M. luteus serine protease for use in the treatment of inflammatory skin conditions

Abstract

A serine protease derived from M. luteus. is disclosed. Also disclosed are compositions comprising the serine protease and associated uses and method of manufacture.

Description

[0001] Medicament

[0002] The present invention relates to a serine protease for use as a medicament, for example, for use in treating or preventing inflammatory conditions. The invention also relates to compositions comprising the serine protease, and to methods for the manufacture and use of such compositions.

[0003] The skin acts as a protective barrier between the internal host and harmful external stressors. Staphylococcus aureus (S. aureus) is the dominant pathogen of human skin, causing the majority of skin and soft tissue infections worldwide. It is also the most frequent cause of infection-induced flares of atopic dermatitis (AD). Recurrent S. aureus skin infections are reported in 39% of patients within 3 months, and in greater than 50% of patients within 6 months of initial infection, necessitating repeated courses of antibiotics and increasing the risk of antibiotic resistance.

[0004] S. aureus has been found to be internalized and survive within keratinocytes without inducing cytotoxicity or simulating the release of the pro-inflammatory cytokine interleukin 33 (IL-33). Many common anti-staphylococcal antibiotics are unable to eradicate internalized S. aureus.

[0005] Antibiotics include a range of powerful drugs that kill bacteria or slow their growth. The overprescription of antibiotics has led to resistant bacteria and some strains that were once very responsive to antibiotics have become more and more resistant. Reducing the use of antibiotics will reduce the rate and prevalence of bacterial resistance.

[0006] Improved antimicrobial strategies are required in order to effectively eradicate S. aureus and prevent recurrent infections and antibiotic resistance.

[0007] The skin is host to a diverse range of microorganisms collectively referred to as the microbiome. Microbiomes can differ significantly between individuals and even between body sites, and may contain various different strains of bacteria, some of which may be pathogenic under certain conditions. In healthy individuals the organisms of the microbiome live in harmony with their human hosts and can provide functions that are beneficial, or even essential, for human health and survival. By colonising the skin these microbes provide the first line of defence against potentially harmful organisms, preventing them from occupying the same biological niche, either directly, for example by inhibiting pathogen adhesion or by bacteriocin production, or indirectly, for example by inducing or regulating a host immune response.

[0008] Keratinocytes are the predominant epidermal cell type and although the main role of these cells is to provide a structural and barrier function, they can also induce inflammation in response to stressors such as S. aureus infection, by recruiting and activating immune cells via the release of chemokines and cytokines. In this way, keratinocytes may promote an inflammatory response. Cytokines including IL-33 and thymic stromal lymphopoietin (TSLP) that are released by keratinocytes are powerful inducers of type 2 inflammation. They are characterised as "alarmins" that are released upon epithelial insult and function to promote proliferation of Th2 cells and initiate cytokine cascades that drive type 2 inflammation.

[0009] Studies in humans have shown that commensals in the skin microbiome may have a protective effect on keratinocytes and reduce inflammation. For example, lactic acid bacteria (LAB) lysates have been found to have anti-inflammatory effects on keratinocytes, and selective killing of S. aureus has also been observed via the production of AMPs from S. epidermidis and Staphylococcus hominis isolated from human skin.

[0010] The present inventors investigated the potential anti-inflammatory effects of skin microbiota and have identified a serine protease in Micrococcus luteus (M. luteus) that is capable of attenuating cutaneous inflammation. M. luteus has not previously been reported to produce or secrete molecules with anti-inflammatory activity.

[0011] Thus, in accordance with a first aspect of the invention, there is provided a serine protease for use as a medicament, wherein the serine protease is derived from M. luteus.

[0012] In accordance with a second aspect of the invention, there is provided a serine protease for use in the treatment or prevention of an inflammatory skin condition, wherein the serine protease is derived from M. luteus.

[0013] The disclosed serine protease may be capable of cleaving IL-33. Cleaved IL-33 is inactive and incapable of acting as a proinflammatory mediator and inducing or propagating inflammation. The disclosed serine protease may, therefore, be capable of reducing the concentration of active IL-33 in an in vitro assay and also at the site of an inflammatory skin condition. Keratinocytes are the predominant cell type in the epidermis and can induce inflammation in response to stimuli by recruiting and activating immune cells via cytokines, making Normal human epidermal keratinocytes (NHEK) cell culture a suitable basis for cell-based models of skin inflammation. Levels of cytokines released from keratinocytes can be measured using an enzyme-linked immunosorbent assay (ELISA) and so are ideal markers of inflammation. The presence and levels of full length and cleaved cytokines may be determined using specific antibodies. A person skilled in the art would be familiar with suitable methods for determining the levels of full-length and cleaved IL-33 in a sample, including using a suitable ELISA.

[0014] The disclosed serine protease may be for use in treating any condition involving IL- 33, or in which IL-33 is contributing factor. Thus, the disclosed serine protease may be for use in treating an inflammatory skin condition in which IL-33 is contributing factor.

[0015] The disclosed serine protease may be for use in treating an inflammatory skin condition by cleaving IL-33, and / or by reducing the concentration of IL-33 present at the site of inflammation.

[0016] Various bacteria present on the skin may promote inflammation by inducing the release of IL-33. Thus, in some embodiments, the inflammatory skin condition with which the serine protease may be used may be a bacterially-induced condition.

[0017] Atopic dermatitis (AD), which is the most common form of eczema, is the most common inflammatory skin disease worldwide affecting up to 20% of children and up to 10% of adults. AD is characterised by dry, itchy red skin rashes that come and go on any part of the body. Scratching can cause the skin to leak fluid and crust over which can eventually lead to lichenification. Scratching can also break the skin making it susceptible to infection further exacerbating the inflammation. Current AD treatment options usually focus on symptomatic amelioration and relief is often transient. Symptomatic management primarily focuses on rehydrating the skin, reducing inflammation, and repairing the skin barrier.

[0018] AD pathogenesis is driven by cytokines released by Th2 lymphocytes and keratinocytes, including IL-33, which cause and promote inflammation in the skin. The disclosed serine protease is capable of cleaving and inactivating IL-33 and thereby reducing this skin inflammation. Thus, in some embodiments, the inflammatory skin condition may be AD, and the disclosed serine protease may be for use in treating AD.

[0019] The disclosed serine protease may be for use in treating AD by cleaving IL-33, and / or by reducing the concentration of IL-33 present at the site of AD, such as at the site of a red skin rash to which the serine protease, such as a composition comprising the serine protease, has been applied.

[0020] S. aureus is frequently detected on AD skin lesions and is suspected to be involved in the clinical symptoms and pathogenesis of skin flare-ups. S. aureus is not commonly found on healthy skin except for in the nares and axilla yet is still found on non-lesional skin of AD patients. The abundance of S. aureus increases greatly during flare-ups and the density of S. aureus correlates with the severity of AD.

[0021] S. aureus has been found to release a unique virulence factor named S. aureus second immunoglobulin-binding protein (Sbi) that stimulates the release of IL-33 and TSLP from Keratinocytes, initiating the type 2 immune response underpinning AD pathophysiology. The S. aureus secretome has been found to disrupt the skin barrier, making filtered S. aureus supernatant (FSA) a suitable stimulator in a model of AD-type inflammation.

[0022] The disclosed serine protease may be capable of cleaving Sbi.

[0023] Cleaved Sbi is inactive and incapable of stimulating the release of IL-33 and TSLP from Keratinocytes. The disclosed serine protease may, therefore, be capable of reducing the concentration of active Sbi. By reducing the concentration of active Sbi, the disclosed serine protease may be capable of reducing the concentration of active IL-33 and / or TSLP released by keratinocytes.

[0024] Since the disclosed serine protease is capable of cleaving and inactivating the pro- inflammatory S. aureus protein Sbi, in some embodiments, the disclosed serine protease may be for use in treating an inflammatory skin condition that is induced or exacerbated by S. aureus.

[0025] The present inventors screened supernatants of bacteria isolated from the skin of healthy volunteers, using ELISA, for the ability to reduce IL-33 and TSLP secretion in NHEK stimulated with FSA. An isolate of M. luteus was identified that completely negated FSA-induced release of IL-33 and TSLP from NHEK (p<0.0001). The disclosed serine protease was identified in the culture medium in which the M. luteus had been cultured.

[0026] The serine protease may be secreted by M. luteus. Micrococcus luteus (M. luteus) is a species of gram-positive, spherical bacteria whose organisms occur in tetrads and in irregular clusters of tetrads but not in chains and belongs to the family Micrococcaceae. M. luteus is part of the normal microbiota of mammalian skin.

[0027] M. luteus has a golden yellow colour when viewed under a microscope, and may be identified on the basis that it is urease & catalase positive but coagulase and gelatinase negative. The skilled person will be familiar with other methods, such as genetic methods, for the identification and characterisation of M. luteus.

[0028] The type strain of M. luteus is available at the National Collection of Type Cultures (NCTC) under number NCTC 2665, and the original strain reference is ATCC 15307. Other collection numbers of M. luteus strains include ATCC 4698, CCM 169, CCUG 5858, CIP A270, DSM 20030, HAMBI 1399, HAMBI 26, IEGM 391, IFO 3333, JCM 1464, LMG 4050, NBRC 3333, NCCB 78001, NRRL B-287, VKM B-1314, ATCC 15307, CN 3475, NCIB 9278, and WDCM 00111.

[0029] Strains of M. luteus from which the disclosed serine protease may be obtained may be identified on the basis of the presence of the serine protease in the culture supernatant in which the M. luteus has been cultured. Thus, the M. luteus may be a strain of M. luteus that, when cultured under growth conditions for at least one hour, provides a culture supernatant that is capable of reducing the concentration of IL-33 in a sample of recombinant IL-33. In addition, or alternatively, the M. luteus may be a strain of M. luteus that, when cultured under growth conditions for at least one hour, provides a culture supernatant that is capable of reducing the concentration of Sbi in a sample of recombinant Sbi. Suitable growth conditions for culturing M. luteus will be known to the skilled person.

[0030] The M. luteus may be the strain of M. luteus that has been deposited at the NCTC depositary institution under Accession Number 22110401. The serine protease, or the culture supernatant in which the M. luteus has been cultured for at least one hour, may be capable of cleaving full-length recombinant IL-33.

[0031] The serine protease, or the culture supernatant in which the M. luteus has been cultured for at least one hour, may be capable of reducing the concentration of IL- 33, for example, in a sample of recombinant IL-33.

[0032] For example, the serine protease, or the culture supernatant in which the M. luteus has been cultured for at least one hour, may be capable of reducing the concentration of full-length IL-33 in a sample of full-length recombinant IL-33. Various suitable methods for measuring the concentration of full-length IL-33 will be known to the skilled person and include, for example, ELISA.

[0033] The serine protease, or the culture supernatant in which the M. luteus has been cultured for at least one hour, may be capable of cleaving full-length Sbi, for example, in a culture supernatant in which S. aureus has been cultured.

[0034] The serine protease, or the culture supernatant in which the M. luteus has been cultured for at least one hour, may be capable of reducing the concentration of Sbi in vitro.

[0035] For example, the serine protease, or the culture supernatant in which the M. luteus has been cultured for at least one hour, may be capable of reducing the concentration of full-length Sbi in a sample of full-length Sbi in vitro. Various suitable methods for measuring the concentration of full-length Sbi will be known to the skilled person and include, for example, ELISA.

[0036] The activity of the serine protease may be inhibited by 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF). The catalytic activity of the serine protease may be reduced or prevented by AEBSF. The protease activity of the serine protease may be reduced or prevented by AEBSF. Suitable methods for determining the inhibition of the serine protease enzyme activity will be known to the skilled person and an example method is described below. For example, the serine protease or supernatant of the culture medium in which the M. luteus has been cultured for at least one hour, is treated, or not, with AEBSF (ImM) for 1 h before adding to a sample of recombinant IL-33 (1500pg / ml). After incubation, the level of full-length and / or cleaved recombinant IL-33 may then be determined by ELISA.

[0037] The serine protease isolated by the inventors from M. luteus has the sequence of SEQ ID NO: 1.

[0038] MRKAEAMPQNPTPARRRRALAAAVAGASLVAAPALAVSATAVELPDGSTVSSPQGEVQV QQQFEDGRYFVVLKDQPSVTAPEAGAVPGAAPKAKFDPSHPRVKNYEAKLQRQQEKVAK THGAKAEISFQRAVNAFVAELTAEEAQEIAKDPAVLGVAPDEQVAPDYSSTEFLGLPGK KGTWKSVYGKAENAGKGVWGVIDSGIHPDNPFIDGQPVQPLKGKAKVGVPYRTADGQI AVLKADGTTATAECETGPDFPASSCDSKLIGAYAFSEDFERFVPVDERAPEERISPLGV FSHGTHVATT I LGNTGVEQT I DGDS FGEGAGVAPAAHL I S YKI CWEDTDPNTGGC YTSA SVAAVEQAIENNVDVLNYSISGSNTSIVDPVAMAFKSAAEAGIFVAASGGNSGPGPNTV NHGSPWLTTVAAETFSNELTATVQFSDGTQLRGASSARTGVGPAEVIHASEVAAGDAEA ARLCLPGGLTDEAAGKIVLCERGVNARTEKSQVVEEAGGVGMILVNTPSGSLDADIHAV PTVHMNDNGVIEKVKSSDLTATIVPGDTTGLPEDPLPQIAGFSSRGPANAVNQELLKPD LAAPGVNVIAGVSPLDPDYHGNTFGLMSGTSMASPNLAGMATLLIGKYPAWSPMAVKSA LMTTAGDVYNADGTVNTDNFATGAGSADPAAAARPGLVYESGKEQWDALLRGDIAGRDV NVPSLAIPDWGSATVTRTVTALENGRWQFSANVPGFEMTASPAVLDLKAGQSADVELT VTRTDAAVNTWTHGSMSWTTAKGKAVPEVTSPVTVKAKSATVTSAVEGSGATGSADVEI TPGVTGELTPQVLGLGKVDSTVATATASNSLVSSALAVSTVTVEEGTKSLVASINAGAA GADWDLYVITPEGKQLSRATAEESETLTIANPTPGAYTWGHLYAANGGKDTGTLETLK LREDAGNLTVSPNPVPVTSGKATEATLSWSGLTSGTWKGLVTWDAGITTDVTVQVP SEQ ID NO: 1 serine protease isolated by the inventors from M. luteus.

[0039] The serine protease may be a variant of SEQ ID NO: 1. Thus, the serine protease may have at least 98% sequence identity to SEQ ID NO: 1.

[0040] The serine protease may have at least 98.5% sequence identity to SEQ ID NO: 1. In some embodiments, the serine protease may have at least 99% sequence identity to SEQ ID NO: 1, such as at least 99.1. 99.2, 99.3, or 99.4% sequence identity to SEQ ID NO: 1. In some embodiments, the serine protease may have at least 99.5% sequence identity to SEQ ID NO: 1, such as at least 99.6, 99.7, 99.8, or 99.9% sequence identity to SEQ ID NO: 1.

[0041] The term "sequence identity" will be understood by the skilled person to refer to the percentage of identical residues between two aligned sequences. The skilled person will understand how to calculate the percentage identity between two amino acid or nucleotide sequences. For example, calculation of percentage identities between two amino acid or nucleotide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and overhangs. The serine protease may have a sequence that is based on SEQ ID NO: 1 but includes one or more amino acid substitutions.

[0042] In some embodiments, the serine protease may consist of or comprise the sequence of SEQ ID NO: 1 with 50 or fewer amino acid substitutions, such as 40 or fewer, 30 or fewer, or 20 or fewer amino acid substitutions. In some embodiments, the serine protease may consist of or comprise the sequence of SEQ ID NO: 1 with 15 or fewer amino acid substitutions, such as 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer amino acid substitutions. The serine protease may consist of or comprise the sequence of SEQ ID NO: 1 with 1 amino acid substitution.

[0043] The amino acid substitutions may comprise conservative amino acid substitutions. In some embodiments, all of the amino acid substitutions relative to the sequence of SEQ ID NO: 1 may be conservative amino acid substitutions.

[0044] As the skilled person would understand, a conservative amino acid substitution refers to the replacement of an amino acid in the protein with another amino acid that has similar biochemical properties such as hydrophobicity, charge, and size. Conservative substitutions are those that are not expected to change the tertiary structure or the function of the protein. Examples of small non-polar, hydrophobic amino acids that may be conservatively replaced for one another include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids that may be conservatively replaced for one another include phenylalanine, tryptophan and tyrosine. Polar neutral amino acids that may be conservatively replaced for one another include serine, threonine, cysteine, asparagine and glutamine. Positively charged (basic) amino acids that may be conservatively replaced for one another include lysine, arginine and histidine. Negatively charged (acidic) amino acids that may be conservatively replaced for one another include aspartic acid and glutamic acid.

[0045] The serine protease may consist of or comprise a fragment of SEQ ID NO: 1.

[0046] The hallmark of serine proteases is that they contain the "classical" catalytic triad of serine (S), histidine (H) and aspartate (D) within the active site, that function together to facilitate catalysis. The disclosed serine protease contains an SHD catalytic triad, as well as an inhibitor 19 domain, peptidase S8 / S53 domain, subtilisin-like protease fibronectin type-III domain and a protease-associated (PA) domain.

[0047] The peptidase S8 / S53 domain is understood to provide the serine protease catalytic properties of the protein. This corresponds to amino acids 186-668 of SEQ ID NO: 1, and the aspartic acid, histidine and serine residues that comprise the catalytic triad occur at position 195, 292 and 615 of SEQ ID NO: 1, respectively.

[0048] Thus, the sequence of the catalytic S8 / S53 domain of the serine protease has the sequence shown below as SEQ ID NO:2.

[0049] KAENAGKGVWGVIDSGIHPDNPFIDGQPVQPLKGKAKVGVPYRTADGQIAVLKADGTT ATAECETGPDFPASSCDSKLIGAYAFSEDFERFVPVDERAPEERISPLGVFSHGTHVAT T I LGNTGVEQT I DGDS FGEGAGVAPAAHL I S YKI CWEDTDPNTGGC YTSAS VAAVEQAI ENNVDVLNYSISGSNTSIVDPVAMAFKSAAEAGIFVAASGGNSGPGPNTVNHGSPWLTT VAAETFSNELTATVQFSDGTQLRGASSARTGVGPAEVIHASEVAAGDAEAARLCLPGGL TDEAAGKIVLCERGVNARTEKSQVVEEAGGVGMILVNTPSGSLDADIHAVPTVHMNDNG VIEKVKSSDLTATIVPGDTTGLPEDPLPQIAGFSSRGPANAVNQELLKPDLAAPGVNVI AGVSPLDPDYHGNTFGLMSGTSMASPNLAGMATLLIGKYPAWSPMAVKSALMTTAGDVY NADGTVNTDN

[0050] SEQ ID NO:2 peptidase S8 / S53 domain of the serine protease isolated by the inventors from M. luteus.

[0051] Thus, in embodiments in which the serine protease consists of or comprises a fragment of SEQ ID NO: 1, the fragment may consist of or comprise the 482 amino acid residue sequence shown in SEQ ID NO:2.

[0052] The protein of SEQ ID NO:2 has a molecular weight of about 49kDa. Thus, the serine protease may have a molecular weight of greater than about 40kDa or 45kDa, such as greater than 50, 60, 70, or 80kDa. The serine protease may have a molecular weight of less than 150, 140, 130, 120, or 115kDa. The serine protease may have a molecular weight of 40-110kDa, such as 45-105kDa. In some embodiments, the serine protease may have a molecular weight of 82-117kDa, such as 85-107kDa, or 87-103kDa.

[0053] The serine protease may consist of or comprise a sequence that is a variant of SEQ

[0054] ID NO:2. The serine protease may consist of or comprise a sequence having at least 98% sequence identity to SEQ ID NO: 2.

[0055] The serine protease may consist of or comprise a sequence having 98.5% sequence identity to SEQ ID NO: 2. In some embodiments, the serine protease may consist of or comprise a sequence having at least 99% sequence identity to SEQ ID NO:2, such as at least 99.1. 99.2, 99.3, 99.4 99.5, 99.6, 99.7, 99.8, or 99.9% sequence identity to SEQ ID NO: 2.

[0056] The serine protease may consist of or comprise the sequence of SEQ ID NO: 2 with 20 or fewer amino acid substitutions, such as 15 or fewer, 12 or fewer, or 10 or fewer amino acid substitutions. In some embodiments, the serine protease may consist of or comprise the sequence of SEQ ID NO:2 with 9 or fewer amino acid substitutions, such as 8, 7, 6, 5, 4, 3, or 2 or fewer amino acid substitutions. The serine protease may consist of or comprise the sequence of SEQ ID NO: 2 with 1 amino acid substitution.

[0057] The amino acid substitutions relative to the sequence of SEQ ID NO:2 may comprise conservative amino acid substitutions. In some embodiments, all of the amino acid substitutions relative to the sequence of SEQ ID NO:2 may be conservative amino acid substitutions.

[0058] In some embodiments, the serine protease may include a serine (S) residue at the position corresponding to position 195 of the sequence shown in SEQ ID NO: 1, a histidine (H) residue at the position corresponding to position 292 of the sequence shown in SEQ ID NO: 1, and aspartate (D) residue at the position corresponding to position 615 of the sequence shown in SEQ ID NO: 1.

[0059] The inventors have found that the type strain of M. luteus, deposited under number NCTC 2665 comprises a number of amino acid substitutions relative to SEQ ID NO: 1, and also a frame shift mutation at the position of amino acid 390, that is predicted to result in two truncated proteins, shown below as SEQ ID NO:3 and SEQ ID NO: 4. Both of the truncated proteins are inactive and incapable of cleaving either IL-33 or Sbi.

[0060] MRKAEAMPQNPTPARRRRALAAAVAGASLVAAPALAVSATAVELPDGSTVSSPQGEVQV QQQFEDGRYFVVLKDQPSVTAPEAGAVPGAAPKAKFDPSHPRVKNYEAKLQRQQAKVAK AHGAKVEISFQRAVNAFVAELTAEEAQAIAKDPAVLGVAPDEQVAPDYSSTEFLGLAGK KGTWKSVYGKAENAGEGVWGVIDSGIHPDNPFIDGQPVQPLKGKAKVGVPYRTADGQI AVLKADGTTATADCETGPDFPASSCDSKLIGAYAFSEDFERFVPVDERAPEERISPLGV FSHGTHVATT I LGNTGVEQT I DGDS FGEGAGVAPAANL I S YKI CWEDTDPDTGGC YTSA SVAAVEQAIENNVDVLNYSISGSNTSIVDPVAMAFKSAAEARWPWPSSPPPRPASSWPP PAATPAPARTP

[0061] SEQ ID NO:3 first truncated protein present in the genome of the M. luteus type strain deposited under number NCTC 2665. MAASGGNSGPGPNTVNHGSPWLTTVAAETFSNELTATVQFSDGTQLRGASSARTGVGPA EVIHASEVAAGDAEAARLCLPGGLTEEAAGKIVLCERGVNARTEKSQWEEAGGVGMIL VNTPSGSLDADIHAVPTVHMNDNGVIEKVKSSDLTATIVPGDTTGLPADPLPQIAGFSS RGPANAVNQELLKPDLAAPGVNVIAGVSPLDPDYHGNTFGLMSGTSMASPNLAGMATLL IGKYPAWSPMAVKSALMTTAGDVYNADGTVNTDNFATGAGSADPAAAARPGLVYESGKE QWDALLRGDIAGRDVNVPSLAIPDWGSATVTRTVTALENGRWQFSANVPGFEVTASPA VLDLKAGQSADVELTVTRTDAAMNTWTHGSMSWTTAKGKAVPEVTSPVTVKAKSATVTS AVEGSGATGSADVEITPGVTGELTPQVLGLGKVDSTVAAATASNSLASSALAVSTVTVE EGTQSLVASINAGAAGADWDLYVITPEGKQLSRATADESETLTIADPVPGAYTWGHLY AANGGKDTGTLETLKLREDAGNLTVSPNPVPVTSGKATEATLSWSGLTSGTWKGLVTWD AGITTDVTVQVP

[0062] SEQ ID NO:4 second truncated protein present in the genome of the M. luteus type strain deposited under number NCTC 2665.

[0063] The inventors have expressed the proteins of SEQ ID NOs:3 and 4 and found them to be inactive, and incapable of cleaving either IL-33 or Sbi.

[0064] Thus, in embodiments in which the serine protease consists of or comprises a fragment of SEQ ID NO: 1, the fragment may consist of or comprise a sequence that is significantly longer than either SEQ ID NO:3 or SEQ ID NO:4.

[0065] Thus, the serine protease may consist of or comprise a fragment of SEQ ID NO: 1 having a length of at least 700, 800, or 900 amino acid residues. In some embodiments, the serine protease may consist of or comprise a fragment of SEQ ID NO: 1 having a length of at least 920 amino acid residues, such as at least 930, 940, 950, 960, 970, or 980 amino acid residues. In some embodiments, the serine protease may consist of or comprise a fragment of SEQ ID NO: 1 having a length of at least 990 amino acid residues, such as at least 991, 992, 993, 994, 995, 996, 997, 998, or 999 amino acid residues.

[0066] The inventors speculated that the protein truncation observed in the M. luteus type strain would split the peptidase S8 / S53 domain and result in the loss of the catalytic triad. In view of this hypothesis, they produced a synthetic protein by removing the frame shift mutation from the sequence present in the type strain of M. luteus. The resulting protein, which has the sequence of SEQ ID NO:5, comprises the amino acid substitutions that are present in SEQ ID NOs:3 and 4 relative to SEQ ID NO: 1, but not the frame shift mutation.

[0067] MPQNPTPARRRRALAAAVAGASLVAAPALAVSATAVELPDGSTVSSPQGEVQVQQQFED

[0068] GRYFWLKDQPSVTAPEAGAVPGAAPKAKFDPSHPRVKNYEAKLQRQQAKVAKAHGAKV EISFQRAVNAFVAELTAEEAQAIAKDPAVLGVAPDEQVAPDYSSTEFLGLAGKKGTWKS VYGKAENAGEGVWGVIDSGIHPDNPFIDGQPVQPLKGKAKVGVPYRTADGQIAVLKAD GTTATADCETGPDFPASSCDSKLIGAYAFSEDFERFVPVDERAPEERISPLGVFSHGTH VATTILGNTGVEQTIDGDSFGEGAGVAPAANLISYKICWEDTDPDTGGCYTSASVAAVE QA I ENNVDVLN YS I S GSNTS I VD P VAMAF KS AAE AG I F VAAS GGNS GPGPNTVNHGS PW LTTVAAETFSNELTATVQFSDGTQLRGASSARTGVGPAEVIHASEVAAGDAEAARLCLP GGLTEEAAGKIVLCERGVNARTEKSQWEEAGGVGMILVNTPSGSLDADIHAVPTVHMN DNGVIEKVKSSDLTATIVPGDTTGLPADPLPQIAGFSSRGPANAVNQELLKPDLAAPGV NV I AGVS PLD PD YHGNT FGLMSGT SMAS PNLAGMATLL I GKY PAWS PMAVKS ALMTTAG DVYNADGTVNTDNFATGAGSADPAAAARPGLVYESGKEQWDALLRGDIAGRDVNVPSLA IPDWGSATVTRTVTALENGRWQFSANVPGFEVTASPAVLDLKAGQSADVELTVTRTDA AMNTWTHGSMSWTTAKGKAVPEVTSPVTVKAKSATVTSAVEGSGATGSADVEITPGVTG ELTPQVLGLGKVDSTVAAATASNSLASSALAVSTVTVEEGTQSLVASINAGAAGADWDL YVITPEGKQLSRATADESETLTIADPVPGAYTVVGHLYAANGGKDTGTLETLKLREDAG NLTVSPNPVPVTSGKATEATLSWSGLTSGTWKGLVTWDAGITTDVTVQVP

[0069] SEQ ID NO:5 synthetic protein sequence derived from truncated proteins present in the genome of the M. luteus type strain deposited under number NCTC 2665.

[0070] When the inventors generated and tested the sequence of SEQ ID NO:5, despite including all three of the D, H, and S residues of the putative catalytic triad, it was nevertheless surprisingly also found to be inactive and incapable of cleaving either IL-33 or Sbi.

[0071] In view of this, in embodiments in which the serine protease consists of or comprises a variant or fragment of sequence SEQ ID NO: 1, the serine protease may nevertheless comprise one or more of the following residues of SEQ ID NO: 1, or at a position corresponding to that of SEQ ID NO: 1 :

[0072] (I) E at position 114;

[0073] (ii) T at position 119;

[0074] (ill) A at position 124;

[0075] (iv) E at position 146;

[0076] (v) P at position 175;

[0077] (vi) K at position 193;

[0078] (vii) E at position 249;

[0079] (viii) H at position 332;

[0080] (ix) N at position 346;

[0081] (x) D at position 483;

[0082] (xi) E at position 564;

[0083] (xii) M at position 747;

[0084] (xiii) V at position 775;

[0085] (xiv) T at position 850;

[0086] (xv) V at position 858; (xvi) K at position 874;

[0087] (xvii) E at position 907;

[0088] (xviii) N at position 916; and / or

[0089] (xix) T at position 918.

[0090] In some embodiments, the serine protease may comprise one or all of the following residues of SEQ ID NO: 1, at a position corresponding to that of SEQ ID NO: 1, which are present in the catalytic S8 / S53 domain of the serine protease:

[0091] (I) K at position 193;

[0092] (ii) E at position 249;

[0093] (ill) H at position 332;

[0094] (iv) N at position 346;

[0095] (v) D at position 483; and / or

[0096] (vi) E at position 564.

[0097] Thus, in embodiments in which the serine protease consists of or comprises a variant of SEQ ID NO:2, the serine protease may comprise one or all of the following residues, at a position corresponding to that of SEQ ID NO: 1 : (I) K at position 193;

[0098] (ii) E at position 249;

[0099] (ill) H at position 332;

[0100] (iv) N at position 346;

[0101] (v) D at position 483; and / or

[0102] (vi) E at position 564.

[0103] In preferred embodiments, the serine protease comprises all six of these specific residues.

[0104] Thus, the serine protease may consist of or comprise the sequence of SEQ ID NO: 5 having one or more of the following amino acid substitutions, or at a position corresponding to that of SEQ ID NO: 1 :

[0105] (i) A108E;

[0106] (ii) A113T;

[0107] (iii) V118A;

[0108] (iv) A140E;

[0109] (v) A169P;

[0110] (vi) E187K;

[0111] (vii) D243E;

[0112] (viii) N326H; (ix) D340N;

[0113] (x) E477D;

[0114] (xi) A558E;

[0115] (xii) V741M;

[0116] (xiii) M769V;

[0117] (xiv) A844T;

[0118] (xv) A852V;

[0119] (xvi) Q868K;

[0120] (xvii) D901E;

[0121] (xviii) D910N; and / or

[0122] (xix) V912T.

[0123] In some embodiments, the serine protease may consist of or comprise the sequence of SEQ ID NO: 5 having one or more of the following amino acid substitutions:

[0124] (i) E187K;

[0125] (ii) D243E;

[0126] (iii) N326H;

[0127] (iv) D340N;

[0128] (v) E477D; and

[0129] (vi) A558E.

[0130] In preferred embodiments, the serine protease comprises all six of these amino acid substitutions.

[0131] Unless otherwise indicated, references to residues at a specific position within a protein sequence are based on the numbering of residues in SEQ ID NO: 1. As the skilled person would appreciate, the exact numbering of residues between related sequences may differ as a result of insertions and / or deletions of residues, or differences in the length of the sequences. Using well-known methods in the art however, the skilled person would be able to align any given sequence with that of SEQ ID NO: 1 and thereby determine which residues correspond to the specific residues of SEQ ID NO: 1 described herein.

[0132] The serine protease may be present in a composition formulated for topical administration.

[0133] The composition may consist of or comprise secreted material from M. luteus. Secreted material refers to material produced within M. luteus cells and subsequently released from the cells into the surrounding medium.

[0134] The secreted material may be a single agent, such as the serine protease. The secreted material may be a mixture of more than one agent. The secreted material may include, in addition to the serine protease, other proteins, carbohydrates, nucleic acids or lipids. Secreted material may consist of or comprise an M. luteus secretome, which is all of the secreted proteins of the strain of M. luteus. It may additionally encompass molecules that are not proteins, such as carbohydrates, lipids and nucleic acid.

[0135] The composition may include the serine protease in or with a carrier. Thus, the serine protease may be formulated in or with a carrier. The carrier may be a solution in which the serine protease is dissolved, suspended, diluted or admixed.

[0136] In some cases, the carrier may consist of or comprise the medium which has been in contact with the M. luteus during culturing. The composition of the medium will have changed during the culture, for example by the secretion of material from the M. luteus.

[0137] Thus, the composition may comprise material secreted from M. luteus in a carrier.

[0138] Thus, the composition may consist or comprise culture medium in which the M. luteus has been grown. Media suitable for culturing M. luteus bacteria is well known to those of skill in the art. As used herein the terms "media" and "medium" encompasses any nutrient containing liquid in which microorganisms such as bacteria may be supported, kept alive, grown and / or expanded. The media may contain the minimal nutrients to support bacterial life, and optionally other nutrients. Exemplary nutrients contained within the media include sugar, magnesium, phosphate, phosphorous and sulphur. The media may be made to, or modified from, a combination of nutrients that is well known in the art.

[0139] Preferably the composition does not contain any M. Luteus. The M. Luteus may have been removed from the media, for example by centrifugation and / or filtration. For example, the bacteria may be removed by sedimenting them from the media in a centrifuge at 15,000 x g for a period of time sufficient for substantially all of the M. Luteus bacteria to sediment from the media. The media may be filtered using a microporous filter with pores of a suitable size to remove substantially all of the bacteria from the media. These methods may remove intact bacteria, and may also remove bacterial debris, such as the remains of any bacteria that have undergone cell lysis.

[0140] The composition may be sterile. Thus, in some embodiments, the media or secreted serine protease may have been subject to a sterilisation process, such as irradiation, heat, chemicals, pressure or filtration, or any combination thereof. This may include autoclaving, x-ray sterilization or UV-light sterilisation.

[0141] In some embodiments, the composition comprises a serine protease secreted from M. luteus but substantially no intact bacteria. The composition may also be substantially free from lysed bacteria or bacterial fragments. The intact bacteria and / or lysed bacteria or bacterial fragments may have been separated from the secreted serine protease and optionally other components of the secretome. Separation may occur by any suitable means known in the art, such as centrifugation or filtration. The term "substantially free from" would be understood by the skilled person to mean that the secreted material contains no or minimal contamination of non-secreted bacterial components, such as whole bacteria, lysed bacteria, or bacterial fragments.

[0142] In some embodiments, the serine protease or secreted material may be present in a composition further comprising a pharmaceutical excipient. The composition may comprise the serine protease or secreted material as a suspension in a pharmaceutically acceptable excipient, diluent or carrier. The serine protease or secreted material may be present in a liposome or other microparticulate. The composition may comprise the serine protease or secreted material dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. Pharmaceutically acceptable ingredients are well known to those skilled in the art, and include, but are not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, carriers, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g. wetting agents), masking agents, colouring agents, fragrance agents and penetration agents.

[0143] The composition may be formulated for topical administration particularly for use or application to, or on, the skin. The composition may be formulated for topical administration in the form of gels, pastes, ointments, creams, sprays, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, cements, glues, and reservoirs. Preferably the composition may be formulated for topical administration in the form of a cream, gel, spray, ointment or oil.

[0144] The composition may be formulated in the form of a cream, gel, spray, ointment or oil. The composition may suitably be in the form of a liquid, solution (e.g., aqueous, non-aqueous), suspension (e.g., aqueous, non-aqueous), emulsion (e.g., oil-in- water, water-in-oil), elixir, syrup, electuary, mouthwash, cavity wash, drops, granules, powders, ampoule, bolus, suppository, pessary, tincture, gel, paste, ointment, cream, lotion, oil, foam, spray, mist, or aerosol. The composition may suitably be provided as part of a patch, adhesive plaster, bandage, dressing, or the like which may be impregnated with one or more active compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. The composition may also suitably be provided in the form of a depot or reservoir.

[0145] Ointments are typically prepared from the composition and a paraffinic or a water- miscible ointment base.

[0146] Creams are typically prepared from the extract and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w / w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

[0147] Emulsions are typically prepared from the heterotrophic skin bacterium and / or a bioactive extract of the heterotrophic skin bacterium and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and / or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Suitable emulsion and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus, the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used.

[0148] The composition may further comprise other active agents, for example antimicrobial agents such as bactericidal and fungicidal agents in order to prevent the composition from spoiling during storage.

[0149] The compositions of the present invention may be formulated as medicaments, that is to say formulated as a medicine, or a medical device. The medicament may include other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g. wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example other therapeutic or prophylactic agents.

[0150] In embodiments in which the composition comprises material secreted from M. luteus, the composition may comprise at least about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% by weight of the secreted material. In embodiments in which the composition comprises isolated serine protease, the composition may comprise, at least about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%, by weight of the serine protease.

[0151] In accordance with a third aspect of the invention, there is provided a composition for use in the treatment or prevention of an inflammatory skin condition, wherein the composition comprises a serine protease derived from M. luteus.

[0152] The composition may be as defined in accordance with the first or second aspect.

[0153] The serine protease may be as defined in accordance with the first or second aspect.

[0154] The M. luteus may be as defined in accordance with the first or second aspect.

[0155] The inflammatory skin condition may be as defined in accordance with the second aspect.

[0156] In accordance with a fourth aspect of the invention, there is provided a method of manufacturing a composition comprising a serine protease, wherein the serine protease is derived from M. luteus, the method comprising culturing M. luteus, obtaining material secreted by the M. luteus comprising the serine protease, and preparing a composition comprising the secreted material.

[0157] The secreted material may consist of or comprise the serine protease.

[0158] The serine protease may be as defined in accordance with the first or second aspect.

[0159] The M. luteus may be as defined in accordance with the first or second aspect.

[0160] The composition may be as defined in accordance with the first or second aspect in the applicable respects.

[0161] The method may comprise culturing M. luteus in culture media under conditions that facilitate growth and expansion of the bacteria. Such conditions are well known to those of skill in the art. For example, the culture may be incubated at 34-39°C, such as 36-38°C, and preferably the culture may be incubated at about 37°C.

[0162] The M. luteus may be cultured in the media for at least one hour. The M. luteus may be cultured in the media for at least two hours. The M. luteus may be cultured in the media for at least four hours. The M. luteus may be cultured in the media for at least six hours. In some embodiments, the M. luteus may be cultured in the media for at least eight hours, at least twelve hours, at least eighteen hours, at least twenty four hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least two weeks or longer.

[0163] The method may further comprise separating the bacteria and bacterial fragments from the media prior to preparing the composition. The method may comprise separating the M. luteus, lysed bacteria and / or bacterial fragments from the media by centrifugation, such as centrifugation at 15000 x g. In addition, or alternatively, the method may comprise separating the M. luteus, lysed bacteria and / or bacterial fragments from the media by filtration.

[0164] The method may comprise the sterilisation of the media, before or after removal of the bacteria. In addition, or alternatively, the method may comprise the sterilisation of the composition. Thus, the media, secreted serine protease, and or composition may be subject to a sterilisation process, such as irradiation, heat, chemicals, pressure or filtration, or any combination thereof. This may include autoclaving, x-ray sterilization or UV-light sterilisation.

[0165] The method may comprise the concentration of the media, such that the proportion of serine protease and optionally additional secreted material increases relative to the total volume of media. Concentration may be conducted by any method known in the art, such as evaporation.

[0166] The method may comprise the separation of the secreted material from the media. Any method of separating material from a carrier solution may be used. For example, the secreted material may be separated from the media by chromatography, crystallisation, distillation, drying, electrophoresis or precipitation. In particular, the secreted material may be separated from the media by a chromatographic separation method. The method may comprise the addition of a carrier to the secreted material.

[0167] In accordance with a fifth aspect of the invention, there is provided a method of manufacturing a composition comprising a serine protease, wherein the serine protease is derived from M. luteus, the method comprising expressing the serine protease in a host cell, extracting the expressed serine protease, and preparing a composition comprising the extracted serine protease.

[0168] The serine protease may be as defined in accordance with the first or second aspect. The M. luteus from which the serine protease is derived may be as defined in accordance with the first or second aspect. The composition may be as defined in accordance with the first or second aspect in the applicable respects.

[0169] For the avoidance of doubt, unless otherwise indicated, references to the serine protease being derived from M. luteus refer to the original identification of the serine protease in M. luteus and the fact that the serine protease is endogenously expressed in strains of M. luteus. Thus ,when the serine protease is obtained by expression in a host cell, the serine protease is obtained from, but not derived from the host cell. Thus, the serine protease that is obtained by expression in a host cell may be considered to be an artificial serine protease, since it has been artificially, rather than naturally, produced. Conversely, serine protease that is obtained by secretion from M. luteus may be considered to be a natural serine protease, since it has been naturally, rather than artificially, produced. The method of the fifth aspect may, therefore, comprise expressing an artificial serine protease in a host cell, extracting the expressed artificial serine protease, and preparing a composition comprising the extracted artificial serine protease.

[0170] The method may comprise culturing the host cell in a suitable culture medium and under suitable conditions. Any medium and conditions suitable for culturing the host cell may be used.

[0171] The method may comprise expressing the serine protease from the host cell, such that the serine protease accumulates in the culture medium. The method may subsequently comprise collecting the culture medium and extracting the serine protease from the culture medium.

[0172] Extraction of the serine protease may comprise isolation and / or purification of the expressed protein. Any suitable method may be used, and suitable methods may depend on the nature of the expression system and / or host used, such as, for example, whether the serine protease is secreted into the culture medium or accumulates intracellularly.

[0173] For example, in methods comprising the intracellular accumulation of the serine protease, the host cells may be collected by centrifugation after the termination of the culture and suspended in an aqueous buffer. The host cells may then be lysed, for example, using an ultrasonic disintegrator, a French press, a Manton-Gaulin homogenizer, or a Dyno-mill, in order to obtain a cell-free extract. The serine protease may then be obtained from a supernatant obtained by centrifuging the cell-free extract using any suitable protein purification method, such as, for example, solvent extraction, salting-out, desalting, precipitation using an organic solvent, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, gel filtration using a molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis.

[0174] In methods comprising the secretion of the serine protease, the host cells may be removed from the culture, for example, by centrifugation and the serine protease obtained from the supernatant by the same isolation and purification method as described above.

[0175] The method may comprise expressing the serine protease from a nucleic acid comprising a nucleic acid sequence encoding the serine protease.

[0176] Thus, in accordance with a sixth aspect of the invention, there is provided a nucleic acid comprising a nucleic acid sequence encoding a serine protease, wherein the serine protease is derived from M. luteus.

[0177] The serine protease may be as defined in accordance with the first or second aspect. The M. luteus from which the serine protease is derived may be as defined in accordance with the first or second aspect.

[0178] Any suitable method may be used to produce the nucleic acid encoding the serine protease. For example, the nucleic acid may be produced by a method in which a nucleotide sequence encoding the serine protease is amplified and cloned by polymerase chain reaction (PCR). The nucleic acid may encode an amino acid sequence comprising the serine protein and a tag. The tag may be arranged to facilitate purification of the serine protease. For example, the tag may comprise a polyhistidine-tag. Thus, the nucleic acid may encode an amino acid sequence comprising a tag amino acid sequence and the serine protease amino acid sequence.

[0179] The nucleic acid of the sixth aspect may be present in an expression vector for use in the method of the fifth aspect to express the serine protease in the host cell.

[0180] Thus, in accordance with a seventh aspect of the invention, there is provided an expression vector comprising a nucleic acid sequence encoding a serine protease, wherein the serine protease is derived from M. luteus.

[0181] The nucleic acid may be as defined in accordance with the sixth aspect. The serine protease may be as defined in accordance with the first or second aspect. The M. luteus from which the serine protease is derived may be as defined in accordance with the first or second aspect.

[0182] The expression vector may further comprise a regulatory sequence that is operably linked to the nucleic acid sequence encoding the serine protease. The regulatory sequence may be a sequence that controls the expression of the serine protease in the host cell. For example, the regulatory sequence may comprise a promoter, an enhancer, a ribosome binding sequence, or a transcription termination sequence. The expression vector may be appropriately selected, for example, based on the nature of the host cell.

[0183] The promoter may comprise an inducible promoter which functions in host cells and is capable of inducing the expression of the serine protease. For example, the inducible promoter may be capable of inducing transcription in the presence of an inducer, in the absence of a repressor molecule, and / or in response to physical factors such as increase or decrease in the temperature, osmotic pressure, pH value, or the like.

[0184] For use in the method of the fifth aspect, the expression vector may be selected as appropriate depending on the nature of the host cell. For example, the expression vector may be a plasmid vector, a viral vector, a cosmid vector, or an artificial chromosome vector. For example, an expression vector which is capable of automatically replicating in a host cell or capable of being incorporated into a chromosome of the host cell may be used. The expression vector may contain a promoter at a position capable of initiating transcription of the nucleic acid encoding the serine protease is suitably used.

[0185] The expression vector of the seventh aspect may be used in the method of the fifth aspect to express the serine protease in the host cell.

[0186] Thus, in accordance with an eighth aspect of the invention, there is provided a host cell comprising an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a serine protease, wherein the serine protease is derived from M. luteus.

[0187] The expression vector may be as defined in accordance with the seventh aspect. The nucleic acid may be as defined in accordance with the sixth aspect. The serine protease may be as defined in accordance with the first or second aspect. The M. luteus from which the serine protease is derived may be as defined in accordance with the first or second aspect.

[0188] The host cell may be a prokaryotic cell or eukaryotic cell.

[0189] Examples of prokaryotic host cells that may be used include bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Brevibacillus such as Brevibacillus agri, the genus Serratia such as Serratia liquefaciens, the genus Bacillus such as Bacillus subtilis, the genus Microbacterium such as Microbacterium ammoniaphilum, the genus Brevibacterium such as Brevibacterium divaricatum, the genus Corynebacterium such as Corynebacterium ammoniagenes, and the genus Pseudomonas such as Pseudomonas putida. Examples of vectors that may be used with a prokaryotic host cell include pBTrp2, pGEX, pUC18, pBluescriptll, pSupex, pET22b, pCold, pUBUO, and pNCO2.

[0190] In terms of eukaryotic cells, the host cell may be, for example, a yeast, a filamentous fungi, an insect cell, an animal cell, or a plant cell. Examples of vectors that may be used with a eukaryotic host cell include YEpl3 (ATCC37115) and YEp24 (ATCC37051).

[0191] Any suitable method for introducing an expression vector into the host cell may be used. For example, methods may include the use of calcium ions, electroporation, lithium acetate, or cell competency methods. The method of the fifth aspect may comprise culturing the transfected host cell in a suitable culture medium and under suitable conditions. Any medium and conditions suitable for culturing the host cell may be used.

[0192] In accordance with a ninth aspect of the invention, there is provided a method of treating or preventing an inflammatory skin condition, the method comprising topically administering a composition comprising a serine protease derived from M. luteus to a site of an inflammatory skin condition on a subject.

[0193] The inflammatory skin condition may be as defined in accordance with the second aspect.

[0194] The serine protease may be as defined in accordance with the first or second aspect.

[0195] The M. luteus may be as defined in accordance with the first or second aspect.

[0196] The composition may be as defined in accordance with the first or second aspect, and / or may be manufactured as defined in accordance with the fourth or fifth aspect.

[0197] The composition may be administered alone or in combination with one or more other substances or compositions.

[0198] Administration is preferably in a prophylactically or therapeutically effective amount, this being an amount sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated or prevented, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &. Wilkins. It will be appreciated by the skilled person that appropriate dosages of the active compounds and compositions comprising the active compounds can vary from patient to patient. The compositions of the present invention may be formulated as medicaments, that is to say formulated as a medicine or a medical device. The medicament may include other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilisers, solubilisers, surfactants (e.g. wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example other therapeutic or prophylactic agents.

[0199] The compositions of the present invention may be formulated as cosmetics, that is to say formulated as a cosmetics product. The cosmetics product may include other cosmetically acceptable ingredients well known to those skilled in the art, including, but not limited to, cosmetically acceptable carriers, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g. wetting agents), masking agents, colouring agents, fragrance agents.

[0200] Examples

[0201] The invention will now be illustrated by reference to specific Examples showing how embodiments may be carried into effect, which are not intended to be limiting.

[0202] The following abbreviations may be used :

[0203] NHEK Normal human epidermal keratinocytes CFCS Cell-free culture supernatant FSA Filtered Staphylococcus aureus supernatant TSLP Thymic stromal lymphopoietin Sbi Second immunoglobulin-binding protein

[0204] Data from the Examples is presented in the Figures, in which:

[0205] Figure 1 shows the effect of skin bacteria CFCS on IL-33 and TSLP release in FSA stimulated NHEK. NHEK were treated with FSA or co-treated with FSA and skin bacteria CFCS for 24 hr before quantifying IL-33 and TSLP in cell culture medium using ELISA. Stimulation of NHK with FSA caused an increase in IL-33 and TSLP release. This was not affected by co-treatment with S. aureus (A), S. capitis (C), S. caprae (D), S. lugdunensis (E), S. epidermidis (F), S. auricularis (G) or S. saprophyticus (H) CFCS. Co-treatment with M. luteus (B) CFCS negated FSA- induced release of IL-33 and TSLP. Data are expressed as mean ± SEM (n>3). P values determined by one-way ANOVA * P < 0.05 ** p < 0.01 **** p < 0.0001 compared with FSA treated NHEK.

[0206] Figure 2 shows the importance of tryptophan in the reduction of FSA-induced IL-33 and TSLP release in NHEK. FSA stimulated NHEK were treated with either CFCS from M. luteus grown in tryptophan-free medium (A) or 0.05% tryptophan medium (B) for 24 hr. IL-33 and TSLP in cell culture medium were quantified using ELISA. CFCS from M. luteus grown in tryptophan-free medium supressed FSA-induced release of IL-33 and TSLP (A). Tryptophan had no effect (B). Data are expressed as mean ± SEM (n>3). P values determined by one-way ANOVA ** p < 0.01 *** P < 0.001 **** p < 0.0001 compared with FSA treated NHEK.

[0207] Figure 3 shows the effect of M. luteus type strain NCTC 2665 on FSA-induced IL- 33 and TSLP release. M. luteus NCTC 2665 was cultured for 96 hr before collecting CFCS. NHEK were co-cultured with FSA and CFCS from M. luteus NCTC 2665 for 24 hr before measuring IL-33 and TSLP in the cell culture medium using ELISA. CFCS from M. luteus NCTC 2665 had no effect on FSA-induced IL-33 and TSLP release in NHEK. Data are expressed as mean ± SEM (n = 3). P values determined by one-way ANOVA *** P < 0.001 **** p < 0.0001 compared with FSA treated NHEK.

[0208] Figure 4 shows the effects of M. luteus CFCS harvested at different time points on TSLP (A) and IL-33 (B) release in FSA stimulated NHEK. M. luteus was cultured for 1, 2, 4, 6 and 24 hr before harvesting CFCS. NHEK were co-cultured with FSA and M. luteus CFCS for 24 hr before measuring IL-33 and TSLP in cell culture medium using ELISA. Data are expressed as mean ± SEM (n>4). P values determined by one-way ANOVA * P < 0.05 ** P < 0.01 *** P < 0.001 **** P < 0.0001 compared with FSA treated NHEK.

[0209] Figure 5 shows the effects of heat treatment (A) and protein precipitation (B) on the bioactivity of the M. luteus secretome. (A) M. luteus CFCS was heat treated to 85 °C for 10 min before testing it for activity against FSA-induced IL-33 and TSLP release in NHEK. (B) Proteins within M. luteus CFCS were precipitated using acetone, then reconstituted in cell culture medium before testing for activity using the same model. Data are expressed as mean ± SEM (n=3). P values determined by one-way ANOVA **** p < 0.0001 compared with FSA treated NHEK.

[0210] Figure 6 shows the effect of M. luteus CFCS on recombinant IL-33 and TSLP. Recombinant IL-33 or TSLP were treated with either M. luteus CFCS or a medium control before quantifying the concentration of remaining IL-33 or TSLP within the sample using ELISA. Data are expressed as mean ± SEM (n = 3). P values determined by unpaired t test ** p < 0.01 compared with untreated IL-33 or TSLP control.

[0211] Figure 7 shows the effect of M. luteus CFCS on IL-33 is blocked by a serine protease inhibitor. M. luteus CFCS was pre-treated with the serine protease inhibitor, AEBSF. Recombinant IL-33 was then treated with either AEBSF treated M. luteus CFCS or untreated M. luteus CFCS. Data are expressed as mean ± SEM (n = 3). P values determined by one-way ANOVA ** p < 0.01 compared with untreated IL-33 control.

[0212] Figure 8 shows western blot analysis of the effect of M. luteus CFCS on Sbi. (A) Purified Sbi was treated for 1 hr with either IX, 0.5X or 0.25X concentration of M. luteus CFCS before performing western blot analysis. (B) The intensity of the Sbi band (51 kDa) was quantified using Image Lab and presented as relative quantity compared to the untreated control band. (C) M. luteus CFCS was pre-treated with the serine protease inhibitor AEBSF before using it to treat purified Sbi for 1 hr. Sbi was also treated with AEBSF for 1 hr as a control. (D) The intensity of the Sbi band was quantified using Image Lab and presented as relative quantity compared to the untreated control band. MLS: M. luteus CFCS. Western blot images A and C are representative of 3 and 4 individual experiments respectively. (B / D) Data are expressed as mean ± SEM (n> 3). P values determined by Kruskal-Wallis test * P < 0.05 compared with the control.

[0213] Figure 9 shows visualisation of M. luteus CFCS protein profile. The total protein content of M. luteus CFCS was separated by gel electrophoresis alongside a Spectra ™ Multicolor Broad Range Protein Ladder. Gels were stained with silver stain to visualise protein bands.

[0214] Figure 10 shows the bioactivity of M. luteus CFCS molecules separated by their molecular weight. SEC was used to separate molecules within M. luteus CFCS into fractions based on their molecular weight. Alternating fractions from 10-38 were assessed for bioactivity against IL-33 (A) and TSLP (B) release in FSA-stimulated NHEK as previously described. Peak activity against both IL-33 and TSLP is seen in fractions 14-18 and 26 (highlighted with rectangles). The data are representative of one out of three repeats. Figure 11 shows the bioactivity of M. luteus CFCS molecules separated by their net charge. IEX was used to separate molecules within M. luteus CFCS into fractions based on their net charge. Fractions from 13-32 were assessed for bioactivity against recombinant IL-33 and IL-33 release in FSA-stimulated NHEK as previously described. The loading sample and flow through were also tested for activity as controls to confirm that the active molecule was present in the loading sample and was eluted from the IEX column into the fractions. Each graph represents one individual experiment.

[0215] Figure 12 shows a representation of the serine protease sequence in the skin isolated M. luteus (SEQ ID NO: 1) and the corresponding sequences in the type strain M. luteus NCTC 2665 (SEQ ID NO:3 and SEQ ID NO:4). The gene encoding serine protease in the M. luteus NCTC 2665 genome contains a frame shift mutation which is predicted to result in two truncated proteins.

[0216] Figure 13 shows the effect of a recombinant serine protease (PADP) designed using the effective skin isolated sequence on IL-33 release compared to a type strain mutant PADP which was designed using the sequence from the ineffective strain of M. luteus including all mutations except for the frame shift mutation.

[0217] Examples

[0218] The inventors have identified members of the skin microbiota able to attenuate cutaneous inflammation. Cell-free culture supernatants (CFCS) of bacteria isolated from the skin of healthy volunteers were screened, using enzyme-linked immunosorbent assay, for the ability to reduce IL-33 and TSLP secretion from NHEK stimulated with filtered S. aureus supernatant (FSA). Surprisingly, an isolate of Micrococcus luteus (M. luteus) completely negated FSA-induced release of IL-33 and TSLP from NHEK (p<0.0001). Treatment of recombinant IL-33 and TSLP with M. luteus CFCS resulted in degradation of IL-33 but not TSLP. Western blot analysis demonstrated the dose-dependent degradation of Sbi when treated with M. luteus CFCS. The activity against IL-33 and Sbi was blocked by a serine protease inhibitor. Liquid chromatography-mass spectrometry analysis of active M. luteus CFCS fractions separated by size exclusion and ion exchange chromatography identified a protein belonging to the subtilisin class of serine proteases. Variant calling of genomic DNA sequences revealed a frame shift mutation is present in the serine protease sequence of an ineffective M. luteus type strain, resulting in a truncated protein. This suggests that the protein is the extracellular serine protease responsible for the activity against IL-33 and Sbi, which ultimately attenuates the secretion of inflammatory cytokines.

[0219] Example 1 : CFCS from M. luteus isolated from human skin negates S. aureus- induced release of IL-33 and TSLP in NHEK

[0220] To investigate the potential anti-inflammatory effects of skin isolates against pathogen-induced / AD type cutaneous inflammation, an NHEK-based model of S. aureus-induced inflammation was used. NHEK were co-treated with FSA and CFCS from a representative panel of eight skin isolates for 24 hr before quantifying IL-33 and TSLP in the cell culture medium by ELISA. The data in Figure 1 demonstrates that treatment of NHEK with FSA causes an increase in IL-33 and TSLP release. No change in IL-33 or TSLP release was observed when NHEK were co-treated with FSA and CFCS from S. aureus, S. auricularis, S. lugdunensis, S. epidermidis, S. saprophyticus, 5. capitis or S. caprae compared to FSA treatment alone. Cotreatment with CFCS from M. luteus negated FSA-induced release of IL-33 and TSLP from NHEK.

[0221] The M. luteus strain was subsequently deposited at the NCTC depositary institution under Accession Number 22110401.

[0222] Example 2: Inhibition of FSA-induced release of IL-33 and TSLP from NHEK by M. luteus CFCS is not tryptophan dependent

[0223] To investigate whether the inhibition of IL-33 and TSLP release observed when treating FSA stimulated NHEK with M. luteus CFCS was tryptophan dependent, CFCS from M. luteus grown in tryptophan-free medium was assessed for activity using the same model. The data in Figure 2 shows that CFCS from M. luteus grown in tryptophan-free medium was able to significantly reduce the release of FSA- induced IL-33 and TSLP from NHEK. Treating FSA-stimulated NHEK with 0.05% tryptophan medium had no effect on IL-33 and TSLP release.

[0224] Example 3: The ability to supress FSA-induced IL-33 and TSLP release in NHEK is not an attribute of all M. luteus strains

[0225] To investigate whether the observed activity was a general attribute of M. luteus or unique to specific strains found on the skin, an M. luteus type strain was tested for activity. M. luteus NCTC 2665 was cultured for 96 hr before collecting CFCS. NHEK were co-cultured with FSA and M. luteus NCTC 2665 for 24 hr before measuring IL- 33 and TSLP in the cell culture medium using ELISA. The data in Figure 3 shows that CFCS from the M. luteus type strain had no effect on FSA-induced released of IL-33 and TSLP in NHEK.

[0226] Example 4: The bioactive molecule is produced in the lag phase of the M. luteus growth cycle

[0227] There are four distinct phases in a bacterial growth cycle: lag phase, the temporary period of non-replication where the bacteria are adjusting to their new environment; exponential phase, where cell numbers increase exponentially; stationary phase, where the rate of cell growth is equal to the rate of cell death; and death phase, where the number of viable cells decreases exponentially. Bacteria generally produce different types of molecules within different phases of growth, and so to understand more about the active molecule, the inventors made investigations into the stage in the M. luteus growth cycle in which it is produced.

[0228] FSA-stimulated NHEK were treated with CFCS collected from M. luteus grown for 1, 2, 4, 6 and 24 hr before measuring IL-33 and TSLP in the cell culture medium using ELISA. The data in Figure 4 shows that CFCS from M. luteus grown for 1 hr supressed IL-33 and TSLP release somewhat, but full inhibition was observed by CFCS from M. luteus cultured for 4 hr or longer.

[0229] To understand what stage of the M. luteus growth cycle this time point corresponds to, a growth curve was generated. Two hundred microlitres of diluted M. luteus culture was added to a 96 well plate in triplicate and cultured in a plate reader for the OD to be read every 0.5 hr for 48 hr. The found that M. luteus is in the lag phase of growth until 7 hr incubation.

[0230] Example 5: the bioactivity of M. luteus CFCS is destroyed by heat treatment and is retained in an acetone precipitate

[0231] To learn more about the identity of the bioactive molecule in M. luteus CFCS, investigations into its properties were made. Possible identities of the bioactive molecule include a protein, lipid, carbohydrate, or a conjugate. These molecules can generally be differentiated by their thermal stability. The majority of proteins in mesophilic bacteria will become denatured by temperatures of 60 °C, and the number of proteins stable past 80 °C is minimal. In contrast, it is believed that lipids and carbohydrates are thermally stable, at least up to temperatures of 100 °C. To investigate the heat stability of the bioactive molecule, M. luteus CFCS was heated treated to 85 °C for 10 min and then tested for bioactivity using the FSA- stimulated NHEK model. The data in Figure 5A shows that the bioactivity of M. luteus CFCS was destroyed after heat treatment.

[0232] Proteins, lipids, and carbohydrates can also be distinguished from one another by their solubility in organic solvents. Lipids are highly soluble in organic solvents such as acetone, whereas proteins and carbohydrates are largely insoluble. Acetone precipitation is a widely used method to separate proteins from organic-soluble contaminants, and yields a higher quantity of protein than alternative methods. The data in Figure 5B shows that the bioactivity of M. luteus CFCS was retained in the precipitate formed by acetone precipitation.

[0233] Example 6: M. luteus CFCS degrades IL-33 but not TSLP

[0234] To investigate the mechanism of action of the observed bioactivity, recombinant IL- 33 and TSLP were independently treated with M. luteus CFCS to investigate whether the reduction of IL-33 and TSLP in FSA-stimulated NHEK was due to M. luteus CFCS acting directly on these cytokines. The data in Figure 6 demonstrates the quantity of IL-33 and TSLP after treatment with M. luteus CFCS compared to untreated controls as determined by ELISA. M. luteus CFCS had no effect on TSLP but significantly reduced the concentration of IL-33. The degradation of IL-33 by M. luteus CFCS is indicative of protease activity.

[0235] Example 7: A putative serine protease of the M. luteus secretome degrades IL-33 Proteolysis was observed when treating IL-33 with M. luteus CFCS. To identify the mechanism of proteolysis and therefore understand more about the identity of the bioactive protein, the role of proteases in the degradation of IL-33 by M. luteus CFCS was investigated..

[0236] M. luteus CFCS was pre-treated with the irreversible serine protease inhibitor AEBSF. Recombinant IL-33 was then treated with either AEBSF treated M. luteus CFCS or untreated M. luteus CFCS before quantifying the IL-33 concentration by ELISA. The data in Figure 7 demonstrates that the degradation of IL-33 treated with M. luteus CFCS is inhibited by AEBSF. Thus, strongly suggesting the role of serine proteases in the degradation of IL-33 by M. luteus CFCS.

[0237] Example 8: A putative serine protease of the M. luteus secretome degrades S. aureus virulence factor Sbi in a dose dependent manner

[0238] To further understand the mechanism of action of the observed bioactivity, the effect of M. luteus CFCS on FSA was investigated. Sbi is the S. aureus secreted virulence factor present in FSA that is responsible for the release of IL-33 and TSLP in NEHK. As protease activity has been identified in the M. luteus CFCS, the inventors tested whether Sbi could also be degraded. Sbi has a molecular weight of ~50 kDa. Sbi was purified from FSA by immunoprecipitation using IgG, as confirmed by the presence of a 51 kDa band by western blot (Figure 8A). Purified Sbi was then treated with different dilutions of M. luteus CFCS for 1 hr before performing western blot analysis. The data in Figure 8A and Figure 8B demonstrate that treatment of Sbi with M. luteus CFCS causes degradation of Sbi in a dose dependent manner. The data in Figure 8C and Figure 8D demonstrate that pre-treatment of M. luteus CFCS with the irreversible serine protease inhibitor AEBSF for 1 hr before treating Sbi inhibited Sbi degradation, thus, strongly suggesting that M. luteus can degrade Sbi via serine protease activity. The bands of a smaller molecular weight in Figure 8A in IX and 0.5X M. luteus CFCS treated Sbi are likely to be cleaved fragments of Sbi due to the proteolytic cleavage by a serine protease.

[0239] Example 9: Visualisation of the M. luteus CFCS protein profile As the data produced thus far suggested that the efficacious molecule was a protein, the proteins within M. luteus CFCS were separated using SDS-PAGE and visualised using Coomassie staining. Initially, no bands were visualised due to the low protein concentration (data not shown). The process was then repeated using CFCS that had been concentrated using acetone precipitation. As the concentration of proteins in the CFCS was still too low to visualise a full protein profile by Coomassie staining, silver staining was also used due to it being a more sensitive method. An image of the protein bands detected using silver staining can be seen in Figure 9. Multiple bands were present, including the ~90 kDa protein previously visualised by Coomassie staining. The ~90 kDa unknown protein band was excised from the gel for LC-MS analysis.

[0240] Example 10: Fractionation of M. luteus CFCS using size exclusion chromatography To understand more about the connection between the activity against IL-33 and TSLP, and help identify the molecule(s) responsible, M. luteus CFCS was separated into fractions based on molecular weight using size exclusion chromatography (SEC). The process of SEC dilutes the sample and so the CFCS was concentrated using acetone precipitation before performing SEC. The elution of proteins was not observed until fraction 10 and the presence of specific proteins generally occurs across neighbouring fractions, so alternating fractions starting from fraction 10 were tested for activity to save time and resources. Each fraction was incubated with FSA-stimulated NHEK to identify any activity against IL-33 and TSLP release. The data in Figure 10 shows that the activity profile of the fractions was similar for both IL-33 and TSLP. A gradient of activity was seen across multiple fractions, but peak activity, similar to that observed in previous experiments, was seen in fractions 14-18 and fraction 26 (highlighted with rectangles). Fractions 14, 15, 26 and 27 were therefore sent for analysis by LC-MS to identify the proteins present.

[0241] Example 11: Identification of proteins within active SEC fractions using LC-MS In an attempt to identify the bioactive protein, LC-MS was used to identify all proteins within the active SEC fractions 14, 15, 26 and 27. In total, 51 proteins were identified within the samples. It was assumed that the bioactive protein must be present in all active fractions. Ten proteins were present in all four active SEC fractions. The identified proteins with the highest number of peptide matches across all samples and therefore the most certain identifications were the disclosed serine protease and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). LC-MS analysis also confirmed the identity of the excised ~90 kDa band from Figure 9 to be the disclosed serine protease.

[0242] Example 12: Fractionation of M. luteus CFCS using ion exchange chromatography To reduce the total number of identified proteins within the active fractions and therefore narrow down the list of possible identities of the bioactive protein, the active SEC fractions were pooled together to further separate the proteins based on their net charge using ion exchange chromatography (IEX). The resulting fractions were then tested for activity against FSA-induced IL-33 and TSLP in NHEK. The process of both SEC and IEX diluted the sample to the extent that no activity was observed in any of the fractions.

[0243] To avoid loss of activity via sample dilution, the whole concentrated CFCS was instead passed through the ion exchange column before assessing activity in FSA- stimulated NHEK. SEC and IEX separate proteins based on different properties, resulting in fractions containing different protein profiles. It was assumed that the bioactive protein must be present within all active SEC and IEX fractions, and so to narrow down the list of possible bioactive protein candidates LC-MS was used to identify the proteins present in active IEX fractions to compare with the protein profile of the active SEC fractions. IEX fractions were tested for activity against recombinant IL-33 and IL-33 release in FSA-stimulated NHEK using ELISA to compare the activity profiles. The data in Figure 11 demonstrates that the activity profile of IEX fractions against FSA-induced IL-33 release in NHEK is identical to activity against recombinant IL-33. Activity was observed in fractions 17-32.

[0244] Example 13: The disclosed serine protease is present in all active M. luteus CFCS fractions

[0245] Of the IEX fractions tested on recombinant IL-33 (Figure 11), fractions 17, 18 and 32 were selected as representative fractions to undergo LC-MS analysis to identify all the proteins eluted in the first and the last active fractions. A total of 32 proteins were identified in the fractions. It was assumed that the bioactive protein must be present within all active IEX fractions. Eight proteins were present in all three active IEX fractions analysed. Other than GAPDH, the putative serine protease was the only protein identified in all active SEC fractions and IEX fractions.

[0246] Example 14: A frame shift mutation is present in the serine protease gene in the inactive M. luteus NCTC 2665 type strain

[0247] To further implicate the disclosed serine protease as the protein responsible for M. luteus CFCS activity, whole genome sequencing was performed for the bioactive skin isolated M. luteus and the inactive type strain M. luteus NCTC 2665 to investigate differences in the genome sequence encoding the serine protease.

[0248] The NCTC 2665 type strain genome sequence was aligned to the skin isolated M. luteus genome sequence as a reference to identify variations by bioinformatics analysis. Multiple non-synonymous coding mutations were identified in the M. luteus NCTC 2665 type strain serine protease encoding gene, as well as a frame shift mutation which is predicted to result in two truncated proteins as shown in Figure 12. The frame shift and non-synonymous coding mutations and the resulting amino acid substitutions are listed in Table 1.

[0249] Table 1

[0250] Table 1 shows a frame shift mutation and multiple non-synonymous coding mutations in the serine protease encoding gene are present in the inactive type strain M. luteus NCTC 2665 genome when compared to the active skin isolated M. luteus strain. The table summarises the resulting amino acid change and location.

[0251] Example 15: A 'type strain mutant PADP' missing the frame shift mutation was ineffective at negating IL-33 release

[0252] A 'type strain mutant PADP' was designed using the sequence from the ineffective strain of M. luteus including all mutations except for the frame shift mutation (which is predicted to result in two truncated proteins). Recombinant IL-33 was then treated with either a recombinant serine protease (PADP) designed using the effective skin isolated sequence or the 'type strain mutant PADP' serine protease, before quantifying the IL-33 concentration by ELISA. The data in Figure 13 demonstrates that recombinant serine protease PADP was capable of degrading IL33. However, the 'type strain mutant PADP' was still ineffective at degrading IL- 33 despite missing the frame shift mutation.

[0253] Materials and Methods

[0254] Bacteria strains

[0255] Micrococcus luteus NCTC 2665 was purchased from the National Collection of Type Cultures (NCTC) (Public Health England, UK) and methicillin-sensitive S. aureus isolated from a chronic wound was gifted from Professor Andrew McBain, The University of Manchester. All other species were obtained from an internal library of bacteria species isolated from the skin of healthy volunteers by Faye Adel Aldehalan as described in a previous doctoral thesis from The University of Manchester (study approved by University of Manchester Ethics committee; 2019-6208-10419) (Aldehalan, 2023). In short, bacteria were collected by swabbing the scalp, forehead, volar forearm, and toe web of five healthy volunteers. Swabs were plated onto Tryptic Soy Agar (TSA) supplemented with 5% sheep's blood (VWR, UK), Mannitol Salt Agar (MSA), MacConkey agar (MAC), Wilkins Chalgren agar (WCA), Lactobacilli MRS agar (MRS), and Nitrofuran containing agar (FTO), to isolate aerobic bacteria, mannitol fermenting Staphylococci, gram-negative bacteria, anaerobic bacteria, Lactobacilli, and Micrococcus and Corynebacterium respectively. Pure colonies were identified using 16S rRNA gene sequencing at the Sanger sequencing facility at The University of Manchester and stored as a collection at - 80°C.

[0256] Storage of bacteria

[0257] Fresh bacterial stocks were created from the library of skin isolates. Bacteria were streaked on Tryptic Soy Agar (TSA) (Sigma-Aldrich, Gillingham, UK) and incubated for 48 hr at 37°C. Sufficient colonies to achieve a 3-4 McFarland standard were used to inoculate the fluid in the Pro-Lab DiagnosticsTM MicrobankTM Bacterial Preservation System (Thermo Fisher Scientific, Loughborough, UK). The vial was inverted 3-4 times and excess fluid aspirated and discarded before storing at - 80°C. Prior to experiments, one bead from the MicrobankTM vial was streaked onto TSA plates and incubated for 48 hr at 37°C to ensure that cultures were pure before selecting single colonies to inoculate liquid cultures.

[0258] Preparation of growth media for bacterial culture

[0259] Bacteria were cultured in either Luria Broth (LB) (Sigma-Aldrich), Nutrient Broth (Thermo Fisher Scientific), Tryptic Soy Broth (TSB) (Sigma-Aldrich) or custom made RPMI 1640 w / o: L-Glutamine, w / o: L-Tryptophan, w: 2.0 g / L NaHCCh, w / o Phenol Red (PAN Biotech UK Ltd, Dorset, UK) supplemented with L-Glutamine 200 mM (300 mg / L) (PAN Biotech UK Ltd) with or without additional 0.05% L- Tryptophan (Sigma-Aldrich).

[0260] Whole genome sequencing

[0261] Whole genome sequencing (WGS) was provided by MicrobesNG

[0262] (http : Z / www. microbesnq . com) . Pure cultures of each strain were grown in TSB to exponential phase. Cells were harvested and resuspended to the equivalent of 8-12 OD 600 nm in a tube containing 0.5 ml DNA / RNA Shield (Zymo Research, USA) following MicrobesNG strain submission procedures, and sent to MicrobesNG for sequencing. To extract DNA, 5 to 45 pl of the cell suspension were lysed with 120 pl of Tris-EDTA (TE) buffer containing lysozyme at a final concentration 0.1 mg / ml, and RNase A (ITW Reagents, Barcelona, Spain) at a final concentration 0.1 mg / ml. The sample was incubated for 25 min at 37°C. Proteinase K (VWR Chemicals, Ohio, USA) at a final concentration O. lmg / mL and SDS (Sigma-Aldrich, Missouri, USA) at a final concentration 0.5% v / v were added and incubatedfor 5 min at 65°C. Genomic DNA was purified using an equal volume of Solid-phase reversible immobilisation (SPRI) beads and resuspended in EB buffer (lOmM Tris-HCI, pH 8.0). DNA was then quantified with the Quant-iT dsDNA HS kit (Thermo Fisher Scientific) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd, United Kingdom) and diluted as appropriate.

[0263] Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) following the manufacturer's protocol with the following modifications: input DNA was increased 2-fold, and polymerase chain reaction (PCR) elongation time was increased to 45 sec. v20230314 1 Genome Sequencing Methods DNA quantification and library preparation was carried out on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland). Libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, USA) using a 250 bp paired end protocol. Reads were adapter trimmed using Trimmomatic version 0.30 with a sliding window quality cutoff of Q15. De novo assembly was performed on samples using SPAdes version 3.7, and contigs annotated using Prokka 1.11. Variant calling against a selected isolate was performed using VarScan to identify genome mutations and single nucleotides polymorphisms (SNPs).

[0264] Normal human epidermal keratinocyte culture

[0265] Primary NHEK (PromoCell, Heidelberg, Germany) were isolated from the epidermis of juvenile foreskin from pooled donors and grown in Keratinocyte Growth Medium 2 (KGM) (PromoCell) supplemented with KGM SupplementMix (Promocell) at 37°C in a humidified atmosphere of 5% CO2. NHEK were maintained in T75 tissue culture flasks and medium was replaced every 2 days to achieve ~90% confluency. For detachment, cells were washed with Dulbecco's Phosphate-Buffered Saline (PBS) (Sigma-Aldrich) then incubated with 6 ml of Trypsin / EDTA 0.04% / 0.03% (PromoCell) at 37°C for 5 min. After cell detachment, 6 ml of Trypsin Neutralising Solution (PromoCell) was added, and cells were harvested by centrifugation at 170 ref for 3 min. Cells were counted using a haemocytometer and seeded into 24-well plates at 5 x 104cells / well. Cells of passages 2-5 were used for experiments.

[0266] Preparation of cell-free culture supernatant

[0267] Single colonies of skin isolates were used to inoculate 5 ml of custom RPMI 1640 with 0.05% tryptophan and incubated in the dark for 96 hr at 37°C with shaking (100 rpm) unless stated otherwise. M. luteus cultures were grown for 24 hr unless stated. Cultures were centrifuged at 2706 ref for 10 min and filter sterilised using 0.22 pm syringe filters. Cell-free culture supernatant (CFCS) was made fresh before each experiment.

[0268] Preparation of FSA

[0269] Nutrient broth was inoculated with a single Microbank™ bead from a stock of methicillin-sensitive S. aureus isolated from a chronic wound and incubated overnight at 37°C. A total of 108CFU were washed with PBS then resuspended in 10 ml KGM. Five ml were added to 15 ml fresh KGM and incubated for 6 hr at 37°C with shaking. After incubation, samples were centrifuged at 1600 ref for 5 min then filter sterilised using 0.22 pm filters (Starlab UK Ltd, England) and stored at -80°C until required for stimulation experiments.

[0270] NHEK inflammation assay

[0271] NHEK cultures were maintained and cultured in 24-well tissue culture plates at a density of 5 x 104cells / well as described in (2.5). Once NHEK reached 70-90% confluence, media was removed, and the cells were stimulated with either 11-10 or FSA to induce an inflammatory response. Stimulated cells were co-cultured with 250 pl bacteria CFCS for up to 24 hr at 37°C in 5% CO2. NHEK culture medium was then removed and centrifuged at 10,000 ref for 15 min to remove cell debris before storing at -20°C for downstream analysis.

[0272] Enzyme-linked immunosorbent assay

[0273] To quantify IL-8, IL-33 and TSLP in NHEK conditioned medium, human IL-8 (R&D systems, Abingdon, UK) human IL-33 (R&D systems) and human TSLP (R8dD Systems) DuoSet ELISA Kits were used according to manufacturer's instructions. Flat bottom 96-well plates (Greiner Bio-One, Kremsmunster, Austria) were coated with either IL-33 or TSLP capture antibody overnight, washed with wash buffer (lx PBS containing 0.05% Tween 20) 3 times with no aspiration using the Tecan HydroFlex Plus microplate washer ( Tecan Group Ltd. Switzerland) and blocked with lx Reagent Diluent (R8iD systems) for 1 hr. Serial dilutions of recombinant standards were prepared in reagent diluent (IL-33: 11.7-1500 pg / ml, TSLP: 15.62- 2000 pg / ml). Standards and samples were added to plates in triplicate and incubated for 1 hr. This was followed by three washes and incubation with either IL- 33 or TSLP detection antibodies for 1 hr. Plates were washed three times, Streptavidin-Horseradish peroxidase (HRP) added and incubated for 20 min avoiding direct light. Plates were washed three times and 3, 3, 5, 5- Tetramethylbenzidine (TMB) liquid substrate solution (Sigma-Aldrich) added for 20 min followed by Sulfuric acid : 1 M H2SO4 (2N) (Honeywell™, Thermo Fisher Scientific) to stop the reaction. The OD for each well was measured at 450 nm using the CLARIOstar plate reader.

[0274] Generation of M. luteus growth curve

[0275] A single colony of M. luteus was inoculated in 5 ml RPMI 1640 and incubated overnight at 37°C with shaking. A 1 : 100 dilution was prepared in fresh RPMI 1640 and 200 pl of this added to a 96 well plate in triplicate alongside blank wells containing RPMI 1640. covered with a Breathe-Easy sealing membrane (Sigma- Aldrich) and incubated at 37°C for 48 hr in a CLARIOstar plate reader. OD was read at 660 nm every 30 min after 5 sec shaking. Data was analysed using GraphPad Prism 10 software (obtained from http: / / www.graphpad.com) (GraphPad Software Inc., California, USA) to plot bacterial growth curves.

[0276] Generation of 5. aureus growth curve in the presence of M. luteus CFC5

[0277] A single colony of S. aureus was inoculated in 5 ml TSB and incubated overnight at 37°C with shaking. A 1 : 10 dilution of overnight culture was prepared in fresh TSB to a total volume of 1 ml, before removing 250 pl and replacing with 250 pl M. luteus CFCS or RPMI 1640 as a control. Of this mixture, 200 pl was added to a 96 well plate in triplicate alongside blank wells containing diluted TSB only, covered with a Breathe-Easy sealing membrane (Sigma-Aldrich) and incubated at 37°C for 24 hr in a CLARIOstar plate reader. OD was read at 660 nm every 30 min after 5 sec shaking. Data was analysed using GraphPad Prism 10 software to plot bacterial growth curves.

[0278] Acetone precipitation of proteins

[0279] 99.9% Acetone (Fisher Scientific) chilled to -20°C was added to bacterial supernatant at a ratio of 4: 1. The sample was then vortexed and incubated at - 20°C for 1 hr. Following incubation, the sample was centrifuged for 10 min at 15,000 ref before decanting the supernatant and allowing the remaining acetone to evaporate. Protein precipitate was resuspended in either NHEK media or PBS to the required concentration.

[0280] Protein separation of M. luteus secretome by gel electrophoresis

[0281] M. luteus CFCS was concentrated via acetone precipitation before quantifying the total protein concentration using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) as per manufacturer's instructions. 10 pg of concentrated M. luteus CFCS was added to 4x NuPAGE™ LDS sample buffer and then heated to 70°C for 10 min before putting on ice. Samples were then loaded in NuPAGE™ 4-12% Bis-Tris gel (Invitrogen), alongside 5pl of Spectra™ Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific). Proteins were run at 200V for 45 min in lx NuPAGE™ MOPS SDS running buffer (Invitrogen). The gels were stained with either InstantBlue (Gentaur UK Ltd, Potters Bar, UK) Coomassie stain overnight or using The SilverQuest Silver Staining Kit (Invitrogen) as per manufacturer's instructions using the basic staining protocol with an overnight fixation step.

[0282] Chromatography fractionation of M. luteus secretome

[0283] Proteins within M. luteus CFCS were concentrated using acetone precipitation in preparation for chromatography. Concentrated supernatant was fractionated by either size exclusion chromatography (SEC) or by ion exchange chromatography (IEX) using Q anion exchange columns at The University of Manchester, Biomolecular Analysis Core Facility according to their proprietary methods.

[0284] Fractions were sterilised using 0.22 pm syringe filters before analysing their activity in FSA-stimulated NHEKs using ELISA (2.13, 2.14).

[0285] Identification of potential active proteins using LC-MS

[0286] In preparation for LC-MS, fractions with activity in FSA-stimulated NHEKs were added to 4x NuPAGE® LDS sample buffer (Novex, Life Technologies), vortexed then heated at 70 °C for 10 min before transferring to ice. Samples were loaded into a NuPAGE™ 10%, Bis-Tris gel and run into the top 5 mm for 5 min at 200V. The gel was stained with Coomassie blue then stored in dH2O at 4 °C overnight before submitting to The University of Manchester, Biological Mass Spectrometry Facility for in-gel digestion and LC-MS analysis.

[0287] In-gel digestion

[0288] Bands of interest were excised from the gel and dehydrated using acetonitrile followed by vacuum centrifugation. Dried gel pieces were reduced with 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide. Gel pieces were then washed alternately with 25 mM ammonium bicarbonate followed by acetonitrile. This was repeated, and the gel pieces dried by vacuum centrifugation. Samples were digested with trypsin overnight at 37 °C.

[0289] LC-MS analysis

[0290] Digested samples were analysed by LC-MS / MS using an UltiMate® 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to a QE HF (Thermo Fisher Scientific, Waltham, MA) mass spectrometer. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile and the column used was a 75 mm x 250 pm i.d. 1.7 mM CSH C18, analytical column (Waters).

[0291] A 1 pl aliquot of the sample was transferred to a 5 pl loop and loaded on to the column at a flow of 300 nl / min for 5 min at 5% B. The loop was then taken out of line and the flow was reduced from 300 nl / min to 200 nl / min in 0.5 min. Peptides were separated using a gradient that went from 5% to 18% B in 34.5 min, then from 18% to 27% B in 8 min and finally from 27% B to 60% B in 1 min. The column is washed at 60% B for 3 min before re-equilibration to 5% B in 1 min. At 55 min the flow is increased to 300 nl / min until the end of the run at 60 min.

[0292] Mass spectrometry data was acquired in a data directed manner for 60 min in positive mode. Peptides were selected for fragmentation automatically by data dependant analysis on a basis of the top 12 peptides with m / z between 300 to 1750Th and a charge state of 2, 3 or 4 with a dynamic exclusion set at 15 sec. The MS Resolution was set at 120,000 with an AGC target of 3e6 and a maximum fill time set at 20 ms. The MS2 Resolution was set to 30,000, with an AGC target of 2e5, a maximum fill time of 45 ms, isolation window of 1.3Th and a collision energy of 28.

[0293] Testing M. luteus CFC5 for serine protease activity

[0294] Whilst performing an IL-33 ELISA as described in 2.14, M. luteus CFCS was pretreated with 1 mM of the serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) (Sigma-Aldrich) for 1 h before adding to 50 pl of 1500 pg / ml IL-33 standard. This was added to ELISA plate wells in triplicate after the block step and incubated for 1 hr before continuing the ELISA.

[0295] Immunoprecipitation of Sbi

[0296] Prior to immunoprecipitation, Sbi was concentrated by fractionating FSA using an Amicon Ultra-15 100 kDa cutoff membrane size exclusion centrifugal filter column (Merck Millipore, Germany) for 5 min at 2000 ref in a swinging bucket rotor and proceeding with the retained fraction. Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen, Loughborough, UK) were used to immunoprecipitate Sbi from FSA using IgG from human serum (Sigma-Aldrich) and magnetic beads as per manufacturer's instructions including antibody crosslinking. Western blot analysis

[0297] Sample preparation: M. luteus CFCS was pre-treated with 10 mM serine protease inhibitor, AEBSF, for 1 hr. Purified Sbi was then pre-treated for 1 hr with either lx, 0.5x, 0.25x concentration M. luteus CFCS or M. luteus CFCS / 10 mM AEBSF mix (5 pl: 1.67 pl) prior to western blot analysis. RPMI 1640 with or without 10 mM AEBSF was added to Sbi as controls.

[0298] Gel electrophoresis and immunoblotting: Samples were prepared by adding 6.67 pl of Sbi sample with 4x NuPAGE™ LDS sample buffer and then heated to 70°C for 10 min before putting on ice. Samples were then loaded in NuPAGE™ 4-12% Bis-Tris gel (Invitrogen), alongside 5pl of Spectra™ Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific). Proteins were run at 200V for 45 min in lx NuPAGE™ MOPS SDS running buffer (Invitrogen).

[0299] Following electrophoresis, semi-wet transfer of proteins to a nitrocellulose membrane was carried out using the Bio-Rad Trans-Blot Turbo Transfer System (Bio-rad, California, USA) according to the manufacturer's instructions. Membranes were then blocked in blocking solution, consisting of IX tris-buffered saline (TBS) with 0.5% (v / v) Tween-20 containing 5% (w / v) non-fat dried milk powder, at room temperature for 1 hr with shaking. Membranes were then incubated with primary antibody (Rabbit anti-Sbi, 1 : 1000, donated by Dr Joanne Pennock (University of Manchester)) diluted in blocking solution on an orbital shaker with gentle agitation at 4°C overnight. The following day, membranes were washed three times for 5 min with blocking solution before incubation with secondary antibody (HRP- conjugated goat anti-rabbit IgG, 1 :5000, BioRad (Hercules, CA, USA)) diluted in blocking solution at room temperature for 1 hr. The membranes were then washed twice for 5 min with blocking solution then washed three times for 5 min with TBS. Membranes were developed by applying enhanced chemiluminescence (ECL) reagent (GE Healthcare Amersham™, UK) for 1 min and visualising protein bands in a ChemiDoc XRS+ System (Bio-rad) using different exposure times. All generated files were then analysed with Image Lab software (Bio-rad).

[0300] Cloning and expression of the disclosed serine protease

[0301] The skin isolated and type strain M. luteus serine protease (PADP) gene sequences were obtained using WGS. A His6-tagged PADP gene was expressed using an Escherichia coli expression vector for 3 hr at 37°C with 400 pM IPTG and purified by the MRC-Protein Phosphorylation &. Ubiquitylation Unit at The University of Dundee. Statistical analysis

[0302] All experiments were carried out with three technical replicates and three or more biological replicates. Graphical data are presented as mean of biological replicates ±SEM using GraphPad Prism 10 software. Results were considered statistically significant if P-values were less than 0.05. All statistical tests were carried out using GraphPad Prism 10 software.

[0303] The Shapiro-Wilk test was used to determine whether a data set was normally distributed. Parametric single-factor data with two groups were analysed using the unpaired t test. Single-factor data with three or more groups was tested using oneway ANOVA followed by multiple comparisons to a control group using Dunnett's post hoc test. If normality could not be assumed, statistical comparisons between three or more groups were determined using the Kruskal-Wallis test with Dunn's multiple comparisons.

Claims

Claims1. A serine protease for use as a medicament, wherein the serine protease is derived from M. luteus.

2. A serine protease for use in the treatment or prevention of an inflammatory skin condition, wherein the serine protease is derived from M. luteus.

3. A serine protease for use as claimed in claim 2, wherein the inflammatory skin condition is a bacterially-induced condition.

4. A serine protease for use as claimed claim 2 or 3, wherein the inflammatory skin condition is Atopic dermatitis (AD).

5. A serine protease for use as claimed in any of claims 2-4, wherein the inflammatory skin condition is induced or exacerbated by S. aureus.

6. A serine protease for use as claimed in any of claims 1-5, wherein the serine protease is capable of cleaving IL-33.

7. A serine protease for use as claimed in any of claims 1-6, wherein the serine protease is secreted by M. luteus.

8. A serine protease for use as claimed in any of claims 1-7, wherein the serine protease has a molecular weight of 40-110kDa.

9. A serine protease for use as claimed in any of claims 1-8, wherein the serine protease has at least 98% sequence identity to SEQ ID NO: 1.

10. A serine protease for use as claimed in any of claims 1-9, wherein the serine protease comprises a sequence having at least 98% sequence identity to SEQ ID NO:2.

11. A serine protease for use as claimed in any of claims 1-10, wherein the serine protease has 15 or fewer amino acid substitutions relative to SEQ ID NO: 1.

12. A serine protease for use as claimed in any of claims 1-11, wherein:(a) the serine protease; or(b) the culture supernatant in which the M. luteus has been cultured for at least one hour, is capable of cleaving full-length S. aureus second immunoglobulin-binding protein (Sbi).

13. A serine protease for use as claimed in any of claims 1-12, wherein the activity of the serine protease is inhibited by 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF).

14. A serine protease for use as claimed in any of claims 1-13, wherein the M. luteus is a strain of M. luteus that, when cultured under growth conditions for at least one hour, provides a culture supernatant that is capable of reducing the concentration of IL-33 in a sample of recombinant IL-33.

15. A serine protease for use as claimed in any of claims 1-14, wherein the M. luteus is the strain of M. luteus that has been deposited at the NCTC depositary institution under Accession Number 22110401.

16. A serine protease for use as claimed in any of claims 1-15, wherein the serine protease is present in a composition formulated for topical administration.

17. A serine protease for use as claimed in claim 16, wherein the the composition comprises:(a) substantially no intact bacteria, lysed bacteria or bacterial fragments; and / or(b) a pharmaceutical excipient.

18. A serine protease for use as claimed in claim 16 or 17, wherein the the composition may be formulated in the form of a cream, gel, spray, ointment or oil.

19. A method of manufacturing a composition comprising a serine protease as claimed in any of claims 1-18, the method comprising culturing M. luteus, obtaining material secreted by the M. luteus comprising the serine protease, and preparing a composition comprising the secreted material.

20. A method of manufacturing a composition comprising a serine protease as claimed in any of claims 1-18, the method comprising expressing the serineprotease in a host cell, extracting the expressed serine protease, and preparing a composition comprising the extracted serine protease.

21. A nucleic acid comprising a nucleic acid sequence encoding a serine protease as claimed in any of claims 1-18.

22. An expression vector comprising a nucleic acid sequence encoding a serine protease as claimed in any of claims 1-18.

23. A host cell comprising an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a serine protease as claimed in any of claims 1-18.

24. A method of treating or preventing an inflammatory skin condition, the method comprising topically administering a composition comprising a serine protease as claimed in any of claims 1-18 to a site of an inflammatory skin condition on a subject.

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