AU2021410995B2 - Anti-viral proteases and methods of use - Google Patents

Abstract

The present invention relates to proteases derived from

Description

ANTI-VIRAL PROTEASES AND METHODS OF USE

1. Field of the Invention

The present invention relates to methods of using anti-viral proteases for the inactivation of viruses. The anti-viral proteases are derived from Carica papaya.

2. Background to the Invention

In the last 20 years, various coronaviruses have increasingly caused significant human disease. Severe Acute Respiratory Syndrome (SARS) first appeared in Asia in November 2002 as atypical pneumonia. The ensuing outbreak lasted 6 months and spread to more than 20 countries in Asia, Europe, South America and North America, with over 8,000 people infected and 774 known deaths. The responsible pathogen is a coronavirus designated SARS CoV. In 2012, Middle East Respiratory Syndrome (MERS) first appeared in Saudi Arabia, and has since been detected in 27 countries in the Middle East, Africa and Asia, with 858 known deaths. The responsible pathogen is another coronavirus designated MERS-CoV. In December 2019, another novel severe respiratory disease was detected in China, and has since pervaded the entire globe, with approximately 248 million confirmed cases so far and over 5 million known deaths so far. The responsible pathogen is yet another coronavirus designated Severe Acute Respiratory Syndrome Coronavirus-2 ("SARS-CoV-2"). The suite of diseases caused by SARS-CoV-2 has been designated COVID-19. While global efforts have seen the rapid development, approval and rollout of numerous vaccines in an effort to combat COVID-19, a combination of manufacturing capacity limitations, vaccine hesitancy, difficulties in cold and ultra-cold supply chain logistics and decreased efficacy against emerging variants has meant that SARS-CoV-2 / COVID-19 remains the single most urgent health issue the world faces.

Accordingly, there is currently an urgent need for compositions and methods that will disable or inactivate the SARS-CoV-2 virus (1) in the atmosphere, including, for example, SARS-CoV 2 in aerosols, (2) on surfaces, including built environment surfaces and the skin of humans and other animals, and (3) in the mouth, eyes, and respiratory tract (including the nose, throat, larynx, trachea, bronchi and lungs) of humans and other animals. Such compositions and methods should be equally effective against any and all possible future variants, thereby providing certainty in treatment rollouts and epidemiological decisions. Such compositions and methods would not only provide immediate health benefits to the world's population, but would also assist in economic recovery after lockdowns and consequent mental health improvement.

3. Summary of the invention

It has been determined that proteases present in the ripe flesh of Carica papaya are effective in inactivating the SARS-CoV-2 virus, such that it is no longer capable of infecting host cells. Such proteases can be delivered so as to prevent COVID-19 by inactivating SARS-CoV-2 in the atmosphere, including, for example, through inactivation of SARS-CoV-2 in aerosols, and on surfaces, including built environment surfaces and the skin of humans and other animals, and in the mouth, eyes, and respiratory tract (including the nose, throat, larynx, trachea, bronchi and lungs) of humans and other animals. The proteases can also be delivered to treat COVID-19 by targeting the respiratory tract of a human or other animals. The proteases target a surface protein of SARS-CoV-2, such as a spike protein, thereby rendering it unable to infect a host cell.

Accordingly, in a first aspect, the present invention provides a method of inactivating a virus, wherein the method comprises administering one or more proteases derived from Carica papaya, or a composition thereof, to the virus.

In a second aspect, the present invention provides a method of preventing an infection in a subject caused by a virus, wherein the method comprises administering one or more proteases derived from Carica papaya, or a composition thereof, to the virus.

In a third aspect, the present invention provides a method of treating an infection in a subject caused by a virus, wherein the method comprises administering one or more proteases derived from Carica papaya, or a composition thereof, to the virus.

In a fourth aspect, the present invention provides use of one or more proteases derived from Carica papaya, or a composition thereof, in the manufacture of a medicament for inactivating a virus.

In a fifth aspect, the present invention provides use of one or more proteases derived from Carica papaya, or a composition thereof, in the manufacture of a medicament for preventing an infection in a subject caused by a virus.

In a sixth aspect, the present invention provides use of one or more proteases derived from Carica papaya, or a composition thereof, in the manufacture of a medicament for treating an infection in a subject caused by a virus.

In a seventh aspect, the present invention provides one or more proteases derived from Carica papaya, or a composition thereof, for use in or when used for inactivating a virus.

In an eighth aspect, the present invention provides one or more proteases derived from Carica papaya, or a composition thereof, for use in or when used for preventing an infection in a subject caused by a virus.

In a ninth aspect, the present invention provides one or more proteases derived from Carica papaya, or a composition thereof, for use in or when used for treating an infection in a subject caused by a virus.

In a tenth aspect, the present invention provides use of one or more proteases derived from Caricapapaya, or a composition thereof, for inactivating a virus.

In an eleventh aspect, the present invention provides use of one or more proteases derived from Carica papaya, or a composition thereof, for preventing an infection in a subject caused by a virus.

In a twelfth aspect, the present invention provides use of one or more proteases derived from Carica papaya, or a composition thereof, for treating an infection in a subject caused by a virus.

In a thirteenth aspect, the present invention provides a kit for use in or when used for inactivating a virus, preventing an infection or treating an infection in a subject caused by a virus, wherein the kit comprises one or more proteases derived from Carica papaya, or a composition thereof.

In one embodiment, the administration is to the atmosphere, including for example to a virus in aerosol form in the atmosphere. In another embodiment, the administration is to a surface, including for example a built environment surface or the skin of a human or other animal. In another embodiment, the administration is to the mouth, eyes, and/or respiratory tract (including the nose, throat, larynx, trachea, bronchi and lungs) of a human subject or other animal subject.

In one embodiment, the one or more protease is a serine protease or a cysteine protease. The one or more proteases may be extracted from ripe Carica papaya flesh. In one embodiment the extraction process comprises an alkali treatment step. In one embodiment, the one or more protease is proteolytically active.

In one embodiment the virus is a coronavirus, such as a SARS-CoV-2 virus. In another embodiment the virus comprises a surface glycoprotein (such as a spike protein). In another embodiment, the spike protein comprises an amino acid sequence having at least 40%, preferably at least 60 or 70%, sequence identity with the SARS-CoV-2 surface glycoprotein amino acid sequence shown in GenBank Accession No. QJX59884.1 (SEQ ID NO: 2).

In a particular embodiment, the one or more proteases derived from Carica papaya, or the composition thereof, is administered using a nebuliser or a dry powder inhaler.

In one embodiment, the composition is a pharmaceutical composition comprising one or more proteases derived from Carica papaya together with a pharmaceutically acceptable compatible carrier, excipient or diluent.

In one embodiment, the present invention provides a liquid pharmaceutical composition for administration to the respiratory tract of an individual using a nebuliser comprising one or more proteases derived from Carica papaya together with pharmaceutically acceptable compatible carriers, excipients or diluents and having a physiologically acceptable pH and osmolality.

In another embodiment, the present invention provides a dry powder composition for inhalation by an individual, said composition comprising particles of respirable or inhalable size wherein the particles comprise one or more proteases derived from Carica papaya.

In another embodiment, the present invention provides a metered dose inhaler composition for inhalation by an individual, said composition comprising one or more proteases derived from Carica papaya and a propellant.

4. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

By "about' is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 %to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

By "active protease" is meant a protease that is proteolytically active.

The term "administering" includes delivering to or "contacting". Hence, a protease of the present invention may be administered to a virus by contacting the virus with the protease.

Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to mean the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed steps or elements are required or mandatory, but that other steps or elements are optional and may or may not be present.

By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

The term "derivable" may be used interchangeably with the term "obtainable".

The term "derived" may be used interchangeably with the term "obtained".

By "isolated' is meant material that is substantially or essentially free from some or all of the components that normally accompany it in its native state. For example, an"isolated protease" as used herein, refers to in vitro isolation and/or partial purification of a peptide or polypeptide protease molecule from its natural cellular environment, and from association with some or all of the components of the cell.

The term "obtainable", when used in relation to proteins or compositions of the present invention, includes proteins or compositions produced not only by a particular specified method, but also the same proteins or compositions however produced, for example, by sourcing proteases from fruits or vegetables, or by recombinant DNA technology or other genetic engineering methods, for example, by using a recombinant expression system.

The terms "patient, "subject and "individual" are used interchangeably and refer to patients, subjects and individuals of human or other animals and include any one for whom it is desired to a treat, prevent, ameliorate, or reduce the severity of a disease, disorder or condition using the invention. However, it will be understood that "patient" does not imply that symptoms are present. Suitable animals, such as mammals, that fall within the scope of the invention include, but are not restricted to, primates (e.g. humans, chimpanzees) livestock animals (e.g. sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g. cats, dogs) and captive wild animals (e.g. foxes, deer, dingoes).

The term "respiratory tract" includes the nose, sinuses, throat, pharynx and larynx (upper respiratory tract); and trachea, bronchial tubes, bronchioles and the lungs (lower respiratory tract).

The terms "wild-type" and "naturally occurring" are used interchangeably to refer to a protein that has the characteristics of that protein when isolated from a naturally occurring source. A wild type protein (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the protein.

The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that prior art forms part of the common general knowledge of the person skilled in the art.

The entire content of all publications, patents, patent applications and other material recited in this specification is incorporated herein by reference.

5. Detailed description

The present invention demonstrates that proteases present in the ripe flesh of Carica papaya are effective in inactivating the SARS-CoV-2 virus, such that it is no longer capable of infecting host cells.

The SARS-CoV-2 virus comprises a single-stranded RNA sense sequence approximately 30Kb in length with interspersed nucleocapsids encased in a spherical viral membrane. The membrane features envelope proteins and spike proteins. The spike proteins are glycoproteins embedded in the spherical membrane as trimers and protrude outwards. These spike proteins include an angiotensin converting enzyme-2 (ACE-2) receptor and a receptor binding domain. Infection of the virus into host cells is facilitated by binding of the viral spike protein to ACE-2 proteins located on the surface of host cells. The spike protein is also crucial to viral assembly, structural stability and immune evasion. It is therefore an ideal target for inactivating the SARS-CoV-2 virus.

The amino acid sequence of the SARS-CoV-2 surface glycoprotein is shown in GenBank Accession No. QJX59884.1 (SEQ ID NO: 6) as follows:

SEQ ID NO: 6

1 mfvflvllpl vssqcvnltt rtqlppaytn sftrgvyypd kvfrssvlhs tqdlflpffs 61 nvtwfhaihv sgtngtkrfd npvlpfndgv yfasteksni irgwifgttl dsktqslliv 121 nnatnvvikv cefqfcndpf lgvyyhknnk swmesefrvy ssannctfey vsqpflmdle 181 gkqgnfknlr efvfknidgy fkiyskhtpi nlvrdlpqgf saleplvdlp iginitrfqt 241 llalhrsylt pgdsssgwta gaaayyvgyl qprtfllkyn engtitdavd caldplsetk

301 ctlksftvek giyqtsnfrv qptesivrfp nitnlcpfge vfnatrfasv yawnrkrisn 361 cvadysvlyn sasfstfkcy gvsptklndl cftnvyadsf virgdevrqi apgqtgkiad 421 ynyklpddft gcviawnsnn ldskvggnyn ylyrlfrksn lkpferdist eiyqagstpc 481 ngvegfncyf plqsygfqpt ngvgyqpyrv vvlsfellha patvcgpkks tnlvknkcvn 541 fnfngltgtg vltesnkkfl pfqqfgrdia dttdavrdpq tleilditpc sfggvsvitp 601 gtntsnqvav lyqdvnctev pvaihadqlt ptwrvystgs nvfqtragcl igaehvnnsy 661 ecdipigagi casyqtqtns prrarsvasq siiaytmslg aensvaysnn siaiptnfti 721 svtteilpvs mtktsvdctm yicgdstecs nlllqygsfc tqlnraltgi aveqdkntqe 781 vfaqvkqiyk tppikdfggf nfsqilpdps kpskrsfied llfnkvtltd agfikqygdc 841 lgdiaardli caqkfngltv lpplltdemi aqytsallag titsgwtfga gaalqipfam 901 qmayrfngig vtqnvlyenq klianqfnsa igkiqdslss tasalgklqd vvnqnaqaln 961 tlvkqlssnf gaissvlndi lsrldkveae vqidrlitgr lqslqtyvtq qliraaeira 1021 sanlaatkms ecvlgqskrv dfcgkgyhlm sfpqsaphgv vflhvtyvpa qeknfttapa 1081 ichdgkahfp regvfvsngt hwfvtqrnfy epqiittdnt fvsgncdvvi givnntvydp 1141 lqpeldsfke eldkyfknht spdvdlgdis ginasvvniq keidrlneva knlneslidl 1201 qelgkyeqyi kwpwyiwlgf iagliaivmv timlccmtsc csclkgccsc gscckfdedd 1261 sepvlkgvkl hyt

The virus targeted by the proteases and compositions of the present invention may therefore be a coronavirus, such as a SARS-CoV-2 virus. The virus may comprise a surface glycoprotein such as a spike protein, wherein the spike protein comprises an amino acid sequence having at least 40%,41%.42%,43%,44%,45%,46%,47%,48%,49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%. 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology (preferably sequence identity) to the SARS-CoV-2 surface glycoprotein amino acid sequence shown in GenBank Accession No. QJX59884.1 (SEQ ID NO: 6).

5.1 Proteases derived from Carica papaya

Proteases derived from Carica papaya can include cysteine proteases and/or serine proteases.

The at least one active serine protease typically comprises a contiguous sequence having at least 95% sequence homology (preferably sequence identity), such as 96, 97, 98 or 99% sequence homology (preferably sequence identity) with amino acids 142 to 618 of SEQ ID NO:1, which is the catalytic domain of the protein containing the key catalytic triad residues Asp, His and Ser (shown in SEQ ID NO: 1 at residues 151, 221 and 558 respectively) and the conserved substrate-binding site Asn at residue 326.

SEQ ID NO: 1

MAVSNPTLYL LSFLLFSISL TPVIASKSSY VVYLGAHSHG LELSSADLDR 50

VKESHYDFLG SFLGSPEEAQ ESIFYSYTKH INGFAAELND EVAAKLAKHP 100

KVVSVFLNKG RKLHTTRSWD FLGLEQNGVV PSSSIWKKAR FGEDTIIGNL 150

DTGVWPESKS FSDEGLGPIP SKWRGICDHG KDSSFHCNRK LIGARFFNRG 200

YASAVGSLNS SFESPRDNEG HGTHTLSTAG GNMVANASVF GLGKGTAKGG 250

SPRARVAAYK VCWPPVLGNE CFDADILAAF DAAIHDRVDV LSVSLGGTAG 300

GFFNDSVAIG SFHAVKHGIV VVCSAGNSGP DDGSVSNVAP WQITVGASTM 350

DREFPSYVLL GNNMSFKGES LSDAVLPGTN FFPLISALNA KATNASNEEA 400

ILCEAGALDP KKVKGKILVC LRGLNARVDK GQQAALAGAV GMILANSELN 450

GNEIIADAHV LPASHISFTD GLSVFEYINL TNSPVAYMTR PKTKLPTKPA 500

PVMAAFSSKG PNIVTPEILK PDITAPGVNV IAAYTRAQGP TNQNFDRRRV 550

QFNSVSGTSM SCPHVSGIVG LLKTLYPSWS PAAIRSAIMT SATTMDNINE 600

SILNASNVKA TPFSYGAGHV QPNQAMNPGL VYDLNTKDYL KFLCALGYSK 650

TLISIFSNDK FNCPRTNISL ADFNYPSITV PELKGLITLS RKVKNVGSPT 700

TYRVTVQKPK GISVTVKPKI LKFKKAGEEK SFTVTLKMKA KNPTKEYVFG 750

ELVWSDEDEH YVRSPIVVKA A 771

Amino acids 1-25 are a putative signal sequence and amino acids 26 to 112 are a putative pro region that is cleaved to form the mature protein.

In one embodiment, the active serine protease is a fragment of SEQ ID NO: 1. The fragment may have an amino acid sequence comprising one or more of the sequences shown in SEQ ID NOs: 2 to 5, such as SEQ ID NOs: 2, 3 and 4, or all four sequences shown in SEQ ID NOs: 2, 3, 4 and 5, respectively.

SFSDEGLGPI PSK SEQ ID NO: 2

GESLSDAVLP GTNFFPLISA LNAK SEQ ID NO: 3

GPNIVTPEIL KPDITAPGVN VIAAYTR SEQ ID NO: 4

TLYPSWSPAA IR SEQ ID NO: 5

Cysteine proteases, also known as thiol proteases and cysteine endopeptidases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad. Cysteine proteases include papain (EC 3.4.22.2).

The proteases derived from Carica papaya may be recombinantly produced or extracted from the fruit. The proteases may be mixtures of one or more serine proteases derived from Carica papaya and one or more cysteine proteases derived from Carica papaya.

One source of the protease is the pulp/flesh from a papaya. Preferably the papaya is ripe. One advantage of ripe papaya as the source material instead of unripe papaya (which is the current commercial source of papain) is that the resulting composition comprising the cysteine protease has much lower levels of latex than typical commercial sources of papain. Latex can cause allergic reactions.

The protease is typically in substantially isolated form. This means that it has been at least partially purified from its biological source material such as Carica papaya or recombinantly produced. In one embodiment the isolated protease is substantially free of insoluble plant materials such as cellulose, lignin and the like. This will typically be as a result of one or more purification steps to remove insoluble materials to leave the soluble active protease in solution.

5.2 Processes for obtaining proteases from Carica papaya

A process can be used to isolate at least one serine protease of the invention from ripe papaya fruit. Ripe papaya fruit is typically yellow over most of the exterior of the fruit. Unripe papaya (from the skin of which latex is harvested to produce papain) is green. Papaya should not be hard when pressed but should give slightly and retain slight indentations. If it is very soft when pressed then it is overripe. In one embodiment papaya flesh is separated from the skin and seeds. In another embodiment the entire fruit is used including the skin and seeds. The fruit, such as the flesh, is then mechanically disrupted to form a pulp (for example in a blender until a smooth pulp is obtained).

The resulting pulp is then treated with an alkali. Suitable alkalis include strong bases such as NaOH and weak bases such as sodium bicarbonate. The alkali can be added as dry powder or in aqueous solution. In one embodiment the alkali is sodium bicarbonate and is typically added as a powder in an amount of at least 3% w/w such as at least 5% w/w. The amount is typically less than 15% w/w, such as less than 12% w/w. In a particular embodiment the amount added is from 5 to 12% w/w.

In one embodiment, the alkali may have a pKa of less than 11. In some embodiments, the alkali may be a bicarbonate or a carbonate or a combination thereof. In some embodiments, the alkali may be a water-soluble alkali metal bicarbonate salt or a water-soluble alkali metal carbonate salt or a combination thereof. In some embodiments, the amount of alkali added may be from about 1% w/w to about 40% w/w. In one embodiment, the amount of alkali added may be from about 1% w/w to about 20% w/w. In a preferred embodiment, the amount of alkali added may be from about 1% w/w to about 15% w/w, such as from about 4% w/w to about 15% w/w.

In one embodiment the alkali is added in an amount such that the final pH of the composition after the alkali has reacted with the pulp is at least 7.9, such as from pH 7.9 to pH 11.5.

Once the alkali has had sufficient time to react with the pulp, the treated pulp is then subjected to a separation step to remove insoluble plant materials (in the case of sodium bicarbonate, the separation step can be performed once the treated pulp is no longer effervescing). For example, the treated pulp can be centrifuged and/or filtered. Centrifugation can be performed at about 5000 to 12000 rpm. In one embodiment the treated pulp is centrifuged and the resulting supernatant is then filtered through a 0.22 pm filter.

The resulting composition can be subject to one or more purification steps to concentrate and/or further separate the proteases of the invention from other cellular components. Suitable techniques include (i) gel filtration (size exclusion) chromatography to separate the proteins specificallyon thebasis of molecular weight e.g. using SephadexG-100 asdescribed in the examples; (ii) ionic exchange chromatography; (iii) dialysis; (iv) ammonium acetate precipitation and/or (v) freeze-drying. Resulting compositions/fractions from chromatography can be tested to confirm the continued presence of protease activity using know assays.

Purification processes may also conveniently be used to adjust the pH and/or salt concentration to levels suitable for the formulation of the composition for use in the methods of the invention.

Methods for purifying proteases used in the present invention are disclosed in international PCT patent application no. PCT/IB2020/062349, published as WO 2021/130675, the entire contents of which is incorporated herein by reference.

5.3 Compositions

The compositions of the present invention are formulated to be suitable to be administered (1) in the atmosphere, including, for example, to viruses suspended in airborne droplets such as aerosols, (2) on surfaces, including built environment surfaces and the skin of humans and other animals, and (3) in the mouth, eyes, and respiratory tract (including the nose, throat, larynx, trachea, bronchi and lungs) of humans and other animals, by combining the proteases with one or more pharmaceutically acceptable carriers, excipients or diluents. Typically, this will be in the form of a nebuliser formulation for administration using a nebulizer (e.g. a jet nebulizer or piezoelectric nebulizer) or as a dry powder for administration using a dry powder inhaler. Compositions may also be administered using a metered dose inhaler (MDI), a spray pump (such as a metered dose spray pump), spray tip (e.g. attached to a syringe), squeeze bottle or dropper.

Accordingly the present invention provides a composition comprising one or more proteases derived from Carica papaya together with one or more pharmaceutically acceptable carriers, excipients or diluents, said composition being suitable for administration to (1) the atmosphere, including, for example, to viruses suspended in airborne droplets such as aerosols, (2) on surfaces, including built environment surfaces and the skin of humans and other animals, and (3) in the mouth, eyes, and respiratory tract (including the nose, throat, larynx, trachea, bronchi and lungs) of humans and other animals.

Liquid compositions of the invention may comprise at least about 1mlU, such as at least about 10mlU or at least about 0.1|U of total protease activity per ml, such as total serine protease activity, total cysteine protease activity or both combined. In one embodiment the compositions comprises from about 1mlU to about 1OIU per ml, such as from about 1mlU to about 1IU or from about 1OmlU to about 1OIU per ml total protease activity per ml, such as total serine protease activity, total cysteine protease activity or both combined. In another embodiment the compositions comprise from about 1 to about 10 IU, about 1 to about 20 IU, about 1 to about 30 IU, about 1 to about 40 IU, about 1 to about 50 IU, about 1 to about 60 IU, about 1 to about 70 IU, about 1 to about 80 IU, about 1 to about 90 IU, about 10 to about 100 IU, such as about 5, 10 or 20 to about 100 IU per ml of total protease activity, such as total serine protease activity, total cysteine protease activity or both combined.

In the case of dry powders, the compositions of the invention may comprise at least 1mlU, such as at least about 1OmlU or at least about 0.1|U of total protease activity per mg of powder, such as total serine protease activity, total cysteine protease activity or both combined. In one embodiment the compositions comprise from about 1mlU to about 101U per mg of powder, such as from about 1mlU to about 1|U or from about 1OmlU to about 1OIU per ml total protease activity per mg of powder, such as total serine protease activity, total cysteine protease activity or both combined. In another embodiment the compositions comprise from about 1 to about 10 IU, about 1 to about 20 IU, about 1 to about 30 IU, about 1 to about 40 IU, about 1 to about 50 IU, about 1 to about 60 IU, about 1 to about 70 IU, about 1 to about 80 IU, about 1 to about 90 IU, about 10 to about 100 IU, such as about 5, 10 or 20 to about 100 IU per mg of powder of total protease activity, such as total serine protease activity, total cysteine protease activity or both combined.

5.4 Liquid Formulations

Liquid nebulizer formulations generally include one or more pharmaceutically acceptable ingredients to regulate pH, ionic strength and drug stability. Hyper- or hypo-osmolar solutions can cause bronchoconstriction, coughing and irritation of the lung mucosa. Thus, to be well tolerated, an aerosol produced from the formulation by the nebulizer should, as far as possible, have a physiologic tonicity or osmolality. Thus, it may be desirable to incorporate into the composition of the invention, an osmotically active excipient to control the osmolality of the aerosol. The content of this excipient (or excipients, if a combination of substances is used) should be selected to yield an osmolality of the aerosol which does not deviate too much from that of physiological fluids, i.e., from about 290 mOsmol/kg. However, in individual cases, a compromise has again to be found between the physical-chemical or pharmaceutical needs on one hand and the physiological requirements on the other hand. In general, an osmolality in the range of about 200 to 550 mOsmol/kg and, in particular, in the range of about 220 or 230 up to about 350 mOsmol/kg may be considered acceptable.

The pH of the formulation also needs to be physiologically tolerable. Preferably, the pH value lies in the slightly acidic to neutral region, i.e., between pH values of about 4 to 8. It is to be noted that deviations towards a weakly acidic environment can be tolerated better than shifts of the pH value into the alkaline region. A pH value in the range of about 4.5 to about 7.5 is particularly preferred. For adjusting and, optionally, buffering pH value, physiologically acceptable acids, bases, salts, and combinations of these may be used. Suitable excipients for lowering the pH value or as acidic components of a buffer system are strong mineral acids, in particular, sulfuric acid and hydrochloric acid. Moreover, inorganic and organic acids of medium strength as well as acidic salts may be used, for example, phosphoric acid, citric acid, tartaric acid, succinic acid, fumaric acid, methionine, acidic hydrogen phosphates with sodium or potassium, lactic acid, glucuronic acid etc. However, sulfuric acid and hydrochloric acid are most preferred. Suitable for raising the pH value or as basic component for buffer system are, in particular, mineral bases such as sodium hydroxide or other alkali and alkaline earth hydroxides and oxides such as, in particular, magnesium hydroxide and calcium hydroxide, ammonium hydroxide and basic ammonium salts such as ammonium acetate, as well as basic amino acids such as lysine, carbonates such as sodium or magnesium carbonate, sodium hydrogen carbonate, citrates such as sodium citrate etc.

In one of the preferred embodiments, the composition of the invention contains a buffer system consisting of two components, and one of the particularly preferred buffer systems contains citric acid and sodium citrate. Nevertheless, other buffering systems may also be suitable.

Not primarily for physiological, but for pharmaceutical reasons the chemical stabilization of the composition by further additives may be indicated. This depends mainly on the kind of the active agent contained therein. The most common degradation reactions of chemically defined active agents in aqueous preparations comprise, in particular, hydrolysis reactions, which may be limited, primarily, by optimal pH adjustment, as well as oxidation reactions. Examples for active agents which may be subject to oxidative attack are those agents that have olefinic, aldehyde, primary or secondary hydroxyl, ether, thioether, endiol, keto or amino groups. Therefore, in the case of such oxidation-sensitive active agents, the addition of an antioxidant, optionally in combination with a synergist, may be advisable or necessary.

Antioxidants are natural or synthetic substances which prevent or interrupt the oxidation of the active agents. These are primarily adjuvants which are oxidizable themselves or act as reducing agents, such as, for example, tocopherol acetate, reduced glutathione, catalase, peroxide dismutase and L-cysteine. Synergistic substances are, for example, those which do not directly act as reactance in oxidation processes, but which counteract in oxidation by an indirect mechanism such as the complexation of metal ions which act catalytically in the oxidation, which is the case, for example, for EDTA derivatives (EDTA: ethylenediamine tetraacetic acid). Further suitable antioxidants are ascorbinic acid, sodium ascorbate and other salts and esters of ascorbinic acid (for example, ascorbylpalmitate), fumaric acid and its salts, malic acid and its salts, butyl hydroxy anisole, propyl gallate, as well as sulfites such as sodium metabisulfite. Apart from EDTA and its salts, citric acid and citrates, malic acid and its salts and maltol (3-hydroxy-2-methyl-4H-pyran-4-one) may also act as chelating agents.

If the active agent and other ingredients contained in the composition give an osmolality below the required or desired value it can be adjusted to the desired value by the addition of one or more suitable osmotically active excipients. Such compounds are, in particular, innocuous mineral salts which react largely neutrally (unless such adjuvants are, at the same time to adjust or buffer the pH value), such as sodium, calcium or magnesium chloride, sulfate or phosphate. One of the particularly preferred members of these is sodium chloride.

Further preferred excipients for this purpose are magnesium and calcium sulfate and chloride. It is known that these calcium and magnesium salts can have a positive or auxiliary influence in the inhalation of active agent solutions, possibly because they themselves counteract the local irritations caused by the administration and exert a bronchodilatory effect which is currently postulated in the clinical literature. Especially magnesium sulfate shows excellent pulmonary tolerance.

The liquid composition may be supplied as a sterile liquid or reconstituted from a dry powder or lyophilisate e.g. by combining with a suitable diluent.

5.5 Dry Powder Formulations

Dry powder forms are often prepared by mixing the active substance in micronized form with the coarse particles of the carrier, obtaining an ordered mixture in which micronized active particles adhere to the surface of the particles of the carrier while inside the inhaler.

The carrier makes the micronized powder less cohesive and improves its fluidity, facilitating the processing of the powder during the manufacturing process (pouring, filling, etc.).

During inhalation, the drug particles separate from the surface of the carrier particles and penetrate into the lower parts of the lungs, while the larger carrier particles are mainly deposited in the oropharynx.

Carriers well-known in the art include lactose and cellobiose.

An alternative approach is to spray dry a liquid formulation comprising the active substance (see for example US20030035778). Such spray-dried powders typically include pharmaceutical excipients that act as carriers. These inventions include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two. Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-p-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

In a particular embodiment, the spray-dried particles are engineered particles, such as perforated particle, e.g. based on the use lipids, such as phospholids (see US 8,877,162 and US2004/0105820). See also US 6,565,885 which is directed to methods of forming dry powders with perforated microstructures. Such methods have the advantage of allowing for higher drug loads than traditional lactose-blend powders.

Where dry powder formulations comprise fine micronized drug particles (e.g. < 5 pm) that are deposited on large carrier particles such as lactose, the carrier particles may for example have a mean geometric particle size of 30 to 90 pm.

For engineered / perforated particles, to maximize dispersibility, dispersion stability and optimize distribution upon administration, the mean geometric particle size is preferably about 0.5-50 pm, more preferably 1-30 pm. It will be appreciated that large particles (i.e. greater than 50 pm) may not be preferred in applications where a valve or small orifice is employed, since large particles tend to aggregate or separate from a suspension which could potentially clog the device. In especially preferred embodiments the mean geometric particle size (or diameter) of the perforated microstructures is less than 20 pm or less than 10 pm, such as less than about 7 pm or 5 pm.

5.6 Metered Dose Inhaler Formulations

Such formulations typically include a propellant such as a hydrofluoroalkane (HFA) e.g. 1,1,1,2-tetrafluoroethane (HFA 134a); 1,1,1,2,3,3,3-heptafluoropropane (HFA 227); and/or 1,1-difluoroethane (HFA 152a). See Myrdal et al., 2014, AAPS PharmSciTech 15(2): 434 455. Proteins and peptides are generally hydrophilic and therefore are poorly soluble in non polar HFA) propellants. Biologics are typically formulated as suspension rather than solution in an MDI formulation. To enhance the stability, the proteases can be incorporated in a particulate carrier to suspend in the propellant. Co-spray drying proteins (e.g., lysozyme, catalase and bovine serum albumin) with polyvinyl alcohol (PVA) and sodium carboxymethylcellulose (NaCMC) can improve the physical stability of protein particles in surfactant-free HFA propellent. Nanoparticles (NPs) have also been reported to improve the stability of proteins such as insulin in pMDI formulation. NPs (fabricated by a bottom-up process) containing lysozyme or thymopentin were readily dispersed in HFA 134a to form a stable suspension with good re-dispersibility and bioactivity.

The formulations of the present invention may conveniently be presented in unit dosage form.

It is preferred that the compounds according to the invention are provided in a kit. Such a kit typically contains an active compound in dosage forms for administration. A dosage form contains a sufficient amount of active compound such that a desirable effect can be obtained when administered to a subject.

Thus, it is preferred that the medical packaging comprises an amount of dosage units corresponding to the relevant dosage regimen. Accordingly, in one embodiment, the medical packaging comprises a pharmaceutical composition comprising a compound as defined above or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable carriers, vehicles and/or excipients, said packaging comprising from 1 to 7 dosage units, thereby having dosage units for one or more days, or from 7 to 21 dosage units, or multiples thereof, thereby having dosage units for one week of administration or several weeks of administration.

The dosage units can be as defined above. The medical packaging may be in any suitable form for respiratory administration, such as intratracheal, intrabronchial, bronchio-alveolar or intraalveolar administration. In a preferred embodiment the packaging is in the form of a vial, ampule, tube, blister pack, cartridge or capsule.

When the medical packaging comprises more than one dosage unit, it is preferred that the medical packaging is provided with a mechanism to adjust each administration to one dosage unit only.

Preferably, a kit contains instructions indicating the use of the dosage form to achieve a desirable affect and the amount of dosage form to be taken over a specified time period.

5.7 Uses

The proteases and compositions comprising proteases from Carica papaya can be used to inactivate/disable viruses such as coronaviruses, for example to both prevent or treat (e.g.

reduce the severity of) viral respiratory infections such as Severe Acute Respiratory Syndrome (SARS). Prevention can be achieved, for example, by administering the proteases and compositions thereof to (1) the atmosphere, including, for example, to viruses suspended in airborne droplets such as aerosols, (2) on surfaces, including built environment surfaces and the skin of humans and other animals, and (3) in the mouth, eyes, and respiratory tract (including the nose, throat, larynx, trachea, bronchi and lungs) of humans and other animals. Treatment can be achieved, for example, by administering the proteases and compositions thereof to the mouth, eyes, and respiratory tract (including the nose, throat, larynx, trachea, bronchi and lungs) of humans and other animals.

The proteases act on the accessible coat proteins of the enveloped viruses e.g. the spike proteins. The spike protein mediates attachment of the virus to the host cell surface receptors and subsequent fusion between the viral and host cell membranes to facilitate viral entry into the host cell. Thus, proteolytic degradation of these coat proteins acts to reduce or prevent the ability of the viruses to engage with their target host cells. Administration of the composition to an infected patient enables the proteases to target viral particles secreted by infected host cells and reduce or prevent the spread of the virus in the respiratory tract.

The compositions can be administered to the respiratory tract of an individual, such as a human patient, using a variety of delivery methods known in the art. Some viruses primarily target the upper respiratory tract; others primarily target the lower respiratory tract; whilst others can target both (e.g. coronaviruses).

The mode of delivery and formulation are selected to allow for particles containing the active substance to be deposited in an effective location within the patient's respiratory tract. Administration can be intranasal or by inhalation.

Examples of modes of delivery include (i) nebulizers, such as ultrasonic or mesh nebulizers (e.g. the Fox@ vibrating mesh, Pari LC Plus@, Pari eRapid @; (ii) jet nebulizers such as the Akita@ JET; (ii) dry powder inhalers, which can be in single or multidose formats, such as the Gyrohaler ; (iv) metered dose inhalers; and (v) a spray pump (such as a metered dose spray pump). Other examples are a spray tip (e.g. attached to a syringe), a squeeze bottle or dropper.

5.8 Dosages

The dose and frequency of dosing can be determined by a physician based, for example, on the stage and severity of the infection, and the age and weight of the patient. The composition may for example be administered daily or twice daily for a period of 2 to 7 days. Administration may be extended depending on the progress of the treatment.

In some embodiments, the compositions of the invention should contain sufficient proteolytic activity to enable at least about 1mlU, such as at least about 1OmlU or at least about 0.1|U of total protease activity to be administered per dose, such as total serine protease activity, total cysteine protease activity or both combined. In one embodiment from about 1mlU to about 101IU is administered per dose, such as from about 1mlU to about 1|U or from about 1OmlU to about 101U. In another embodiment from about 1 to about 10 IU, about 1 to about 20 IU, about 1 to about 30 IU, about 1 to about 40 IU, about 1 to about 50 IU, about 1 to about 60 IU, about 1 to about 70 IU, about 1 to about 80 IU, about 1 to about 90 IU, about 10 to about 100 IU, such as about5,10or20toabout 100 IU of total protease activity to be administered per dose, such as total serine protease activity, total cysteine protease activity or both combined.

Serine protease activity can be measured by the L-BApNA assay described in the examples with the use of a serine protease inhibitor during the assay to determine the serine protease component. Alternatively, the benzoyl-L-arginine ethyl ester (BAEE) assay described in GB 2,440,117 may be used.

The compositions of the invention may also contain a similar level of cysteine protease activity. Cysteine protease activity can be measured by the L-BApNA assay described in the examples with the use of a cysteine protease inhibitor during the assay to determine the cysteine protease component.

5.9 Timing of Therapies

Typically, in therapeutic applications, treatment would be for the duration of the disease state.

Those skilled in the art will appreciate that the proteases and compositions thereof disclosed herein may be administered as a single agent or as part of a combination therapy approach to the methods disclosed herein, at diagnosis or subsequently thereafter, for example, as follow-up treatment or consolidation therapy as a compliment to currently available therapies for such treatments. The proteases and compositions thereof disclosed herein may also be used as preventative therapies for subjects who are genetically or environmentally predisposed to developing such diseases.

The proteases and compositions thereof may be administered regularly for as long as is needed, such as until an improvement in the condition is seen. Therefore, they may be administered hourly, multiple times a day, daily, multiple times a week, weekly, monthly or at whatever frequency is deemed appropriate.

5.10 Kits

Kits of the present invention facilitate the employment of the methods and uses of the present invention. Typically, kits for carrying out a method or use of the invention contain all the necessary reagents and means to carry out the method. For example, in one embodiment, the kit may comprise a protease or composition thereof of the present invention and, optionally, means to administer the protease or composition such as devices for point of care methods.

Typically, the kits described herein will also comprise one or more containers. In the context of the present invention, a compartmentalised kit includes any kit in which proteases or compositions are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of proteases or compositions from one compartment to another compartment whilst avoiding cross-contamination of proteases or compositions, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion.

Typically, a kit of the present invention will also include instructions for using the kit to perform the appropriate methods and uses.

Methods, uses, compositions and kits of the present invention are equally applicable to any animal, including humans, for example including non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline and canine species. Accordingly, for application to different species, a single kit of the invention may be applicable, or alternatively different kits, for example containing proteases or compositions specific for each individual species, may be required.

The person skilled in the artwill understand and appreciate that different features disclosed herein may be combined to form combinations of features that are within the scope of the present invention.

The present invention will now be illustrated with reference to the following examples, which are illustrative only and non-limiting.

6. Examples

Example 1 - Evaluation of proteases derived from Carica papaya on SARS-CoV2

This example demonstrates the efficacy of proteases derived from Caricapapaya in targeting and inactivating the SARS-CoV-2 Spike S1 protein. The experimental strategy, discussed in detail below, employed a time course assay assessing exposure of the spike protein to proteases derived from C. papaya, followed by a Western blot analysis of spike protein.

Materials and Methods

Recombinant SARS-CoV-2 Spike S1 -100pg (#bs-46004P-BSS-100pg Stratech) protein was resuspended in 100pl sterile water and aliquoted in 10pl aliquots. Two aliquots were used for the experiment while the rest stored at -20°C.

A 1OpI solution of proteases derived from Carica papaya (produced according to the methods described in PCT patent application no. PCT/IB2020/062349, published as WO 2021/130675), or 10pl PBS were mixed with 10pg (10pL) Spike S1 protein for the test and control reactions, respectively. At time point zero, 5pl of mix were taken out of both reactions and stored at 20°C. The remaining 15pl of the test and control reactions were incubated at 37°C for 24 hours. At time point 8, 16 and 24-hours, 5pl reaction aliquots were collected from both reactions and stored at -20°C.

Western Blot

Immediately after the final 24-hour time point collection, all eight reaction tubes were diluted 5 times with sterile water and mixed with 4x LDS sample buffer (#B0007, ThermoFisher Scientific) and 50mM DTT (#R0861, ThermoFisher Scientific). All tubes were incubated at 70°C for 10 minutes. A total 0.75 pg protein (10pl) from each reaction tube and precision plus protein marker (#1610375, Biorad) were loaded onto a Bolt 4-12% Bis-Tris 12 well protein gel (#NW04122BOX, ThermoFisher Scientific) as shown below.

Lane 1: 2pl protein marker

Lane 2: Empty

Lane3: Control Timepoint 0

Lane4: Control Timepoint 8

Lane5: Control Timepoint 16

Lane6: Control Timepoint 24

Lane7: Test Sample Timepoint 0

Lane8: Test SampleTimepoint 8

Lane9: Test Sample Timepoint 16

Lane10: Test Sample Timepoint 24

Lanel1: Empty

Lane12: 4pl protein marker

After running the protein samples for 35 minutes at 180V, the proteins were transferred onto a nitrocellulose membrane using a Trans Blot Turbo transfer system (#1704150, Biorad) and a preassembled transfer pack (#1704158, Biorad). The membrane was washed with sterile water and subjected to red ponceau (#P7170, Sigma-Aldrich) staining for 5 minutes (Figure 1, showing lanes 1-12 from left to right).

After washing the red ponceau staining with sterile water, the membrane was blocked with 5% BSA for 1 hour at room temperature. The membrane was then incubated with 5pg Anti-6X-His tag monoclonal antibody (#MA1-135, ThermoFisher Scientific) diluted in 5ml 5% BSA at +4 on a rocking platform. The next day, after washing the membrane three times for 10 minutes each with 1X TBS-Tween 0.1% (TBST), the membrane was incubated with donkey anti-mouse IgG HRP (#Ab6820, Abcam) antibody diluted 1:10000 in 5% BSA at room temperature for 1 hour.

The membrane was then washed three times with 1X TBST and bound secondary antibody was detected by enhanced chemiluminescence with ECL substrate (#RPN2232, GE Healthcare) and analysed with a Chemidoc system.

Results

In the control reactions, Western blot analysis showed a strong band of the SARS-CoV-2 Spike S1 protein at 120KDa in the 0, 8, 16 and 24 hour time points with a minor decrease in intensity. However, the Spike S1 protein band was totally undetectable in the test sample reactions incubated with the proteases at all time points (Figure 2). The protease solution is highly effective at degrading the SARS-CoV-2 Spike S1 protein. The absence of any detectable Spike S1 protein even at time point 0 in the test sample reaction suggests that the protease solution degrades the SARS-CoV-2 Spike S1 protein within minutes.

Example 2 - Evaluation of proteases derived from C. papaya on SARS-CoV2

This example employs a suspension test of virucidal activity to test whether a composition containing proteases derived from C. papaya had virucidal activity against SARS-CoV2 by incubating 2x10 6 infectious units of SARS-CoV2 with 4 volumes of undiluted protease formulations.

Materials and Methods

2x10 6 infectious units of SARS-CoV2 were incubated with 4 volumes of undiluted protease formulations. After different contact times (1 minute, 5 minutes, or 1 hour), an excess of cold media was added, and the infectious virus present in the mixture was quantified through a serial dilution on a monolayer of Vero cells. Five days after infection, virus titre was quantified by determining the dilution at which half of the cells displayed virus-induced cytopathic effect (TCID50).

A parallel test where the same dilutions of formulation were added to the cells in the absence of virus was included to determine any cytotoxic effect of the formulation on the assay cells.

Results and Conclusions

Exposure of SARS-CoV2 to a composition of the invention comprising proteases extracted from C. papaya led to a substantial reduction in the level of active virus. A negative control (papaya juice) showed no activity.

Example 3 - Methods for Measuring Protease Activity

L-BApNA (N-benzoyl-L-arqinine 4-nitroanilide hydrochloride) Assays

A 1.5 mM stock solution of L-BApNA (B-3133 sigma) was prepared by dissolving 63 mg of L-BApNA in 1.5 ml dimethyl sulfoxide (DMSO), and then made up to 100 ml with water. Hydrolysis of the L-BApNA at the bond between the arginine and the p-nitroaniline moieties releases the chromophore p-nitroaniline, which can be detected by spectroscopy at an absorbance of 410 nm in International Units (IU). IU is defined as the amount of enzyme that causes an increase in absorbance of 0.01 units/min under excess substrate conditions (zero order kinetics). Solids specific activity is expressed in IU/mg, while in the case of liquid formulations, the activity is expressed in IU/mL.

To determine the optimal substrate concentration to maintain zero order kinetics throughout experiments, a series of titration runs were carried out with a range of substrate concentrations. The minimum concentration thus found is 0.2 mM or 1:6 dilution of the stock 1.5 mM solution. Table 1 shows reagent doses used for all kinetic experiments. Phosphate buffer at pH 6 was used in the L-BApNA assays because it gave the highest enzymatic activity reading compared to other buffers.

Table 1: L-BApNA assay final reagent concentrations

Content Reference Sample

pH 6.0 Buffer 333 333 Water 501 333 Substrate (L-BApNA) 166 166 Zero at 410 nm Enzyme Preparation 0 166 Record A41 0

To verify substrate purity, a controlled total hydrolysis catalyzed by alkali was carried out under the following conditions.

Table 2: L-BApNA substrate purity assay compositions

Reference cell (pl) Sample Cell (pl) 5M NaOH 0 966 Water 966 0 Zero at 41Onm Substrate 33 33 Record A410

Thus [substrate] = A41o X 30 (dilution factor)

8800 (molar coefficient of extinction of p-nitroanaline at 410 nm)

Results confirmed 99% purity of the substrate as stated by manufacturer.

Inhibitors

E-64(IUPAC name: (1S,2S)-2-(((S)-1-((4-Guanidinobutyl)amino)-4-methyl-1-oxopentan 2-yl) carbamoyl) cyclopropane carboxylic acid) is a strong and irreversible specific inhibitor of cysteine proteases. The trans-epoxysuccinyl group (active moiety) of E-64 irreversibly binds to the active thiol group of cysteine proteases, thereby inhibiting them. E-64 does not react with non-protease enzymes and does not inhibit serine proteases.

2,2'-dipyridyldisulfide (2DPS) is used as a thiol-specific reversible inhibitor for cysteine proteases, including papain, bromelain, and ficin.

All references mentioned herein are incorporated by reference. While this invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. Features and embodiments in different sections can be combined mutatis mutandis.

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<400> 3 <400> 3

Gly Glu Ser Leu Ser Asp Ala Val Leu Pro Gly Thr Asn Phe Phe Pro Gly Glu Ser Leu Ser Asp Ala Val Leu Pro Gly Thr Asn Phe Phe Pro 1 5 10 15 1 5 10 15

Leu Ile Ser Ala Leu Asn Ala Lys Leu Ile Ser Ala Leu Asn Ala Lys 20 20

<210> 4 <210> 4 <211> 27 <211> 27 <212> PRT <212> PRT <213> Carica papaya <213> Carica papaya

<400> 4 <400> 4

Gly Pro Asn Ile Val Thr Pro Glu Ile Leu Lys Pro Asp Ile Thr Ala Gly Pro Asn Ile Val Thr Pro Glu Ile Leu Lys Pro Asp Ile Thr Ala 1 5 10 15 1 5 10 15

Pro Gly Val Asn Val Ile Ala Ala Tyr Thr Arg Pro Gly Val Asn Val Ile Ala Ala Tyr Thr Arg 20 25 20 25

<210> 5 <210> 5 <211> 12 <211> 12 <212> PRT <212> PRT <213> Carica papaya <213> Carica papaya

<400> 5 <400> 5

Thr Leu Tyr Pro Ser Trp Ser Pro Ala Ala Ile Arg Thr Leu Tyr Pro Ser Trp Ser Pro Ala Ala Ile Arg 1 5 10 1 5 10

<210> 6 <210> 6 <211> 1273 <211> 1273 <212> PRT <212> PRT <213> SARS‐CoV‐2 virus <213> SARS-CoV-2 virus

<400> 6 <400> 6

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Thr Asp Ala Gly Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Thr Asp Ala Gly 820 825 830 820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 1265 1270

Claims (25)

1. A method of inactivating a virus, wherein the method comprises administering one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, to the virus.

2. A method of preventing an infection in a subject caused by a virus, wherein the method comprises administering one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, to the virus.

3. A method of treating an infection in a subject caused by a virus, wherein the method comprises administering one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, to the virus.

4. Use of one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, in the manufacture of a medicament for inactivating a virus.

5. Use of one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, in the manufacture of a medicament for preventing an infection in a subject caused by a virus.

6. Use of one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, in the manufacture of a medicament for treating an infection in a subject caused by a virus.

7. One or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, for use in or when used for inactivating a virus.

8. One or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, for use in or when used for preventing an infection in a subject caused by a virus.

9. One or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, for use in or when used for treating an infection in a subject caused by a virus.

10. Use of one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, for inactivating a virus.

11. Use of one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, for preventing an infection in a subject caused by a virus.

12. Use of one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof, for treating an infection in a subject caused by a virus.

13. A kit for use in or when used for inactivating a virus, preventing an infection or treating an infection in a subject caused by a virus, wherein the kit comprises one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, or a composition thereof.

14. The method of either one of claims 1 or 2, wherein the administration is to a virus in aerosol form in the atmosphere, to a surface including a built environment surface or the skin of a human or other animal, or to the mouth, eyes, and/or respiratory tract of a subject.

15. The method of claim 3, wherein the administration is to the mouth, eyes, and/or respiratory tract of a subject.

16. The method of any one of claims 1-3, 14 or 15, the use of any one of claims 4-6 or 10-12, the one or more proteolytically active serine proteases or a composition thereof of any one of claims 7-9, or the kit of claim 13, wherein the virus is coronavirus SARS-CoV-2.

17. The method of any one of claims 1-3, wherein the administering comprises a nebuliser or a dry powder inhaler.

18. The method of any one of claims 1-3 or 14-17, the use of any one of claims 4-6, 10 12 or 16, the one or more proteolytically active serine proteases or a composition thereof of any one of claims 7-9 or 16, or the kit of claim 13 or 16, wherein the composition is a pharmaceutical composition comprising the one or more proteolytically active serine proteases extracted from ripe Carica papaya flesh, together with a pharmaceutically acceptable compatible carrier, excipient or diluent.

19. The method of any one of claims 1-3 or 14-18, the use of any one of claims 4-6, 10 12, 16 or 18, the one or more proteolytically active serine proteases or a composition thereof of any one of claims 7-9, 16 or 18, or the kit of any one of claims 13, 16 or 18, wherein the protease is extracted from ripe Carica papaya flesh using an alkali treatment.

20. The method, use, one or more proteases or a composition thereof, or kit, of claim 19, wherein the alkali is a weak alkali.

21. The method, use, one or more proteases or a composition thereof, or kit, of claim 20, wherein the weak alkali has a pKa of less than 11.

22. The method, use, one or more proteases or a composition thereof, or kit, of claim 20 or 21, wherein the weak alkali is sodium bicarbonate.

23. The method, use, one or more proteases or a composition thereof, or kit, of any one of claims 19-22, wherein soluble protease is separated from insoluble plant material after the alkali treatment.

24. The method of any one of claims 1-3 or 14-23, the use of any one of claims 4-6, 10 12, 16 or 18-23, the one or more proteolytically active serine proteases or a composition thereof of any one of claims 7-9, 16 or 18-23, or the kit of any one of claims 13, 16 or 18-23, wherein the protease is obtainable from ripe Carica papaya flesh.

25. The method of any one of claims 1-3 or 14-24, the use of any one of claims 4-6, 10 12, 16 or 18-24, the one or more proteolytically active serine proteases or a composition thereof of any one of claims 7-9, 16 or 18-24, or the kit of any one of claims 13, 16 or 18-24, wherein the composition further comprises one or more cysteine proteases extracted from ripe Carica papaya flesh.

Figure 1

150kDa

100kDa 75kDa

50kDa

37kDa

26kDa 20kDa

15kDa 10kDa

Empty Control Control Control Control Sample Sample Sample Sample Empty

24hr 16hr 8hr Ohr Ohr 8hr 16hr 24hr

Size markers shown (descending) are: 150kDa, 100kDa, 75kDa, 50kDa, 37kDa, 26kDa, 20kDa, 15kDa and 10kDa.

1/2

SUBSTITUTE SHEET (RULE 26)

Figure 2

200kDa 150kDa Spike S1

100kDa 75kDa

50kDa

37kDa

Service

26kDa 19. 20kDa Company

15kDa I I I 10kDa

Empty Control Control Control Control Sample Sample Sample Sample Empty

Ohr 8hr 16hr 24hr Ohr 8hr 16hr 24hr

Size markers shown (descending) are: 200kDa, 150kDa, 100kDa, 75kDa, 50kDa, 37kDa, 26kDa, 20kDa, 15kDa and 10kDa.

2/2

SUBSTITUTE SHEET (RULE 26)