of the Samples HZI 2-09 and HZI 2-05

[0119] 1200 mg Bromelain (Merck, Order #. 1.01651, Batch #K38171251719) are dissolved in 30 ml buffer A (50 mm TRIS, 500 mM NaCl, pH 7.2) at 40 mg/ml and are centrifuged at 9000 g for 15 min. A 5 ml aliquot of this solution is loaded at a flow of approximately 2 ml/min on a D-mannose agarose chromatography column of the dimensions 10?50 mm (pack volume 4 ml) linked covalently that had previously been equalized with buffer A. The column is flushed with at least 25 CV (column volumes) until the original base line has again been reached or a constant base line has been obtained. The elution of the anlec now takes place by switching to buffer B (50 mM TRIS, 500 mM NaCl, 1 M D-mannose, pH 7.2); the fraction is collected. This application in 5 ml aliquots is repeated multiple times, e.g. five times. The eluated and collected fractions are pooled and are filled with MWCO 3.5 kDa in a dialysis tube. The fraction size of an elution amounts to approximately 7 ml. The dialysis takes place against 2 l buffer C (50 mM TRIS, 150 mM NaCl, pH 7.2) under steady stirring at room temperature or at 4? C. with multiple buffer changing until the mannose concentration by dialysis amounts to ?1 mM. Flocculation is separated by centrifuging or filtration after the dialysis. The excess is reduced to a smaller volume by means of an ultrafiltration membrane having MWCO 5 kDA or less so that the original protein concentration after the dialysis is increased to a multiple. The protein concentration in a first approximation by means of the UV absorption can be calculated at 280 nm, e.g. by means of a theoretically calculated molar extinction coefficient for anlec in accordance with www.uniprot.org, as was done here. Typical UV/VIS spectra for exemplary batches are shown in FIG. 8, a detailed evaluation by forming the second derivation is shown in FIG. 9.

[0120] The results of an electrophoretic analysis by means of SDS-PAGE exemplary anlec batches are shown in FIG. 10. The two batches show a main band at approximately 15 kDA and a plurality of subbands; these subbands can in particular be recognized by the heavy gel overload in lanes 3 and 7.

[0121] An RP-HPLC analysis of a representative batch is shown in FIG. 11; the main mass 1 was determined by LC-MS coupling and a calculation via deconvolution at 15.388 Da (approximately 15.38 kDa). +162 Da species could furthermore also be measured that indicate glycated proteins, see Gross et al. (2020) J. Pharm. Biomed. Anal. 181, 113075.

EXAMPLE 3

[0122] Examination of the Binding of Bromelain with Respect to the Spike Protein of the SARS CoV-2 Virus.

1. Sample Preparation

1.1 Cloning, Expression, and Purification of the SARS-COV-2 Spike Protein

[0123] The nucleotide sequence of the extracellular domains of the SARS-COV-2 spike protein (1-1213) was acquired as a synthetic gene from Eurofins MWG. The gene was amplified by means of PCR and was provided with the restriction interfaces 5-BamHI/XhoI-3. The gene was subsequently tied up by sticky end cloning by means of T4 DNA ligase in the eucaryotic expression vector pCAGGS (BamHI tied up again). The expression of the spike protein was carried out in HEK293 cells. For this purpose, HEK293 cells were completely cultivated in a hyperflask (growth area 1720 cm.sup.2) up to a confluence of 80-90% in DMEM (37? ? C., 5% CO.sub.2). The transfection was subsequently carried out with a 1:2 ratio of pCAGGS spike protein:polyethyleneimine (linear, average molecular weight 25,000 Da). The DNA-PEI solution was removed after 5 hours and completely replaced with DMEM. The cells were cultivated for a further 48 h at 37? C. and 5% CO.sub.2 before the culture excess was harvested.

[0124] The culture excess was applied to a 5 ml GHisTrap HP column to purify the SARS-COV-2 spike protein. After a thorough washing with lysis buffer (200 mM NaCl, 20 mM TRIS pH 8.0, 20 mM imidazole), the protein was eluated with elution buffer (200 mM NaCl, 20 mM TRIS pH 8.0, 500 mM imidazole). The acquired protein was subsequently further separated via a HiLoad 16/600 Superdex 200 PG and the peak was collected with an elution volume of 60-70 ml.

1.2 SPR Binding and Affinity Test

[0125] The binding kinetics and affinity tests of lectins were carried out on a Biacore X100 system (GE Healthcare). The purified extracellular domain of the spike protein (1-1213) and of the ACE-2 receptor (Sigmal Aldrich, #SAE0064) was covalently immobilized at a CM5 sensor chip via aminocoupling in 10 mM sodium acetate buffer (pH 4.5) for a final 9600 for the spike and 4000 for the ACE-2 receptor. Surface plasmon resonance spectroscopy (SPR) assays were carried out at a flow rate of 30 ?l/min in 1?HBS-EP (150 mM NaCl, 10 mM HEPES pH 7.4, 3 nM EDTA, 0.005% Tween-20). Increasing concentrations of lectin (1.25 to 20 M) for spike were injected for single cycle measurements (120 s contact time, 180 s dissociation time).

1.3 SARS-COV-2 SDS-PAGE and LC-MS Analytics

[0126] The glycolization of the recombinantly acquired spike was demonstrated enzymatically: 20 ?g SARS-COV-2 spike in 200 mM NaCl solution and 20 mM TRIS pH 8.0. Buffering was carried out with 2 ?l (1000 IU) PNGAse F and the associating buffering was carried out in accordance with the manufacturer's protocol of New England Biolabs?, Inc. and incubated at 37? C. for 6 h. The lyophilizate of the bromelain inhibitor (HZI 2-07) was buffered and suspended in 200 mM NaCl solution with 20 mM TRIS pH 8.0. The Eppendorf vessel was centrifuged with the suspension for the pelletization of the insoluble components and the content of the solution was photometrically determined. To detect the proteolytical degradation of the recombinant SARS-COV-2 spike and of the peptide product fragment sizes produced therefrom and of the inhibitory function of the HZI 2-07, recombinantly produced SARS-COV-2 spike was mixed with bromelain protease HZI 2-08 and without inhibitor HZI 2-07 in stoichiometric ratios and was incubated at 37? C. The sample extraction for SDS-PAGE or LC-MS analytics took place as follows:

[0127] The sample was admixed with SDS dye (1.2 g SDS, 6 mg bromophenol blue, 4 ml glycerol, 0.6 ml 1 M TRIS pH 8.0, 5.4 ml H.sub.2O heated to dissolve all the components; 930 mg dithiotheitol (DTT) were then added to acquire a 6?SDS dye buffer) and immediately boiled at 100? ? C. for 2-3 min.

[0128] Sample extraction for LC-MC analytics: The sample was immediately frozen in liquid nitrogen, stored at ?80? C., and thawed on ice a few minutes before the LC-MS measurement.

[0129] The SDS-PAGE with 12% v/v polyacrylamide portion was loaded with 10 ?l of the prepared samples. 6 ?l PageRuler Prestained Protein Ladder (Thermo Fisher Scientific) was loaded to monitor the progress of the SDS-PAGE and to estimate the approximate amount of the separated proteins after the dyeing of the gel. The electrophoresis was carried out in a Mini-PROTEAN? Tetra System (BIO RAD) with an SDS Laemmli buffer at a voltage of 140 V for 90 min. After the termination of the electrophoresis the SDS-PAGE was heated in a microwave oven with a dye solution (Coomassie Blue 0.05% m/v, methanol 50% v/v, pure acetic acid 7% v/v, water 43% v/v) and stored in water for 24 h before the photodocumentation took place.

[0130] The direct intact protein UPLC-ESI-MS analysis was carried out over an UltiMate 3000 UPLC system coupled with a Maxis4G Q-TOF mass spectrometer with an Apollo II ESI source. Measurement took place in the positive mode. The samples were separated via an Aeris Widepore XB-C8 column (3.6 ?m, 150?2.1 mm; Phenomenex). The separation took place at a flow rate of 0.3 ml/min (eluent A: deionized water with 0.1% V/V acetic acid, eluent B: distilled acetonitrile with 0.1% v/v acetic acid) at 45? C. with a gradient of 2% B for 30 s, followed by a linear gradient up to 75% B in 10 min and a constant of 75% B for a further 3 min. The flow rate was throttled to 75 ?l/min before entering the ion source. Mass spectra were generated in the centroid mode of 150-2500 m/z at 2 Hz. The mass spectrometry ion source parameters amounted to: 500 V end plates offset, 4000 V capillary voltage, 1.1 bar nebulizer gas pressure, 6 l/min dry gas flow, and 180? C. drying temperature. Protein masses were summed via the total ion chromatograph from the retention time period 6.5 min to 9 min for the bromelain protease fraction (HZI 2-08) assays with and without bromelain inhibitor (HZI 2-07) for the total ion chromatographs to demonstrate the glycolsylization of 7.0-7.6 min and were deconvoluted with an instrument resolving power of 8000 to the maximum entropic deconvolution algorithm at high resolution with Compass DataAnalysis to natural masses.

2. Results

2.1 SARS-COV-2 Spike Protein Binding Assay

[0131] SARS-Co-2 spike protein binding assays were carried out with the aid of SPR in vitro for the lectins differently isolated from the bromelain. Purified SARS-COV-2 spike protein was immobilized by aminocoupling on a Cm5 chip for this purpose. The jacalin-related lectins were subsequently tested for their binding; their dissociation constant and their binding kinetics were determined. In this respect, different binding characteristics and on-off rates were observed, which is shown in more detail in the following.

[0132] Different batches of the bromelain isolated lectin (HZI 2-05, HZI 2-06, HZI 2-09, HZI 2-10, and Acm-JRL 2) were examined as to their characteristics within the binding assay by means of SPR. In this respect, slight differences of the batches were able to be determined, with above all the association and dissociation rates being changed. It was able to be observed here that the bound lectin at HZI 2-09 and Acm-JRL 2 dissociated more slowly from the spike protein than an association was able to be measured, whereby the measured values on the chip increased at a constant rate. The measured values of the different injections of the respective lectin batches are shown in dependence on the time in FIG. 12. The surface containing the bound spike protein was regenerated with 80% ethylene glycol between the individual measurements.

[0133] The sensorgram of the SPR analysis of the binding kinetics and the affinity test is shown for SARS-COV2 spike protein and different lectin batches isolated from bromelain (HZI 2-05, HZI 2-06, HZI 2-09, HZI 2-10, Acm-JRL 2) in increasing concentrations. The K.sub.D values and association constants and R.sub.max values were calculated using Biacore X100 software (Table 3). We were able to show that the lectin batches HZI 2-09, HZI 2-10, and Acm-JRL 2 have a high affinity to the SARS-COV-2 spike protein in the micromolar range. A fast binding (K.sub.a value 1031 1/Ms) and slow dissociation (K.sub.a value 0.003 1/s) of the spike protein could furthermore above all be observed.

TABLE-US-00005 TABLE 3 Surface plasmon resonance spectroscopy (SPR binding kinetics and affinity test for SARS-CoV2 spike protein and different lectin batches isolated from bromelain. The values calculated with the Biacore X100 software are shown. Lectin K.sub.a (1/Ms) K.sub.d (1/s) K.sub.D (mol/l) R.sub.max (RU) HZI 2- 05 132.6 0.005 4.12 ? 10.sup.?5 746.9 HZI 2- 06 358.7 0.008 2.288 ? 10.sup.?5 369.3 HZI 2- 09 331.0 0.003 8.249 ? 10.sup.?6 627.6 HZI 2- 10 285.5 0.003 9.999 ? 10.sup.?6 373.6 Acm-JRL-2 1031 0.003 2.626 ? 10.sup.?6 660.0

[0134] In a further trial, the SPR assay was examined for the analysis of the binding kinetics and the affinity test of the lectin at the ACE-2 receptor (FIG. 13). Lectin concentrations between 3.125 and 50 UM were used for this purpose.

[0135] The sensorgram of the SR analysis of the binding kinetics and of the affinity test for the ACE-2 receptor and the lectin batch HTI 2-9 is shown in increasing concentrations in FIG. 13. A conclusion can be drawn from the sensorgram that a dissociation constant of 100 UM or higher is present. In addition, different binding characteristics with respect to the on-off rate are shown. The K.sub.D cannot be calculated using the measurement since it is outside the measured concentrations.

2.2 SARS-COV-2 Spike SDS-PAGE and LC-MS Analytics

[0136] The glycolization of the spike protein was demonstrated by means of LC-MS analytics. The deconvoluted mass spectograms are shown in FIG. 14. PNGase F removes N bound oligosaccharides from glycoproteins. In this respect, splitting takes place between the innermost GIC-NAc and asparagine residues of oligosaccharides. After the digestion of the recombinant SARS-COV-2 spike protein using PNGase F, a mass shift in the mass spectrograph can be recognized, triggered by the loss of oligosaccharides from the spike protein.

[0137] SDS-PAGE and fluid chromatography coupled with mass spectrometry was used for the analytical demonstration of the proteolysis activity of the bromelain protease fraction HZI 2-08 and of the inhibitory function of the peptide HZI 2-07 acquired from bromelain. The fragmentation of the recombinant SARS-COV2 spike and the inhibitory function of HZI 2-07 were able to be demonstrated via SDS-PAGE (FIGS. 15-17). The mass spectra and the neutral masses deconvoluted therefrom can likewise demonstrate the fragmentation (FIGS. 18-21).

[0138] The proteolytic degradation of the SARS-COV-2 spike protein by the protease (HTI 2-08) can be recognized in lanes 2 to 7 in FIG. 15. The main mass of approximately 150 kDa of the intact spike cannot be demonstrated. However, a main degradation product can be recognized at approximately 24 kDa (dashed rectangle in FIG. 15). After the addition of HZI 2-07 (=inhibitor), the inhibitory function becomes visiblea degradation of the spike protein is inhibited. This results in bands at approximately 150 kDa in lanes 8-10 (rectangle in FIG. 15).

[0139] The inhibitory action of the inhibitor on the protease (HZI 2-08) is maintained over a longer trial time period; testing took place up to 120 min. This can be recognized at the intact spike protein in FIG. 16 that was marked by a rectangle.

[0140] The kinetics of the proteolytic degradation of the spike protein is show in FIG. 17. At a spike:protein ratio of 10:1, a significant band at approximately 150 kDa (row 2) can be recognized after a 10 minute incubation, which indicates an intact spike protein. After a longer incubation of 120 min (lane 5), it can be recognized that the band has already substantially disappeared, the fragmentation increases. This is not the case at a ratio of 100:1 (lanes 3 and 6). The intact protein can also be seen here after a 120-minute incubation, which indicates an insufficient action of the protease HZI 2-08. An optimum ratio of spike:protease of 1:1 thus results with the results that are shown in FIG. 15 and FIG. 16. It can furthermore be concluded that the ratio of 1:1:0.5 (spike:protease:inhibitor) is unfavorable since the degradation of the spike was unable to be inhibited. The ratio 1:1:1 appears optimum here (FIG. 16).

[0141] The total ion chromatograph (TIC) and the mass spectrum of the SARS-COV-2 spike protein are shown in FIG. 18. After addition of the protease (HZI 2-08; ratio spike:protease: 100:1) no mass peak could be demonstrated at 150 kDa after 10 min (arrow). The fragments that occur indicate an effective degradation of the spike protein after a protease addition. This confirms the results that were already represented by means of SDS-PAGE (FIG. 15).

[0142] No mass peak could also be demonstrated at 150 kDa (right arrow, FIG. 19) after a 120 min. incubation with the protease (HZI 2-08; ratio spike:protease:100:1). This indicates that the proteolytic action of the protease remains constant over a time period of at least 120 min. In addition, a mass peak is now visible at m/z 24.392, which represents the main degradation product of the spike protein. This confirms the results that were already represented by means of SDS-PAGE (FIG. 15).

[0143] The proteolytic action of the protease (HZI 2-08) again becomes clear in FIG. 17). A spike with a protease is here present in a ratio of 1:1: The peak of the degradation product (left arrow) is very pronounced after 120 min incubation.

[0144] After addition of the inhibitor HZI 2-07, the intact spike protein becomes visible at m/z 145.196 (FIG. 21). The degradation product at 24 kDA cannot be detected. This confirms the result of FIG. 15 where it has already been able to be shown that the inhibitor inhibits the protease (HZI 2-08). The spike protein can accordingly not be degraded.

[0145] FIG. 22 shows an SDS-PAGE analysis of recombinant SARS-COV-2 spike protein after 1 h digestion with bromelain base powder at 37? C. (material quantity ratio protein:protease approximately 1:1, lanes 5 and 6). SARS-COV-2 spike protein not incubated with bromelain is also shown for comparison. (lanes 7 and 8). The results show that bromelain base powder is also able to digest the spike protein.

[0146] It was thus able to be shown that bromelain can bind to the spike protein of coronaviruses and that the lectin contained in bromelain (5% of the total bromelain) can bind both to the spike protein and to mannose, but not to the ACE-2 receptor of the host with a comparable K.sub.D.