FIELD OF THE INVENTION

The embodiments of the present invention relate to fabrics into which enzymes, including psychrophilic and/or cold active enzymes, have been incorporated such that upon contact they inactivate aerosolized pathogenic microbes including enveloped viruses such as Coronavirus and Influenza virus and Gram negative bacteria. The fabrics of the invention may be used in the production of personal protective equipment (PPE) including disposable face masks, surgical gowns and head coverings, shoe coverings, clothes and bedding.

BACKGROUND

Many human illnesses are transmitted from one individual to another by aerosols or by fomites. Viruses, bacteria and prions are the causative agents of many serious diseases which cause public health emergencies and consequent economic devastation.

The 1918 Spanish flu pandemic was caused by an H1N1 influenza virus and caused about 65,000 deaths globally. Severe acute respiratory syndrome (SARS) was caused by the SARS coronavirus (SARS-CoV), and had a 9.6% fatality rate. COVID-19 first emerged in 2019 and is caused by a novel coronavirus, a positive-sense single-stranded enveloped RNA virus, the same as the SARS and MERS virus. The person-to-person transmission of viruses by aerosols and fomites is of great concern due to widespread mortality and morbidity.

Gram-negative bacilli are also a major cause of nosocomial infection in ICUs and hospitals. In 2003, gram-negative bacilli were associated with 23.8% of bloodstream infection, 65.2% of pneumonia episodes, 33.8% of surgical site infection, and 71.1% of urinary tract infection. Hospital workers need PPE that is effective in preventing the spread of nosocomial infections from patient to patient, as well as being convenient, safe, affordable, disposable, and biodegradable. See Todorova, V. et al. Gram-negative nosocomial infections in a general ICU: emerging new clues. Crit. Care 15, P224 (2011), hereby incorporated by reference.

Typical face-masks and other PPE act as fomites. Fomites are objects that when contaminated with a pathogen, can transfer it to a new host. Fabrics used in PPE have large microscopic surface areas, and act as fomites, especially in a hospital situation. When contaminated with viruses they act as a reservoir, transferring virus to hands of the user. Masks are ideal fomites. In health-care environment, self-sterilizing materials will significantly reduce nosocomial infection—a major cause of death.

Current masks concentrate particles on their surfaces, increasing the probability of introduction of an infectious dose to the user if they touch the mask and subsequently touch their nose, mouth or eyes. Breathing through a mask can become tiresome due to inherent air resistance through the fabric and moisture buildup. Air resistance and discomfort leads to frequent desire to remove or adjust the mask; therefore the mask is frequently touched, adjusted, removed, pocketed and refitted, leading to frequent handling of the contaminated outer surface.

Reducing the pore size of disposable facemasks, so that they would remove smaller virus-containing droplets, would impact the ability of the wearer to breathe, encouraging removal or adjustment of the mask. This solution would therefore be counterproductive.

Fabrics with antimicrobial additives are known. These include additives based on silver, copper, and zinc, and so called “Organic Antimicrobial Additives” that include phenolic biocides, quaternary ammonium compounds and fungicides (thiabendazole). Copper and silver-containing anti-microbial facemasks that are currently being produced reduce growth of bacteria and fungi in the mask material, but are not efficient at killing or inactivating a significant proportion of viruses entering or passing through the mask. Any killing effect that is theoretically possible is only provided on contact with the metal, and the vast majority of the surface area of such masks does not incorporate an effective proportion of metal ions. Making these masks anti-viral using this approach would require adding a proportion of metal to the material that would make the mask both extremely uncomfortable to wear and prohibitively expensive. Consequently, this solution is impractical.

There are also masks designed recently by Dibakar Bhattacharyya at the University of Kentucky that incorporate proteolytic enzymes that specifically bind to attach to the spike proteins of the coronavirus and kill the virus. Although this design is theoretically possible, there are several potential difficulties and disadvantages in the design. Firstly, these are enzymes that bind specifically to the spike-protein. These enzymes are not commercially available easily or cheaply or in large quantities. They must be created and manufactured by complex and expensive biological processes. Certainly they are not readily available in 2021 during the present COVID-19 outbreak. This contrasts with the enzymes (proteases) of the present invention which have been developed over many years for use in washing detergents or the food industry which are cheap, well-researched and dermatologically tested. Secondly, the University of Kentucky mask, when in use, will not necessarily create an appropriate chemical and osmotic environment for the proteases to adopt the correct confirmation that will be required for enzyme activity. Thirdly the University of Kentucky (UK) mask only uses specific protease enzymes that bind only to the coronavirus spike protein. It does not employ non-specific enzymes, and it does not comprise multi-enzyme blends as does the present invention. Fourth, the enzymes used in the UK mask are not and have not been designed to be active at low temperatures, such as at room temperatures, for example 10-20 degrees centigrade. Therefore they will not function efficiently as room temperatures.

Trypsins are serine proteases that cleave the peptide bond on the C-terminal side of arginine and lysine residues. They are active at neutral and alkaline pH and usually have a molecular weight of about 22-30 kDa. Trypsins have esterase and amidase activity which may be important for inactivation of viruses. Esterases cleave the ester bonds of arginine or lysine amidase—hydrolyses the C-terminal amide bond of peptides.

Cold-active trypsins are produced by many psychrophiles. Such trypsins may be isolated from the pyloric cecum and intestines of fish and the viscera of crustaceans that live in permanently cold environments. These enzymes may be described in the literature as psychrophilic and/or cold active or cold-adapted. Temperature profiles show significant activity at low temperatures, below 15, 10, 7, 5 or 2 Centigrade. While some trypsins from cold-adapted fish/crustacea may have temperature optima close to those of mammalian (mesophilic) trypsins, they retain greater activity at lower temperatures. Cold-active trypsins retain activity at below 10° C. Any individual species may produce several different trypsin isoenzymes. E.g. Atlantic cod produces several type I and type III trypsins. Trypsin I is the major trypsin, but may only be active to 10° C. Trypsins Y and ZT are active down to 2° C. and have temperature optima below those of mesophilic trypsins. Using trypsin isolated from a fish such as Atlantic cod as an anti-viral is ideal because a trypsin preparation contains a range of different trypsin isoenzymes. Trypsin sources are plentiful: Mixtures of trypsin isozymes can be extracted from cold-adapted fish or crustacean viscera. Recombinant trypsins may also be used.

The genusGadus(e.g., Cod) includes psychrophiles thriving permanently at near-zero temperatures which synthesize cold-active enzymes to sustain their cell cycle. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold.

These enzymes are already used in many biotechnological and commercial applications requiring high activity at low temperatures. However they have not been incorporated into fabrics for PPE.

The fabrics of the present invention may also be used for dressings for wound debridement, or in other embodiments, specifically exclude use for wound debridement.

A number of particularly relevant disclosures and publications are set out below, and are all incorporated by reference for all purposes. U.S. Pat. Nos. 4,801,451 and 4,963,491 disclose a mixture of exo- and endopeptidases isolated from Antarctic hill (Euphasia superba) and the use of this mixture as a cleaning solution. U.S. Pat. No. 4,801,451 discloses the use of such enzymes to remove foreign matter and dead tissue from wounds. WO 85/04809 discloses the use of hill enzymes as a digestion-promoting agent. EP-A1-0170115 discloses the use of krill enzymes to dissolve blood clots. WO96/24371 discloses the use of a hill-derived multifunctional proteolytic enzyme and a family of crustacean and fish derived proteolytic enzymes having structural similarity to the multifunctional enzyme derived from Antarctic hill. The document also relates to cosmetic and other uses of the enzyme. WO2000078332 discloses the use of cod derived trypsins and chymotrypsins in pharmaceutical compositions or medicaments for local and topical application. The serine proteinases disclosed in WO 2000078332 are proteinases that have at least 90% amino acid sequence homology with trypsin I, trypsin II, trypsin III, trypsin IV derived from Atlantic cod and proteinases that are chymotrypsin having at least 90% amino acid sequence homology with any of chymotrypsin A and chymotrypsin B isolated from Atlantic cod. Spilliaert and Gudmundsdottir, 1999, Mar Biotechnol (NY) 1: 598-607 discloses a cDNA encoding trypsin Y isolated from an Atlantic cod cDNA library. Cod trypsin Y has approximately 45% identity to the two Atlantic cod trypsin I and X (WO 2000078332). The native trypsin Y and recombinant forms of trypsin Y have previously briefly been described by Palsdottir and Gudmundsdottir, 2007, Protein Expr Purif 51: 243-252, and Palsdottir and Gudmundsdottir, 2008, Food Chemistry 111: 408-414. WO2018138292A1 (to Enzymatica Ab) describes a marine serine protease that is a trypsin, for example trypsin I, trypsin X, trypsin Y or trypsin ZT. Three major isozymes of trypsin were originally characterized from Atlantic cod, designated Trypsin I, II and III (see Asgeirsson et ai, 1989, Eur. Priority 2017-Jan.-26⋅Filed 2018-Jan.-26⋅Published 2018-Aug.-2. WO2018138292A1 discloses polypeptides having protease activity for use in the treatment or prevention of otitis. In one embodiment, the polypeptide consists of an amino acid sequence of trypsin I from Atlantic cod (Gadus morhua).

Other particularly relevant patent applications include: US2020/0197288 (Novel trypsin isoforms and their use) and US2016/0339087 which discloses a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 (see published application) or a fragment, variant, derivative or fusion thereof which retains the trypsin activity. Other related applications and patents include U.S. Ser. No. 15/115,065 and PCT/GB2015/050212. A publication of particular relevance is Stefannson, B., A. Gudmundsdottir and M. Clarsund. (2017) A medical device forming a protective barrier that deactivates four major common cold viruses. Virol. Res. Rev. 1(5):1-3. Other relevant disclosures include the following: 20210130852, Device and method for increasing the organic yield of a bioliquid; 20200353060, Peptides having protease activity for use in the treatment or prevention of coronavirus infection; 20200197288, Novel trypsin isoforms and their use; 20200085921, Combination therapies; 20190375664, Methods of processing municipal solid waste (msw) using microbial hydrolysis and fermentation; 20190343932, Novel treatments; 20160339087, Novel treatments; and 20170107503, which discloses recombinantly expressed mutants of trypsin I from Atlantic cod, which mutants exhibit improved stability and/or catalytic properties relative to the wildtype trypsin purified from cod. These and all other patents, applications and publications herein are incorporated by reference.

There is a long-felt need, with a particular new urgency, for fabrics that are self-sterilizing, and that will inactivate enveloped pathogenic viruses and Gram negative bacteria on contact. Doctors, nurses and other hospital workers need PPE that is effective in preventing the spread of nosocomial infections from patient to patient, as well as being convenient, safe, affordable, disposable, and biodegradable. The fabrics of this invention meet this long felt need and may be used in the production of protective facemasks. The present application incorporates proteases into fabrics that may be used for PPE applications. Specifically this application employs cold active trypsins incorporated into fabrics.

SUMMARY OF THE INVENTION

The invention provides self-sterilizing fabrics incorporating low temperature enzymes, specifically psychrophilic and/or cold active enzymes, which inactivate pathogenic viruses and Gram negative bacteria and prions on contact, including Coronavirus (e.g. Covid19), and Influenza viruses. The fabrics of the invention may be used in the production of protective facemasks and PPE. In a specific preferred embodiments the material incorporates cold active trypsin enzymes derived for organisms that live in cold climates such as (but not limited to) deep sea cod (genusGadus), King crabs (red, particularly), Dungeness, and Snow crabs (also called Tanner crabs).

