ANTIMICROBIAL PROTEIN

Abstract

The inventors provide a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof for use in a method of treatment of the human or animal body by therapy or prophylaxis, such as for use in a method of treating or preventing an infection in or on a subject by a microorganism. Also provided is the use of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof to kill, or inhibit the growth of, a microorganism that is pathogenic to a human or an animal at a site that is not on or in the human or animal body.

Claims

1-17. (canceled)

18. A method of treating a human or animal comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an antimicrobial polypeptide comprising the Blad sequence shown in SEQ ID NO:4 or an active variant thereof, wherein said active variant has antimicrobial activity and comprises a sequence which has at least 60% amino acid identity to either SEQ ID NO:4 or a fragment of SEQ ID NO:4.

19. The method according to claim 18 which is carried out to treat or prevent an infection in or on the subject by a microorganism.

20. The method according to claim 18 wherein the composition further comprises a pharmaceutically acceptable carrier or diluent.

21. The method according to claim 18 wherein the composition further comprises a chelating agent.

22. The method according to claim 18 wherein the subject has a compromised immune system or is critically ill.

23. A method of killing, or inhibiting the growth of, a microorganism that is pathogenic to a human or an animal at a site that is not on or in the human or animal body, said method comprising administering to said site a composition comprising an effective amount of an antimicrobial polypeptide comprising the Blad sequence shown in SEQ ID NO:4 or an active variant thereof, wherein said active variant has antimicrobial activity and comprises a sequence which has at least 60% amino acid identity to either SEQ ID NO:4 or a fragment of SEQ ID NO:4.

24. The method according to claim 23 wherein said composition is used to disinfect, with respect to a human or animal pathogenic microorganism, an article that is to be ingested by, or placed directly on or in, a human or animal, or a surface that is in need thereof.

25. The method according to claim 24 wherein said article is a foodstuff or a medical device or instrument.

26. The method according to claim 24 wherein said surface is located within an environment where: (a) medical examination, diagnosis or treatment is to take place; (b) a foodstuff is to be prepared or otherwise handled or stored; (c) personal washing and/or sanitation is to take place; and/or (d) a person at particular risk of (i) acquiring an infection by a microorganism; and/or (ii) being unable to clear a microbial infection without medical intervention; is situated.

27. The method according to claim 23 wherein said composition further comprises a chelating agent.

28. The method according to claim 18 wherein the microorganism is a bacterium or a fungus.

29. The method according to claim 28 wherein the bacterium is a pathogenic species from one of the following genera: Pseudomonas, Listeria, Staphylococcus and Salmonella.

30. The method according to claim 28 wherein the fungus is a pathogenic species from one of the following genera: Candida, Aspergillus, Alternaria, Fusarium, Cryptococcus and Trichosporon.

31. The method according to claim 30 wherein the fungus can cause invasive fungal infection, preferably Candida albicans, Aspergillus fumigatus or Alternaria alternata.

32. A method of preventing or treating an infection by a microorganism comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an antimicrobial polypeptide comprising the Blad sequence shown in SEQ ID NO:4 or an active variant thereof, wherein said active variant has antimicrobial activity and comprises a sequence which has at least 60% amino acid identity to either SEQ ID NO:4 or a fragment of SEQ ID NO:4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The invention will be described with reference to the accompanying drawings, in which:

[0050] FIG. 1A shows time-kill curves for Listeria monocytogenes;

[0051] FIG. 1B shows time-kill curves for Pseudomonas Aeruginosa;

[0052] FIG. 2 shows inhibition halos for

[0053] BLAD against (top left) Staphylococcus aureus, (top right) Bacillus subtilis, (bottom left) Pseudomonas aeruginosa, and (bottom right) Listeria monocytogenes;

[0054] FIG. 3A shows a time-kill curve for C. albicans;

[0055] FIG. 3B shows a growth curve for C. albicans;

[0056] FIG. 4 shows a Growth curve for BLAD with Candida albicans in PDB pH 7 medium;

[0057] FIG. 5 shows Inhibition halo data for (top left and top right) BLAD and (bottom) amphotericin B or fluconazole against Candida albicans;

[0058] FIG. 6 shows Inhibition halo data for BLAD against Cryptococcus neoformans on (top row) PDA and (bottom row) PDA pH 7.5 (3-day incubation);

[0059] FIG. 7 shows Inhibition halo data for BLAD against Aspergillus fumigatus on (top row) Mueller-Hinton 10 medium (rule M44-A), (middle row) PDA or (bottom row) PDA pH 7.5 (3 day incubation);

[0060] FIG. 8 shows Inhibition halo data for (top row) BLAD and (bottom) amphotericin B or fluconazole against Aspergillus fumigatus on PDA pH 7.5 (6-day incubation);

[0061] FIGS. 5 to 8 collectively show inhibition halos for C. albicans, Cryptococcus neoformans and A fumigatus;

[0062] FIG. 9A shows a time-kill curves for BLAD and/or EDTA with Listeria monocytogenes;

[0063] FIG. 9B shows a time-kill curves for BLAD and/or EDTA with Pseudomonas aeruginosa;

[0064] FIG. 9C shows a time-kill curves for BLAD and/or EDTA with Candida albicans

[0065] FIG. 10 shows the Lupinus albus β-conglutin precursor encoding sequence (SEQ ID NO: 1); and

[0066] FIG. 11 shows the internal fragment of the β-conglutin precursor encoding sequence that corresponds to Blad (SEQ ID NO: 3).

DETAILED DESCRIPTION OF THE INVENTION

Blad

[0067] Blad (“banda de Lupinus albus doce”—band from sweet L. albus) was the name given to a stable and intermediary breakdown product of β-conglutin, the major storage protein present in seeds of the Lupinus genus. It was characterised as a 20 kD polypeptide, composed of 173 amino acid residues, and encoded by an internal fragment (519 nucleotides, deposited in GenBank under the accession number ABB13526) of the gene encoding the precursor of β-conglutin from Lupinus (1791 nucleotides, published in GenBank, under the accession number AAS97865). When primers encoding Blad terminal sequences are used to amplify a sequence from genomic Lupinus DNA, a ˜620 bp product is obtained, indicating the presence of an intron in the gene fragment encoding Blad. Naturally-occurring Blad is the main component of a 210 kD glycooligomer which accumulates exclusively (following intensive limited proteolysis of β-conglutin) in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Whilst said oligomer is glycosylated, naturally-occurring Blad is non-glycosylated. The Blad-containing glycooligomer is composed of several polypeptides, the major ones exhibiting molecular masses of 14, 17, 20, 32, 36, 48 and 50 kD. The 20 kD polypeptide, Blad, is by far the most abundant polypeptide within the oligomer and appears to be the only one with lectin activity. Naturally-occurring Blad constitutes approximately 80% of the total cotyledonary protein in 8-day old plantlets.

[0068] The L. albus β-conglutin precursor encoding sequence (SEQ ID NO: 1) is given in FIG. 10. The β-conglutin parent subunit coding sequence is located at residues 70 to 1668. The encoded, 533 amino acid residue β-conglutin parent subunit (SEQ ID NO: 2) is:

TABLE-US-00001 MGKMRVRFPTLVLVLGIVFLMAVSIGIAYGEKDVL KSHERPEEREQEEWQPRRQRPQSRREEREQEQEQG SPSYPRRQSGYERRQYHERSEQREEREQEQQQGSP SYSRRQRNPYHFSSQRFQTLYKNRNGKIRVLERFD QRTNRLENLQNYRIVEFQSKPNTLILPKHSDADYV LVVLNGRATITIVNPDRRQAYNLEYGDALRIPAGS TSYILNPDDNQKLRVVKLAIPINNPGYFYDFYPSS TKDQQSYFSGFSRNTLEATFNTRYEEIQRIILGNE DEQEYEEQRRGQEQSDQDEGVIVIVSKKQIQKLTK HAQSSSGKDKPSDSGPFNLRSNEPIYSNKYGNFYE ITPDRNPQVQDLNISLTYIKINEGALLLPHYNSKA IYVVVVDEGEGNYELVGIRDQQRQQDEQEEKEEEV IRYSARLSEGDIFVIPAGYPISINASSNLRLLGFG INADENQRNFLAGSKDNVIRQLDRAVNELTFPGSA EDIERLIKNQQQSYFANGQPQQQQQQQSEKEGRRG RRGSSLPF

[0069] The internal fragment of the β-conglutin precursor encoding sequence that corresponds to Blad (SEQ ID NO: 3) is given in FIG. 11. The Blad polypeptide (SEQ ID NO: 4) is:

TABLE-US-00002 RRQRNPYHFSSQRFQTLYKNRNGKIRVLERFDQRT NRLENLQNYRIVEFQSKPNTLILPKHSDADYVLVV LNGRATITIVNPDRRQAYNLEYGDALRIPAGSTSY ILNPDDNQKLRVVKLAIPINNPGYFYDFYPSSTKD QQSYFSGFSRNTLEATFNTRYEEIQRIILGNED

[0070] The invention relates to a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof. It therefore relates to a composition comprising an antimicrobial polypeptide comprising the polypeptide sequence of SEQ ID NO: 4 or an active variant thereof. In alternative embodiments, the composition consists essentially of an antimicrobial polypeptide comprising Blad or an active variant thereof and/or the antimicrobial polypeptide consists essentially of Blad or an active variant thereof. In further embodiments the antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof may be used in isolated form.

[0071] An active variant of Blad is a variant of Blad that retains the ability to act as an antimicrobial (i.e. has antimicrobial activity—see below for a description of the level of such activity and how to measure it). “An active variant of Blad” includes within its scope a fragment of SEQ ID NO: 4. In preferred embodiments, a fragment of SEQ ID NO: 4 is selected that is at least 10% of the length of SEQ NO: 4, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of SEQ NO: 4. Blad or a variant thereof generally has a length of at least 10 amino acid residues, such as at least 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160 or 173 amino acid residues.

[0072] “An active variant of Blad” also includes within its scope a polypeptide sequence that has homology with SEQ ID NO: 4, such as at least 40% identity, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, and most preferably at least 99% identity, for example over the full sequence or over a region of at least 20, preferably at least 30, preferably at least 40, preferably at least 50, preferably at least 60, preferably at least 80, preferably at least 100, preferably at least 120, preferably at least 140, and most preferably at least 160 or more contiguous amino acid residues. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as “hard homology”).

[0073] The homologous active Blad variant typically differs from the polypeptide sequence of SEQ ID NO: 4 by substitution, insertion or deletion, for example from 1, 2, 3, 4, 5 to 8 or more substitutions, deletions or insertions. The substitutions are preferably ‘conservative’, that is to say that an amino acid may be substituted with a similar amino acid, whereby similar amino acids share one of the following groups: aromatic residues (F/H/W/Y), non-polar aliphatic residues (G/A/P/I/L/V), polar-uncharged aliphatics (C/S/T/M/N/Q) and polar-charged aliphatics (D/E/K/R). Preferred sub-groups comprise: G/A/P; I/L/V; C/S/T/M; N/Q; D/E; and K/R.

[0074] An antimicrobial polypeptide comprising Blad or an active variant thereof (as described above) may consist of Blad or an active variant thereof with any number of amino acid residues added to the N-terminus and/or the C-terminus provided that the polypeptide retains antimicrobial activity (again, see below for a description of the level of such activity and how to measure it). Preferably, no more than 300 amino acid residues are added to either or both ends of Blad or an active variant thereof, more preferably no more than 200 amino acid residues, preferably no more than 150 amino acid residues, preferably no more than 100 amino acid residues, preferably no more than 80, 60 or 40 amino acid residues, most preferably no more than 20 amino acide residues.

[0075] An antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof (as described above) may be utilised in the invention in the form of a purified (e.g. removed from a plant, animal or microbial source) and/or recombinant protein. Production of a recombinant form enables the production of active variants of Blad.

[0076] Methods of purifying naturally-occurring Blad are already described in the art (e.g. Ramos et al. (1997) Planta 203(1): 26-34 and Monteiro et al. (2010) PLoS ONE 5(1): e8542). A suitable source of naturally-occurring Blad is a plant of the Lupinus genus, such as Lupinus albus, preferably a cotyledon of said plant, preferably harvested between about 4 and about 14 days after the onset of germination, more preferably harvested 6 to 12 days after the onset of germination (such as 8 days after the onset of germination). Methods are disclosed in the art for a total protein extraction leading to a crude extract comprising Blad, and for a protein purification of such an extract leading to a partially purified extract, e.g. comprising the Blad-containing glycooligomer that comprises Blad.

[0077] To isolate Blad itself one can then use SDS-PAGE and/or, preferably, reverse phase (RP)-HPLC on a C-18 column.

[0078] An alternative way of obtaining a partially purified extract comprising the glycooligomer that comprises Blad is to utilise the chitin binding activity of Blad. The glycooligomer binds in a very strong manner to a chitin column as part of a chitin affinity chromatography purification, being eluted with 0.05 N HCl. Details of an example of this purification method are as follows: [0079] Cotyledons from eight-day old lupin plants were harvested and homogenized in Milli-Q plus water (pH adjusted to 8.0), containing 10 mM CaCl.sub.2 and 10 mM MgCl.sub.2. The homogenate was filtered through cheesecloth and centrifuged at 30,000 g for 1 h at 4° C. The pellet was subsequently suspended in 100 mM Tris-HCl buffer, pH 7.5, containing 10% (w/v) NaCl, 10 mM EDTA and 10 mM EGTA, agitated for 1 h at 4° C., and centrifuged at 30,000 g for 1 h at 4° C. The total globulin fraction, contained in the supernatant, was precipitated with ammonium sulphate (561 g/1), left stirring in the cold for 1 h and centrifuged at 30,000 g for 30 mM at 4° C. The pellet obtained was dissolved in 50 mM Tris-HCl buffer, pH 7.5, desalted in PD-10 columns equilibrated in the same buffer and passed through a chitin-affinity chromatography column pre-equilibrated in the same buffer. The column was washed with 50 mM Tris-HCl buffer, pH 7.5, and the bound proteins eluted with 0.05 N HCl. The eluted fractions were immediately neutralized with 2 M Tris and the peak fractions pooled, lyophilized and analyzed by SDS-PAGE. [0080] For the preparation of the chitin column, crude chitin was obtained from Sigma and processed as follows: the chitin sample was washed extensively with Milli-Q plus water, followed by 0.05 N HCl. It was then washed with 1% (w/v) sodium carbonate and then with ethanol, until the absorbance of the wash was less than 0.05. Chitin was then packed into a pipette tip and equilibrated with 50 mM Tris-HCl buffer, pH 7.5.

[0081] Methods of producing recombinant proteins are well known in the art. Such methods as applied here will involve inserting the polynucleotide encoding a polypeptide comprising Blad or an active variant thereof into a suitable expression vector—enabling the juxtaposition of said polynucleotide with one or more promoters (e.g. an inducible promoter, such as T7lac) and with other polynucleotides or genes of interest—introducing the expression vector into a suitable cell or organism (e.g. Escherichia coli), expressing the polypeptide in the transformed cell or organism and removing the expressed recombinant polypeptide from that cell or organism. To assist such purification the expression vector may be constructed such that the polynucleotide additionally encodes, for example, a terminal tag that can assist purification: e.g., a tag of histidine residues for affinity purification. Once the recombinant polypeptide is purified, the purification tag may be removed from the polypeptide, e.g., by proteolytic cleavage.

[0082] In a composition comprising an antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof, said polypeptide is preferably in partially purified form, more preferably in purified form. Said polypeptide is partially purified when it is present in an environment lacking one or more other polypeptides with which it is naturally associated and/or is represented by at least about 10% of the total protein present. Said polypeptide is purified when it is present in an environment lacking all, or most, other polypeptides with which it is naturally associated. For example, purified Blad means that Blad represents at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the total protein in a composition.

[0083] In a composition comprising an antimicrobial polypeptide comprising (or consisting essentially of) Blad or an active variant thereof, the Lupinus protein content may consist essentially of the Blad-containing glycooligomer that comprises a polypeptide that comprises (or consist essentially of) Blad or an active variant thereof.

[0084] A composition comprising an antimicrobial polypeptide comprising (or consisting essentially of) Blad may also be a formulation comprising another compound(s) added to the composition by the skilled person. In preferred embodiments, such a formulation is a pharmaceutical formulation comprising an antimicrobial polypeptide comprising (or consisting essentially of) Blad and a pharmaceutically acceptable carrier or diluent.

Microbial Targets

[0085] The invention relates to the use of Blad as an antimicrobial compound, i.e. to inhibit the growth of or kill microorganisms that are pathogenic to humans or animals. Such microorganisms include, in particular, bacteria and fungi. Such pathogenic microorganisms are capable of causing infectious disease or any other ill-health (e.g. food poisoning, allergy) in humans and/or animals, and may affect or infect, for example, the eyes, the skin, burns, wounds, the upper respiratory tract, the lungs, the gastrointestinal tract, the genitourinary tract, the kidneys, the liver, the nervous system and/or the cardiovascular system (e.g. the bloodstream). Such pathogenic microorganisms may be inherently pathogenic or may be opportunistic (i.e. do not cause disease in a healthy host but can do in a host with a compromised immune system). Such pathogenic microorganisms may additionally or alternatively cause ill-health by releasing compounds that are toxic to humans or animals.

[0086] Blad can be used as an antimicrobial against both Gram-positive and Gram-negative bacterial pathogens. Particularly preferred bacterial targets include pathogenic Pseudomonas species, such as P. aeruginosa, Pseudomonas oryzihabitans and Pseudomonas plecoglossicida (most preferably P. aeruginosa), pathogenic Listeria species, such as L. monocytogenes and Listeria ivanovii (most preferably L. monocytogenes), pathogenic Bacillus species such as B. subtilis, Bacillus anthracis and Bacillus cereus (most preferably B. subtilis), pathogenic Staphylococcus species, such as

[0087] S. aureus (including Methicillin-resistant Staphylococcus aureus [MRSA]), Staphylococcus pseudintermedius, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus lugdunensis, Staphylococcus schleiferi and Staphylococcus caprae (most preferably S. aureus), pathogenic Salmonella species, such as Salmonella enterica subspecies such as Salmonella arizonae, Salmonella choleraesuis, Salmonella enteritidis, Salmonella paratyphi A, Salmonella paratyphi B, Salmonella typhi, Salmonella typhimurium, Salmonella dublin, Salmonella typhisuis and Salmonella brandenburg (most preferably S. enteritidis or S. typhi) and pathogenic Campylobacter species such as Campylobacter jejuni and Campylobacter coli (most preferably C. jejuni). In preferred embodiments Blad is used against pathogens that can cause generalised inflammation and sepsis (e.g. P. aeruginosa), Cholera (e.g. V. cholerae), Meningitis (e.g. L. monocytogenes, Haemophilus influenzae type b, Neisseria meningitidis, or Streptococcus pneumoniae), Pneumonia (e.g. S. pneumoniae, Streptococcus agalactiae or S. aureus), Shigellosis (e.g. Shigella boydii, Shigella dysenteriae, Shigella flexneri or Shigella sonnei), Strep throat (e.g. Streptococcus pyogenes), Tuberculosis (e.g. Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti and Mycobacterium microti), Typhoid (S. typhi), or food poisoning (e.g. pathogenic species from one of the following genera: Listeria, Staphylococcus and Salmonella).

[0088] Blad can be used as an antimicrobial against both unicellular (yeast) and multicellular (filamentous, mold) fungal pathogens. Particularly preferred fungal targets include pathogenic Candida species, such as C. albicans, Candida glabrata, Candida lusitaneae, Candida parapsilosis, Candida tropicalis, Candida krusei and Candida dubliniensis, pathogenic Alternaria species, such as A. alternata and Alternaria molesta, pathogenic Aspergillus species, such as A. fumigatus, Aspergillus niger, Aspergillus flavus and Aspergillus clavatus, pathogenic Fusarium species, such as Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, and Fusarium proliferatum, pathogenic Cryptococcus species, such as Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus albidus and Cryptococcus gattii, and pathogenic Trichosporon species, such as Trichosporon ovoides, Trichosporon inkin, Trichosporon asahii, Trichosporon mucoides, Trichosporon asteroides, and Trichosporon cutaneum (all previously considered under the general name of Trichosporon beigelii), and Trichosporon dermatis, Trichosporon dohaense and Trichosporon loubieri. In preferred embodiments Blad is used against pathogens that can cause invasive fungal infection (IFI), which is usually defined as a systemic, generalized and visceral fungal infection that is often severe and/or life-threatening (in contrast to superficial, local, benign, self-limiting fungal diseases). Particularly preferred IFI causing fungi include pathogenic Candida, Aspergillus or Alternaria species as defined above, preferably C. albicans, A. fumigatus or A. alternata, most preferably C. albicans or A. fumigatus.

[0089] The skilled person will be able to identify, through routine methods, a suitable concentration with which to use an antimicrobial polypeptide comprising (or consisting essentially of) Blad (or an active variant thereof) as an antimicrobial in any particular setting. Preferably, for example, Blad is used at a concentration of at least 1 μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 20 μg/ml, at least 50 μg/ml, or at least 100 μg/ml, and up to 500 μg/ml, up to 600 μg/ml, up to 1 mg/ml, up to 2.5 mg/ml, up to 5 mg/ml or up to 10 mg/ml. Preferably the concentration of Blad selected is between 10 μg/ml and 5 mg/ml, more preferably between 50 μg/ml and 2.5 mg/ml, more preferably between 100 μg/ml and 1 mg/ml, and even more preferably between 100 μg/ml and 600 μg/ml (such as about 250 μg/ml). The inventors have provided evidence (see Examples 4 and 5) that Blad is non-toxic to the host up to at least 400 μg/ml.

[0090] The inventors have surprisingly found that a combination of Blad with a chelating agent (e.g. EDTA) produces a synergistic antimicrobial effect. Therefore, preferably, a chelating agent is used to improve the antimicrobial activity of a polypeptide comprising (or consisting essentially of) Blad (or an active variant thereof), and the use of such a chelating agent may decrease the concentration of said antimicrobial polypeptide required to achieve a particular level of antimicrobial activity. A chelating agent (also known as a chelant, a chelator or a sequestering agent) is any compound that binds to a metal ion to form a non-covalent complex and reduces the ion's activity. Suitable chelating agents include polyamino carboxylates such as EDTA (ethylenediaminetetraacetic acid) and EGTA (ethyleneglycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid). Preferably, EDTA is used as the chelating agent, preferably at a concentration of at least 10 μg/ml, at least 50 μg/ml, or at least 100 μg/ml, and up to 500 μg/ml, up to 1 mg/ml, up to 5 mg/ml, up to 10 mg/ml, or up to 20 mg/ml. Preferably, EDTA is used at a concentration of between 0.1 mg/ml and 1 mg/ml.

Outcomes

[0091] The antimicrobial polypeptide comprising (or consisting essentially of) Blad (or an active variant thereof) may be used to inhibit the growth of a human/animal pathogenic microorganism (meaning that it has microbistatic activity) and/or to kill said microorganism (meaning that it has microbicidal activity). The skilled person will be able to identify a suitable dose and/or concentration to obtain a particularly desired growth inhibition or killing of the microorganism.

[0092] Preferably, when used as a microbistatic agent, the antimicrobial polypeptide reduces the rate of growth by 10%, more preferably by 50%, more preferably by 75%, more preferably by 90%, more preferably by 95%, more preferably by 98%, more preferably by 99%, and even more preferably by 99.9% in comparison to equivalent conditions where the antimicrobial polypeptide is not present. Most preferably the antimicrobial polypeptide prevents any growth of the microorganism.

[0093] Preferably, when used as a microbicidal agent, the antimicrobial polypeptide kills 10% of the population of the microorganisms, more preferably 50% of said population, more preferably 75% of said population, more preferably 90% of said population, more preferably 95% of said population, more preferably 98% of said population, more preferably 99% of said population, and even more preferably by 99.9% of said population in comparison to equivalent conditions where the antimicrobial polypeptide is not present. Most preferably the antimicrobial polypeptide kills all of the population of the microorganism.

[0094] When used to prevent or treat an infection in or on a human or animal the antimicrobial polypeptide is preferably used in a therapeutically effective amount, that is to say an amount that provides a level of growth inhibition and/or killing of a microorganism such that a clinically detectable level of infection prevention or abrogation is achieved. Preferably, the therapeutically effective amount of the antimicrobial polypeptide is non-toxic to the human or animal subject. It is intended that said therapeutically effective amount of the antimicrobial polypeptide is therapeutically effective when administered as part of a composition comprising the antimicrobial polypeptide.

[0095] The inventors have surprisingly found that, at similar concentrations (by mass), Blad is approximately as potent as amphotericin B and more potent than fluconazole against C. albicans and A. fumigatus (in terms of fungicidal and fungistatic activity). This is a striking result given (i) the much greater molecular mass of Blad in comparison to the relatively small organic molecules of amphotericin B and fluconazole and (ii) the non-toxic and edible nature of Blad to humans and other animals

Medical Uses and Methods

[0096] The inventors provide a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof for use in a method of treatment of the human or animal body by therapy or prophylaxis. To this end they also provide a method of treating a human or animal comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an antimicrobial polypeptide comprising Blad or an active variant thereof.

[0097] The inventors also provide a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof for use in a method of preventing or treating an infection in or on a human or animal subject by a microorganism. To this end they also provide: [0098] a method of preventing or treating an infection by a microorganism comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an antimicrobial polypeptide comprising Blad or an active variant thereof; and [0099] use of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof in the manufacture of a medicament for preventing or treating an infection in or on a human or animal subject by a microorganism.

[0100] Said composition may be administered by injection (such as intradermal, subcutaneous, intramuscular, intravenous, intraosseous, and intraperitoneal), transdermal particle delivery, inhalation, topically, orally or transmucosally (such as nasal, sublingual, vaginal or rectal).

[0101] Preferably, said composition comprises a pharmaceutically acceptable carrier or diluent. Such a pharmaceutical composition may be formulated as a conventional pharmaceutical preparation. This can be done using standard pharmaceutical formulation chemistries and methodologies, which are available to those skilled in the art. For example, an antimicrobial polypeptide comprising Blad (or an active variant thereof) can be combined with one or more pharmaceutically acceptable carriers or diluents to provide a liquid preparation. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may also be present.

[0102] The carriers, diluents and auxiliary substances are generally pharmaceutical agents which may be administered without undue toxicity and which will not in themselves induce an immune response in the individual receiving the composition. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. It is also preferred, although not required, that the preparation will contain a pharmaceutically acceptable carrier that serves as a stabilizer, particularly advantageous for a composition comprising a polypeptide like Blad. Examples of suitable carriers that also act as stabilizers for polypeptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEGs), and combination thereof.

[0103] Once formulated, the composition can be delivered to a subject in vivo using a variety of known routes and techniques. For example, the liquid preparations can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, intradermal, intramuscular, intravenous, intraosseous or intraperitoneal injection using a conventional needle and syringe, or using a liquid jet injection system. Liquid preparations can also be administered topically to the eyes, to skin, hair or mucosal tissue (e.g. nasal, sublingual, vaginal or rectal), or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques. In preferred embodiments the antimicrobial polypeptide is formulated into a composition suitable as a topical lotion, hand-cream, eye-drop solution, shampoo or conditioner.

[0104] The subject in need of the antimicrobial polypeptide may be any human or animal individual. In preferred embodiments the antimicrobial polypeptide may be used to prevent infection in subjects at particular risk of acquiring an infection by a microorganism and/or to treat infection in subjects at particular risk of being unable to clear a microbial infection without medical intervention, such as the young (such as an individual below the age of 16 years, such as an individual below the age of 5 years, 3 years, 2 years, 1 year, 6 months or 1 month), the elderly (such as an individual above the age of 70 years, such as an individual above the age of 80 years or 90 years), those with a compromised immune system (such as those with a primary immunodeficiency, those with an acquired immunodeficiency (e.g. those with AIDS) and those with a suppressed immune system as a result of treatment such as chemotherapy or immunosuppressive drug regimes), those who are critically ill, or those who might have a particularly high exposure to pathogenic microorganisms (e.g. medical professionals).

Other Antimicrobial Uses and Methods

[0105] The inventors also provide the use of a composition comprising an antimicrobial polypeptide comprising Blad or an active variant thereof to kill, or inhibit the growth of, a microorganism that is pathogenic to a human or an animal at a site that is not on or in the human or animal body. To this end they also provide a method of killing or inhibiting the growth of a microorganism that is pathogenic to a human or an animal at a site that is not on or in the human or animal body, said method comprising administering to said site a composition comprising an effective amount of an antimicrobial polypeptide comprising Blad or an active variant thereof. Said effective amount is an amount that provides a level of growth inhibition and/or killing of a microorganism such that a detectable level of prevention or abrogation of microbial colonisation is achieved. Preferably, the effective amount of the antimicrobial polypeptide is non-toxic to the human or animal subject. It is intended that said effective amount of the antimicrobial polypeptide is effective when administered as part of a composition comprising the antimicrobial polypeptide.

[0106] In these embodiments it is intended that the antimicrobial polypeptide is used as a disinfectant to prevent the growth of and/or kill a pathogenic microorganism on an article that is to be ingested by, or placed directly on or in, a human or animal, or a surface that is in need thereof (e.g. a surface that may, directly or indirectly, come into contact with a human or animal) so that the risk is of:

[0107] (i) a human or animal becoming infected with said pathogenic microorganism; or

[0108] (ii) a human or animal coming into contact with a toxin released by a pathogenic microorganism; is reduced.

[0109] In preferred embodiments the antimicrobial polypeptide is used within or on a foodstuff to prevent the growth of a human/animal pathogenic microorganism on or within that foodstuff or to kill a human/animal pathogenic microorganism already present on or within that foodstuff. In this way the antimicrobial polypeptide can be used to reduce the risk of a human or animal becoming infected with a pathogenic microorganism, or of a human or animal ingesting a toxin released by a pathogenic microorganism, as a result of ingesting that foodstuff. In these embodiments it is particularly preferred that said pathogenic microorganism is capable of causing food poisoning (e.g. directly or via a released toxin). By foodstuff it is intended to mean any liquid or solid substance intended for consumption for nutritional or pleasurable reasons. The composition comprising the antimicrobial polypeptide can for example be mixed with other components of the foodstuff during the preparation for the foodstuff or may for example be applied to the surface of the foodstuff (for example as a liquid film or a spray). Particular foodstuffs considered in these embodiments include water, soft drinks such as fruit juices, alcoholic drinks, raw meat, cooked poultry meat, eggs, milk, cream, ice-cream, cheese, raw vegetables and fruits, processed foods (particularly relevant to e.g. L. monocytogenes, V. cholerae, pathogenic Staphylococcus species, pathogenic Salmonella species and pathogenic Campylobacter species), and nuts and starchy foods such as bread, rice and potatoes (particularly relevant for pathogenic Aspergillus species).

[0110] In alternative preferred embodiments the antimicrobial polypeptide is used within or on a medical device or instrument—any device placed on or within the body to carry out a diagnostic, therapeutic or surgical function—such as artificial body tissue, pacemakers, stents, scaffolds, valves, thermometers, syringes, hypodermic needles, monitoring equipment, ventilators, cardiac defibrillators, heart lung machines, EEG and ECG units, ultrasound devices, drills, saws, knives, scalpels, tongues, scissors, clips and stitches and the like. In such a way the antimicrobial polypeptide can be used to prevent infection of a body that comes into contact with a device or instrument during a medical procedure.

[0111] In alternative preferred embodiments the antimicrobial polypeptide is used on a surface that is in need thereof (e.g. a surface that may, directly or indirectly, come into contact with a human or animal). The surface to which the antimicrobial polypeptide may be applied may be located within an environment where:

[0112] (a) medical examination, diagnosis or treatment is to take place;

[0113] (b) a foodstuff is to be prepared or otherwise handled or stored;

[0114] (c) personal washing and/or sanitation is to take place; and/or

[0115] (d) a person at particular risk of [0116] (i) acquiring an infection by a microorganism; and/or [0117] (ii) being unable to clear a microbial infection without medical intervention;

[0118] is situated (and examples of such persons are described above).

[0119] Examples of such surfaces include any within an industrial food factory and shelves/benches within a food supermarket.

[0120] The surface to which the antimicrobial polypeptide may be applied may be a floor or wall of a building (or a room thereof) or a surface of an article within said room or building. Particular buildings envisaged include hospitals and other healthcare buildings, schools and other child-care centres, elderly care buildings, restaurants and other eateries, places of food preparation, processing and/or storage (e.g. markets, foodstores, supermarkets, and industrial food factories), and private dwellings. Particular rooms envisaged include all of those within a healthcare setting, especially operating theatres, accident and emergency departments, intensive care and patient wards, as well as kitchens, bathrooms, toilets, restaurants and food preparation/processing halls.

EXAMPLES

[0121] In the following Examples BLAD denotes the naturally-occurring Blad-containing glycooligomer comprising the 20 kD Blad polypeptide, purified as per Ramos et al. (1997) Planta 203(1): 26-34: see “Plant material and growth conditions” and “Purification of proteins” parts of the Materials and Methods section of that document.

Definitions

[0122] MIC—Minimum Inhibitory Concentration: the lowest concentration of an antimicrobial that inhibits the visible growth of a microorganism. [0123] MFC/MBC—Minimum Fungicidal/Bactericidal Concentration (or Minimal Lethal Concentration): the lowest concentration of an antimicrobial agent needed to kill 99.9% of the initial inoculum after 24 h under a standardized set of conditions. [0124] Time-kill curves—Determination of the “killing” of an isolate over time by one or more antimicrobial agents under controlled conditions is known as the time-kill method. It is a broth based method where the rate of killing of a fixed inoculum is determined by sampling control (organism, no drug) and antimicrobial agent-containing tubes or flasks, at certain time intervals, and determining the survivor colony count (cfu/ml) by spreading each sample onto an agar plate.

Example 1—Bactericidal Activity of BLAD

MIC and MBC of BLAD for Various Bacterial Species (Using Mueller-Hinton Medium):

[0125]

TABLE-US-00003 Bacterial Species MIC (μg/ml) MBC (μg/ml) Pseudomonas aeruginosa 32−256 128−256 Listeria monocytogenes 8 >512 Bacillus subtilis 4 >512 Staphylococcus aureus 8 >512 Salmonella thyphimurium 64 128

[0126] Time-kill curves for BLAD with (A) Listeria monocytogenes and (B) Pseudomonas aeruginosa: see FIG. 1.

[0127] Against L. monocytogenes and P. aeruginosa BLAD is bacteriostatic at 100 μg/ml and bactericidal at 250 μg/ml.

[0128] Inhibition halo data for BLAD against (A) on top left Staphylococcus aureus, (B) on top right Bacillus subtilis, (C) on bottom left Pseudomonas aeruginosa, and (D) on bottom right Listeria monocytogenes: see FIG. 2.

[0129] Growth of all tested bacterial species on PCA was increasingly inhibited with increasing BLAD amounts on the treatment disks, from 20 μg (lower right disks) to 100 μg (lower left disks) and to 200 μg (top disks) (incubation 24 h—the effects were seen for several days).

Example 2—Fungicidal Activity of BLAD

[0130]

TABLE-US-00004 MIC and MFC of BLAD for Candida species (using RPMI medium) Candida Species MIC (μg/ml) MFC (μg/ml) Candida albicans 16−32 256 Candida dubliniensis 32−64 256 Candida glabrata 1-2 >512 Candida lusitaneae 32−64 >512 Candida parapsilosis 32 >512 Candida tropicalis 16−32 >512

TABLE-US-00005 MIC and MFC of BLAD for Candida species (using PDB medium at pH 7.5) Candida Species MIC (μg/ml) MFC (μg/ml) Candida albicans 2−4 4−8 Candida dubliniensis 2−4 8 Candida glabrata 2 16−64 Candida lusitaneae 2−4 8−32 Candida parapsilosis 2−4 64 Candida tropicalis 4 4−16

TABLE-US-00006 MIC and MFC of BLAD for various filamentous fungi (using RPMI medium) Fungal Species MIC (μg/ml) MFC (μg/ml) Alternaria sp. 64 >512 Aspergillus fumigatus 32 >512 Aspergillus niger 32−64 >512 Botrytis cinerea 128 512 Colletotrichum acutatum 64 >512 Colletotrichum gloesporioides 64 >512 Fusarium oxysporum 64 >512 NB − MIC for Cryptococcus neoformans measured at 0.25−1.0 ug/ml.

[0131] Time-kill curve (A) and growth curve (B) for BLAD with Candida albicans in PDB medium: see FIG. 3.

[0132] Against C. albicans BLAD is fungistatic at 10 μg/ml and fungicidal at 100 μg/ml.

[0133] Growth curve for BLAD with Candida albicans in PDB pH 7 medium: see FIG. 4.

[0134] Against C. albicans BLAD and amphotericin B are fungistatic at 10 μg/ml. At 100 μg/ml fluconazole merely delays growth.

[0135] Inhibition halo data for (A and B) (top row) BLAD and (C) (bottom row) amphotericin B or fluconazole against Candida albicans: see FIG. 5.

[0136] Growth of C. albicans on Potato Dextrose Agar (PDA) pH 7.5 was inhibited with increasing BLAD amounts on the treatment disks, from 20 μg (A, lower disk) to 50 μg (B, lower disk) to 100 μg (B, upper disk) and to 200 μg (A, upper disk) (incubation 3 days). This compares very favourably with the inhibition achieved with 20 μg amphotericin B (C, upper disk) and 25 μg fluconazole (C, lower disk).

[0137] Inhibition halo data for BLAD against Cryptococcus neoformans on (A) on top row PDA and (B) on bottom row PDA pH 7.5 (3 day incubation): see FIG. 6.

[0138] Growth of C. neoformans was inhibited on both media with increasing BLAD amounts on the treatment disks, though with greater efficacy on PDA. I—top disks 200 μg, bottom disks 10 μg; II—upper left disks 50 μg, upper right disk 20 μg, lower disk 100 μg.

[0139] Inhibition halo data for BLAD against Aspergillus fumigatus on (A) on top row Mueller-Hinton medium (rule M44-A), (B) on middle row PDA or (C) on bottom row PDA pH 7.5 (3 day incubation): see FIG. 7. Left panel shows plates seen from above; right panel shows plates seen from below.

[0140] Growth of A. fumigatus was inhibited on all media with increasing BLAD amounts on the treatment disks, though with greatest efficacy on PDA pH 7.5. I—top disks 200 μg, bottom disks 10 μg; II—upper left disks 50 μg, upper right disk 20 μg, lower disk 100 μg.

[0141] Inhibition halo data for (A and B) on top row BLAD and (C) on bottom row amphotericin B or fluconazole against Aspergillus fumigatus on PDA pH 7.5 (6 day incubation): see FIG. 8.

[0142] Growth of A. fumigatus on PDA pH 7.5 was inhibited with increasing BLAD amounts on the treatment disks, from 20 μg (A, lower disk) to 50 μg (B, lower disk) to 100 μg (B, upper disk) and to 200 μg (A, upper disk). This compares very favourably with the inhibition achieved with 10 mg amphotericin B (C, upper disk) and 100 mg fluconazole (C, lower disk). Very similar results were seen for Trichosporon cutaneum (data not shown).

Example 3—Synergistic Effect of EDTA with BLAD with Respect to Bactericidal/Fungicidal Activity Against Human Pathogens

[0143] Time-kill curves for BLAD and/or EDTA with (A) Listeria monocytogenes, (B) Pseudomonas aeruginosa and (C) Candida albicans: see FIG. 9.

[0144] Against L. monocytogenes neither BLAD at 10 μg/ml nor EDTA at 0.1 mg/ml inhibits growth but a combination of the two is bacteriostatic. Against P. aeruginosa BLAD at 50 μg/ml or EDTA at 1 mg/ml inhibits growth (i.e. both are bacteriostatic) but a combination of the two is bactericidal. Against C. albicans BLAD at 10 μg/ml or EDTA at 0.1 mg/ml inhibits growth (i.e. both are fungistatic) but a combination of the two is fungicidal.

Example 4—Dermal Toxicity Study of BLAD in Guinea Pigs

[0145] Confidential study carried out at the Faculty of Veterinary Medicine, Technical University of Lisbon, on behalf of Instituto Superior de Agronomia (Jul. 18, 2006—Aug. 1, 2006) using OECD Guideline for testing of chemicals, No. 402, Acute Dermal Toxicity. The study was conducted in accordance with good laboratory practice and animal welfare.

[0146] The acute dermal toxicity of BLAD was evaluated after single dose exposure in guinea pigs, which are widely accepted as suitable animals for dermal toxicity studies. BLAD was applied to the glabrous skin in two groups of 10 animals each, with dosing at 200 μg/ml and 400 μg/ml respectively. After exposure the animals were kept under observation for a period of 15 days, during which body mass, morbidity and mortality were recorded.

Materials and Methods—

1. Materials

[0147] Test item: BLAD was supplied at 5 mg/ml (yellowish opaque liquid, 0-4° C.) and stored at −80° C.

[0148] Animals: albino guinea pigs; strain: Dunkin Hartley (HsdPoc: DH) by Harlan Iberica, Barcelona.

[0149] Number of animals used: 30; body weight: 400-449 g; age: 6 weeks.

[0150] Lodging: the animals were individually placed in polyethylene boxes with sterilized wood shavings (Lignocel).

[0151] Ambient conditions:

[0152] a) Photoperiod: cycles of light/dark for 12 h in 12 h.

[0153] b) Controlled environment: an average temperature of 19/22° C. and average humidity of 60%.

[0154] Adaptation: the animals were kept under environmental conditions of the test for seven days before the start of the test.

[0155] Food: Global Diet 2014, Rodent Maintenance Diet supplied by Harlan Iberica, Barcelona; water ad libitum.

2. Methods

[0156] Administration: animals were shaved 48 h before the test and only animals that had lesion-free skin were taken forward in the study. An aliquot of 1 ml (at either 200 μg/ml or 400 μg/ml) was applied to the shaved skin of each animal.

[0157] Study design: the 30 animals of the study were divided into four groups, two groups of ten animals each and two groups with five animals each. A group of ten animals was exposed to BLAD at 200 μg/ml (test group 1) and another group of ten animals was exposed to BLAD at 400 μg/ml (test group 2). The two groups of five animals served as controls: one group was exposed to water (1 ml aliquot) whilst another group was not subjected to any administration but handled as per all the other groups.

[0158] Outcomes: after exposure the animals were observed daily for 15 days to record any signs of morbidity or even death. In terms of morbidity particular attention was paid to possible appearance of skin lesions at the site of exposure and possible signs of general toxicity such as changes in normal behavior patterns. Body weight was individually assessed before exposure and at the end of test period.

Results—

[0159] At neither concentration of BLAD were there signs of any physical changes in the dermal administration area or changes in drinking/feeding or general behavior. No adverse reactions or death occurred upon BLAD administration. Increase in body mass was similar in all groups (and was consistent with the increase expected from developing animals of such young age).

[0160] Conclusions—BLAD at concentrations up to 400 μg/ml (and possibly higher) does not show dermal toxicity.

Example 5—Oral Toxicity Study of BLAD in Albino Rats

[0161] Confidential study carried out at the Faculty of Veterinary Medicine, Technical University of Lisbon, on behalf of Instituto Superior de Agronomia, using OECD Guideline for testing of chemicals, No. 401, Acute Oral Toxicity. The study was conducted in accordance with good laboratory practice and animal welfare.

[0162] The acute oral toxicity of BLAD was evaluated after single dose exposure in rats, which are widely accepted as suitable animals for oral toxicity studies. BLAD was administered by gavage in two groups of 10 animals each, with dosing at 200 μg/ml and 400 μg/ml respectively. After exposure the animals were kept under observation for a period of 15 days, during which body mass, morbidity and mortality were recorded. After the observation period the animals were euthanized and underwent necropsy.

Materials and Methods—

1. Materials

[0163] Test item: BLAD was supplied at 5 mg/ml (yellowish opaque liquid, 0-4° C.) and stored at −80° C.

[0164] Animals: Rattus norvegicus, strain: Wistar Hannover, acquired by the vivarium of the Faculty of Veterinary Medicine of Lisbon from Harlan Iberica, Barcelona.

[0165] Number of animals used: 30; body weight: 250-300 g; age: 10 weeks.

[0166] Lodging: the animals were individually placed in polyethylene boxes with sterilized wood shavings (Lignocel).

[0167] Ambient conditions:

[0168] a) Photoperiod: cycles of light/dark for 12 h in 12 h.

[0169] b) Controlled environment: an average temperature of 19/22° C. and average humidity of 60%.

[0170] Adaptation: the animals were kept under environmental conditions of the test for seven days before the start of the test.

[0171] Food: Global Diet 2014, Rodent Maintenance Diet supplied by Harlan Iberica, Barcelona; water ad libitum.

2. Methods

[0172] Administration: an aliquot of 1 ml (at either 200 μg/ml or 400 μg/ml) was applied to each animal by oral (oro-esophageal) intubation, commonly known as gavage. The administration was carried out with a metal probe appropriate to the species of animal used. The animals were subjected to fasting for 18 h prior to administration and fed 3 h following administration.

[0173] Study design: the 30 animals of the study were divided into four groups, two groups of ten animals each and two groups with five animals each. A group of ten animals was exposed to BLAD at 200 μg/ml (test group 1) and another group of ten animals was exposed to BLAD at 400 μg/ml (test group 2). The two groups of five animals served as controls: one group was exposed to water (1 ml aliquot) whilst another group was not subjected to any administration but handled as per all the other groups.

[0174] Outcomes: after administration the animals were observed daily for 15 days to record any signs of morbidity or even death. Body weight was individually assessed before exposure and at the end of test period. After the observation period the animals were euthanized (by asphyxiation in an atmosphere saturated with carbon dioxide) for subsequent post-mortem examination.

Results—

[0175] At neither concentration of BLAD were there signs of any physical changes or changes in drinking/feeding or general behavior. No adverse reactions or death occurred upon BLAD administration. Increase in body mass was similar in all groups (and was consistent with the increase expected from developing animals of such young age). Necropsy/macroscopic observation of the organs of the thoracic and abdominal cavity revealed no changes thereto.

[0176] Conclusions—BLAD at concentrations up to 400 μg/ml (and possibly higher) does not show oral toxicity.