NZ736735B2 - A method of in vivo treatment - Google Patents

Abstract

The present disclosure teaches the treatment or prophylaxis of infection by a microorganism including a fungus or bacterium which is infecting or colonizing an in vivo tissue, surface or membrane. The method comprising administering to the subject with the infection or directly to the site of infection a plant-derived defensin or a functional variant or derivative thereof. ion a plant-derived defensin or a functional variant or derivative thereof.

Description

A METHOD OF IN VIV0 TREATMENT BACKGROUND FIELD The present disclosure relates generally to the control of in viva microbial infection in humans and animals. Agents and natural and synthetic ations and extracts useful for the control of microbial infection of non—external tissues, surfaces and membranes are also encompassed by the subject disclosure.

DESCRIPTION OF RELATED ART Bibliographic s of the ations referred to by author in this specification are collected etically at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of tion that this prior art forms part of the common general knowledge in any y.

Microbial infection can lead to significant health issues in humans and animals.

Examples include fungal and bacterial infection of mucosal tissue, atory es, wounds and deep tissue.

Although antimicrobial agents including antibiotics and chemical microbicides have been successful in human and veterinary ne, there is a range of environmental and regulatory concerns with the continued use of these microbicides for a host of reasons including the widespread development of resistance. There is clearly a need to develop alternative mechanisms of controlling infection in humans and animals by microbial pathogens or at least to complement existing antimicrobial agents.

The most prevelant human fungal pathogen is Candida. Candida is a commensal organism and part of the normal flora for 30—50% of the population but can cause disease in patients who are immunocompromised or whose natural microflora is disrupted such as through the use of antibiotics. Several species of Candida can cause infections in humans ing Canida albicans, C. glabrata, C parasilosis, C. tropicalis and C. .

Common Candida infections include infections of the mucosal membranes such as oral and vaginal thrush. Oral candidiasis (oropharyngeal) is one of the most common infections observed in those suffering HIV (Singh et al. (2014) Journal of Oral and Maxillofacial Pathology 18(Supp1):SSl—SSS). More serious Candida infections include systemic blood stream infections (Candidemia) and biofilms on implanted medical devices. Candidemia is responsible for between 2 and 8 of every 1000 hospital intensive care admissions and has a —day mortality rate of ~30%. Candidemia is terised by spreading of Candida cells to the entire body creating abscesses in almost all vital organs, inducing their failure and g to a morbidity rate of 50%. Antifungal agents belonging to the e, azole and echinocandin family have been used to treat Candidiasis, however, all have unwanted side effects such as toxicity, drug—drug interactions and resistance. Candidiasis is commonly observed in animals and often caused by C. albicans. Although C. albicans is a normal inhabitant of animal species, it can be an opportunistic en. Birds are most commonly affected. Superficial infections have been described in pigs and foals. Systemic candidiasis has been observed in cattle, calves, sheep and foals following prolonged re to antibiotics.

Another serious fungal infection is illosis. Aspergillosis comprises a large spectrum of fungal es caused by Aspergillus that primarily affect the lungs, although other organ systems can be ed. Clinical manifestations of lung aspergillosis are ic bronchopulmonary aspergillosis, chronic necrotizing aspergillosis, illoma and the most severe, ve aspergillosis (IA). IA is the most common filamentous fungal infection in immunocompromised patients (Patterson et al. (2000) Medicine 79(4):250—60). The genus Aspergillus includes over 185 species, which are ubiquitous in nature, especially common in soil and decaying vegetation. The most common ens that cause disease in humans are A.fumigatus, A. flavas, A. terreas, A. niger and A tabingensis. Reports have shown that attributable ity of IA exceeds 80% (Fisher et al. (1981) The American Journal of Medicine 7](4):571—7). Current treatments involve members of polyene and azole family, including amphotericin b and isavuconazole respectively. These treatments display unwanted side effects such as toxicity, drug—drug interactions and for isavuconazole, the mortality rate remains at 18%. Aspergillosis is also a common fungal ion observed in s. It is caused by several Aspergillas species such as A. fumigatus, A. terreus, A. niger, A. nidulans, A. viridnutans, A. flavus and A. felis.

Animals that are affected include birds, , dogs, cats and .

Cryptococcosis is another ious disease caused by fungal pathogens. The manifestation may range from asymptomatic to mild bronchopneumonia to life—threatening infections of the central s system (CNS; Mitchell & Perfect (1995) Clinical Microbiology Reviews 8(4):515—48). Furthermore, cryptococcal itis is one of the most widely observed infections in patients suffering from AIDS (Vibhagool et al. (2003) Clinical Infectious Diseases 36(10):1329—31). The major causative agents of cryptococcosis are from the Cryptococcas family, generally C. neoformans and C. gatti.

The most serious type of cryptococcal disease arises from uncontrolled pulmonary cryptococcosis, which sses to cryptococcal meningitis. This progression tends only to occur in patients that are immunosuppressed. The most common treatment for cryptococcal meningitis is ericin B in combination with flucytosine. Early appropriate treatment has reduced the mortality rate from 14—25% to 6%, however, toxic side effects from amphotericin B are common including hypotension, anorexia, vomiting, headache and multiple organ damage. Cryptococcosis is also ed in s. It is most common in cats but has also been reported in dogs, cattle, horses, sheep, goats, birds and wild animals. Transmission occurs via inhalation of spores or by contamination of wounds.

Penicilliosis is another common opportunistic infection in patients with HIV and 9.36% of patients with HIV develop Penicilliosis. The causative pathogen of penicilliosis is Penicilliam marnefi‘ei (also known as myces marnefi‘ei). AIDS patients suffering from P. marnefi‘ei infection display symptoms of fever, anaemia, weight loss, lymphadenopathy, respiratory signs and skin lesions. The mortality for those that do not undergo treatment is at least 75% (Supparatpinyo et al. (1996) Lancet 15): 1 10—3).

Murcomycoses are the second most frequent mold infections observed in immunosuppressed patients. Most mucormycoses are life threatening and the most WO 91790 common presentation is severe ion of the sinuses, which may extend to the brain.

Infectious agents belong to the order Mucorales with the most recognized causative agent belonging to the Genus Rhizopus (Rhizopus oryzae). However, additional mucormycosis causing species have been identified including Apophysomyces elegans, Cunninghamella bertholletiae, Saksenaea vasiformis, Rhizomucor pusillus, Syncephalastrum racemosum, Cokeromyces recurvatus, mucor elegans (Gomes et al. (2011) Clinical Microbiology Reviews 24(2):4ll—45). The mortality rates are very high between 40% and 85%. The cutaneous forms of the infection represent the lowest mortality rate at 15% with the disseminated disease carrying a mortality rate approaching 100%. Treatments currently e amphotericin B, surgical procedures and azole treatments such as posaconazole.

Pythiosis is an emerging, life threatening infectious disease. It commonly affects horses and has been found in other s such as dogs, cats and cattle causing granulomatous ions in the skin, intestines and arteries (Wanachiwanawin et al. (2004) Vaccine 22(27-28):3613—21). The first report of a human infection was reported in Thailand in 1985 and has since been reported in tropical and subtropical countries. The disease can manifest as a localized form, including eye infections, corneal ulcers with cutaneous or aneous involvement. However, it can also present as a systemic or vascular infection and this is usually the most severe (Thianprasit et al. (1996) t Topics in Medical Mycology 7(1):43—54). Clinical features are limb ischemia and gangrene and if the infection reaches a main artery, the patient will often die from systemic pythiosis. The major pathogen is Pythium insidiosum, a fungus—like aquatic organism from the phylum Oomycota. ngal ents such as amphotericin B and various azoles display poor efficacy for systemic and ar phythiosis and the most effective ent is considered to be amputation of the site of ion.

Mycotic keratitis has been reported in many parts of the world, particularly tropical areas. There are two major forms, one caused by filamentous fungi such as Fnsarium and Aspergillus and another form caused by yeast—like pathogens such as Candida. Traumatic injury is a major predisposing factor for keratitis caused by filamentous fungi. The major filamentous fungal species involved are F. , F. oxysporum, A fumigatus and A flavus (Thomas (2003) Eye (London, England) I7(8):853—62). Topical natamycin and amphotericin B are used as a first line of ent, however if deep lesions are present oral ketoconazole, itraconazole or fluconazole may be administered with reasonable response rates (Thomas (2003) . In cases where medical treatments fail, surgical intervention may be necessary.

Histoplasmosis is a fungal infection that contracted after inhalation of the spores of the thermally dimorphic fungus Histoplasmosis capsulatum. This pathogen is found in North America, South America, Africa and Asia. There are several types of histoplasmosis, with the mildest form producing little or any symptoms at all. However, infants or those with compromised immune systems may develop progressive disseminated histoplasmosis, which can be fatal if left untreated (Wheat et al. (1990) Medicine 69(6):36l—74). Symptoms include fever, chills, headache, muscle aches and a dry cough.

For severe infections, amphotericin B is recommended for l — 2 weeks followed by itraconazole. ericin B displays very high, some , levels of ty.

Histoplasmosis has also been described in many animal species, however infection is rare in dogs and cats. Infection usually arises following aerosol exposure to the respiratory tract.

Blastomycosis is caused by the dimorphic fungus Blastomyces dermatitidis.

Infection is most common among dogs and cats but it has also been ed in horses, ferrets, wolves, deer, lions and dolphins. It is limited to North a and transmission is thought to be via tion of aerosolised conidia.

Plant defensins are small (45—54 amino acids), basic proteins with four to five disulfide bonds (Janssen et al. (2003) Biochemistry 42(27):82l4—8222). They share a common ide bonding pattern and a common structural fold, in which a triple— stranded, antiparallel B—sheet is tethered to an d—helix by three disulfide bonds, g a cysteine—stabilized dB motif. A fourth disulfide bond also joins the N— and C—termini leading to an extremely stable structure. A variety of functions has been uted to defensins, including anti—bacterial activity, protein synthesis inhibition and oc—amylase and protease inhibition (Colilla et al. (1990) FEBS Lett 270(1-2): 191— 194; Bloch and Richardson (1991) FEBS Lett 279(1): 101—104), generally in the context of plants.

The structure of defensins consists of seven 'loops', defined as the regions between cysteine residues. Loop 1 encompasses the first B—strand (1A) as well as most of the flexible region that connects this B—strand to the d—helix (1B) between the first two ant cysteine residues. Loops 2, 3 and the beginning of 4 (4A) make up the d—helix, while the remaining loops (4B — 7) make up B—strands 2 and 3 and the flexible region that connects them rpin region) (van der Weerden et al. (2013) Cell Mol Life Sci 70 (19): 3545—3570). Loop 5 of the plant defensins is known to be essential for antifungal activity and an important determinant for mechanism of action of these proteins (Sagaram et al., (2011) PLoS One 6.4: ).

Plant defensins lly share eight completely ved ne residues.

These es are commonly referred to as “invariant cysteine residues”, as their presence, location and connectivity are conserved amongst defensins. Based on sequence similarity, plant defensins can be categorized into different groups. Within each group, sequence homology is relatively high whereas inter—group amino acid similarity is low (van der Weerden et al. (2013) Fungal Biol Rev 26:121—131). Plant defensins belonging to different groups generally have different biological activities or different mechanisms of action for the same biological activity.

There are two major classes of plant defensins. Class I defensins consist of an endoplasmic reticulum (ER) signal sequence followed by a mature defensin domain. Class II defensins are produced as larger precursors with C—terminal pro—domains or pro—peptides (CTPPs) of about 33 amino acids. Most of the Class II defensins fied to date have been found in Solanaceous plant species.

There is a need to p protocols to more effectively manage microbial infection in humans and animals. Whilst some defensins have antifungal ties, their activities across different fungal pathogens vary significantly and the majority of demonstrated activity has been toward plant fungal pathogens.

SUMMARY Amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>l (SEQ ID NOzl), <400>2 (SEQ ID NO:2), etc. A y of the sequence identifiers is ed in Table l. A sequence listing is provided after the claims.

The present disclosure teaches a method for inhibiting infection by a rganism in in viva tissue in a subject the method comprising contacting the in vivo tissue with an effective amount of a plant defensin or a functional natural or synthetic derivative or variant thereof, the plant defensin, derivative or variant selected from the list consisting of SEQ ID NO:1 through 47 or a plant defensin having at least about 80% amino acid sequence similarity to any one of SEQ ID NO:1 through 47 after l alignment for a time and under conditions sufficient to eradicate or otherwise l microbial growth or colonization. Despite evolving to target plant pathogens, the plant defensins d herein have potent activity t rganism with no or medically acceptable minimal off target ty on ian cells. More surprisingly, many of the defensins have a microbicidal activity on contact.

The defensins defined by SEQ ID NO:1 through 47 are defined in Tables 1 and 2.

The present invention encompasses the treatment of any internal surface tissue or membrane which is not external skin, hair or nails. In an embodiment, the defensin is defined by the consensus amino acid sequence SEQ ID NO:24 or a functional natural or synthetic derivative or variant thereof which includes a defensin having at least 80% similarity to SEQ ID NO: 24 after optimal alignment wherein SEQ ID NO:24 has an optional N—terminal alanine residue. Examples include SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and their variants with an N—terminal alanine residue (SEQ ID NO: 25 though 27, respectively). Reference to "external skin" does not exclude subcutaneous layers of external skin or a e wound.

Examples of microbial infection include infection by fungi and bacteria. These include ions by Candida such as of mucosal membranes (e.g. thrush), the gastrointestinal tract and the blood stream, infections by Cryptococcus such as meningitis, infections by Aspergillus such as respiratory ions, subcutaneous infections such as mucorymycosis, bacterial gastroenteritis, respiratory ion, wound ion and ion leading to a sexually transmitted disease. Also included are infections by Ecoli, such as diarrhoea and urinary tract infections and by Bacillus spp. such as ear ions, meningitis, urinary tract infections and septicaemia.

The defensins contemplated for use in the treatment protocol are defined by the amino acid sequence selected from SEQ ID NO:l through 47 listed in Table l and further characterized in Table 2 as well as a defensin having at least 80% similarity to any one of the SEQ ID NO:l through 47 after optimal alignment. For the sake of brevity, reference to “SEQ ID NO:l through 47” hereinafter includes defensins having at least 80% similarity to any one of SEQ ID NO:l through 47. In an embodiment, the defensin is a permeabilizing defensin or a functional l or synthetic derivative or variant thereof. Examples of synthetic ts include where a LooplB from a Class I defensin replaces the LooplB from the Solanaceous Class II defensin. These include HXP4, HXP34 and HXP35. Other variants or derivatives include a defensin listed in Table 2 or having at lest 80% similarity to a defensin listed in Table 2 wherein the defensin ses an alanine residue at its N— terminus (i.e. SEQ ID NO:25 h 47). The addition of an alanine at the N—terminus of a defensin allows the peptide to be produced recombinantly in the Pichia pastoris expression system without the need for the STEl3 protease site. The STEl3 site normally allows for efficient proessing of the 0t—mating factor secretion signal by KEX2. However, under high expression loads the STEl3 protease cleavage can be inefficient g to Glu— Ala s remaining on the N—terminus of the peptide. The additional negative charge red by these s can be detrimental to the activity of plant defensins. The STEl3 protease site can be ed with an alanine to prevent incomplete processing (Cabral er al., (2003) Protein Expres Purif 3](]):llS—l22). The presence of an N—terminal alanine can also decrease the ability of the defensin to lyse red blood cells (WO201 1/ 16074).

In an embodiment, a defensin listed in Table 2 (SEQ ID NO:l through 47) is modified to enhance the stability of the peptide. In a further embodiment this is achieved by replacing amino acids that are susceptible to deamidation such as asparagine and glutamine, or amino acids that are susceptible to isomerization such as aspartic acid, with amino acids that are not susceptible to cations. In a particular embodiment, the defensins HXLOOS, HXL035 or HXL036 are modified at positions 18, 36 or 42.

In an embodiment, a defensin listed in Table 2 (SEQ ID NO:1 through 47) is modified to se the positive charge of the peptide. Positive charge is known to be important for the ty of antimicrobial es, including plant defensins am er al., (2011) PLoS One 6.4: ). In an embodiment the increase in positive charge is achieved by replacement of a negatively charged residue such as glutamatic acid or ic acid with a neutral amino acid. In a preferred embodiment the l amino acid is an alanine or a glycine. In another embodiment, the increase in positive charge is achieved by replacing a neutral amino acid with a positively charged residue such as lysine or arginine.

In an embodiment, the treatment es a defensin in combination with a non— defensin peptide, a proteinase inhibitor, another defensin or a proteinaceous or non— proteinaceous (i.e. chemical) icide including an antibiotic.

In an embodiment, the defensin is provided by any means including by oral, inhalation, intravenous, sublingual, intraperitoneal, rectal or subcutaneous administration.

Alternatively, it is topically applied to an internal tissue, surface or membrane or to a wound. The defensin can also be administered via a slow release patch or implant (e.g. a subcutaneous implant). The defensins can also be coated on the surface of medical devices such as catheters and ts or condoms or other sheaths.

Further taught herein is a formulation or extract comprising a plant defensin selected from SEQ ID NO:1 h 47. The formulation or t may further comprise another active agent or a combination of formulations or extracts wherein at least one formulation or extract comprises a defensin as defined herein which are admixed prior to use or used sequentially in either order. The plant defensins or extracts comprising same as defined herein may be used such as in the form of systemic or local formulations, coatings, gels, ointments, cream, spray, foam, capsule, tablet, oral formulations, inhalable or atomized formulations and the like ing herbal formulations and plant extracts.

Enabled herein is a use of a plant defensin as defined by SEQ ID NO:1 through 47 in the manufacture of a medicament for the treatment or prophylaxis of microbial infection of in vivo tissue in a subject. Also taught herein is a plant defensin as defined by SEQ ID NO:1 through 47 for use in the treatment or laxis of microbial infection of in vivo tissue in a subject. Further taught herein is a plant defensin as d by SEQ ID NO:1 through 47 When used in the treatment of microbial infection of in vivo tissue in a subject.

In an ment, the defensin has no adverse ty or medically able minimal activity against mammalian cells. Activity against mammalian cells will increase the likelihood of dermal irritation and other side effects.

As indicated above, the in may also be a onal natural or synthetic derivative or variant of a defensin as defined by any one of SEQ ID NO: 1 through 47.

The subject may be a human or non—human animal subject.

Further contemplated herein is an isolated microorganism engineered to express a in as defined herein for use in the manufacture of compositions comprising the microorganisms. In an embodiment, the microorganism is a yeast such as but not limited to Pichia. Such compositions are useful in the treatment of humans and animals such as in the form of a probiotic. Alternatively, the in is provided as a cell extract including a plant extract or microbial extract.

A kit in compartmental form comprising a plant defensin or a functional natural or synthetic derivative or variant thereof, the plant defensin selected from the list consisting of SEQ ID NO:1 through 47, is also taught herein. In an optional embodiment, another compartment comprises a second active agent and optionally bly in a further compartment or together in an existing compartment, a pharmaceutically or veterinarily acceptable diluent, carrier or excipient. In an embodiment, the defensin is defined by the consensus amino acid sequence set forth in SEQ ID NO: 24. Examples include HXLOOS (SEQ ID NO: I), HXL035 (SEQ ID NO: 2), HXL036 (SEQ ID NO: 3) and the N—terminal alanine forms, SEQ ID NO: 25 though 27, respectively.

In an ment, a defensin contemplated for use herein may or may not comprise an N—terminal e residue. This is particularly the case with some recombinant defensins which comprise the N—terminal alanine e. Encompassed by the definition of a "defensin" herein is any of SEQ ID NO:1 though 23 with an N—terminal alanine. These are ented as SEQ ID NO:25 through 47. The consensus amino acid sequence, SEQ ID NO: 24, has an al N—terminal alanine residue.

The addition of an alanine at the N—terminal of a defensin allows the peptide to be produced predominantly in Pichia pastoris expression system without need for the STEl3 se site. (Cabral er al. (2003) Protein Express Purif 3](]):llS—l22). The presence of the N—terminal alanine can also decrease the ability of a in to lysine red blood cells.

In an embodiment, a defensin listed in Table 2 (SEQ ID NO:1 through 47) is modified to enhance the stability of the peptide. In a r embodiment, this is achieved by replacing amino acids that are susceptible to deamidation such as asparagine and glutamine, or amino acids that are susceptible to isomerization such as aspartic acid, with amino acids that are not susceptible to modifications. In a particular embodiment, the defensins HXLOOS, HXL035 or HXL036 are modified at positions 18, 36 or 42.

In an ment, a defensin listed in Table 2 (SEQ ID NO:1 h 47) is modified to increase the positive charge of the peptide. Positive charge is known to be important for the ty of antimicrobial peptides, including plant defensins. In an embodiment the increase in positive charge is achieved by replacement of a negatively d residue such as glutamatic acid or aspartic acid with a neutral amino acid. In an embodiment, the neutral amino acid is an alanine or a glycine. In another embodiment, the increase in positive charge is achieved by replacing a neutral amino acid with a positively d residue such as lysine or arginine Conservative amino acid changes are also contemplated herein.

Table 1 Summary ence identifiers SEQUENCE DESCRIPTION ID NO: _—Amino acid sequence of HXLOOS protein _—Amino acid sequence of HXL035 protein _—Amino acid sequence of HXL036 protein _—Amino acid sequence of HXL001 protein _—Amino acid sequence of HXL002 protein Amino acid sequence of HXL003 protein _—Amino acid sequence of HXL004 protein Amino acid ce of HXLOOS protein Amino acid ce of HXL009 protein >—‘ 0 Amino acid sequence of HXL012 protein _—Amino acid sequence of HXL013 protein t—‘ l\) Amino acid sequence of HXL015 protein _—Amino acid sequence of HXL032 protein y—A 4; Amino acid ce of HXL033 protein >—‘ L1] Amino acid sequence of HXL034 protein >—‘ O’\ Amino acid sequence of Nle n >—‘ \1 Amino acid sequence of NsD2 protein >—‘ 00 Amino acid sequence of NaDl protein >—‘ \0 Amino acid sequence of NoD173 protein l\)0 Amino acid sequence of DmAMPl protein l\) >—‘ Amino acid ce of HXP4 protein l\)l\) Amino acid sequence of HXP34 protein l\) U) Amino acid sequence of HXP35 protein l\)4; Consensus SCunl’lCC [\) LII Amino acid sequence of HXLOOS protein + N—terminal alanine l\) O’\ Amino acid sequence of HXL035 protein + N—terminal alanine WO 91790 SEQUENCE DESCRIPTION ID NO: l\)\1 Amino acid sequence of HXL036 protein + N—terminal alanine l\) 00 Amino acid sequence of HXLOOl protein + N—terminal alanine l\) \0 Amino acid sequence of HXL002 protein + N—terminal alanine L») 0 Amino acid sequence of HXL003 protein + N—terminal alanine _—Amino acid sequence of HXL004 protein + N—terminal alanine U) l\) Amino acid sequence of HXLOOS protein + N—terminal alanine b) b) Amino acid sequence of HXL009 protein + N—terminal alanine b) 4; Amino acid sequence of HXL012 n + N—terminal alanine DJ LII Amino acid sequence of HXLOl3 protein + N—terminal alanine _—Amino acid sequence of HXLOlS protein + N—terminal alanine o acid sequence of HXL032 protein + N—terminal alanine b) 00 Amino acid ce of HXL033 protein + N—terminal alanine U) \0 Amino acid sequence of HXL034 protein + N—terminal alanine 4;0 Amino acid sequence of Nle protein + N—terminal e _—4; Amino acid sequence of NsD2 protein + N—terminal alanine 4; l\) Amino acid sequence of NaDl protein + inal alanine 4; b) Amino acid sequence of NoDl73 protein + inal alanine 4;4; Amino acid sequence of DmAMPl protein + N—terminal alanine 4; L11 Amino acid ce of HXP4 protein + N—terminal alanine 4; O’\ Amino acid sequence of HXP34 protein + N—terminal alanine 4;\1 Amino acid sequence of HXP35 protein + N—terminal alanine Amino acid sequences for HXL proteins are recited in US Patent No. 6,911,577 and related patent family members. _ 14 _ Table 2 es ofplant defensins Type 1 (Class I, Class II Reference Pentide Source variant) HXL008 Picramnia Class I SEQ ID N021 HXL035 Picramm'a Class I SEQ ID NO:2 pentarzdra HXL036 Picramnia Class I SEQ ID N033 penlandra 1 1 HXLOOI Zea mays l Class I SEQ ID NO:4 HXLOOZ Triticum aestivum Class I g SEQ ID NO:5 1HXL003 Triticum aestiva—C Class I l SEQ ID N016 m'iconana l Class I SEQ ID NO:7 benthamiana HXL005 Taraxcum kok- —fiass I SEQ ID N0z8 saghyz HXL009 Zea mays Class I SEQ ID N029 ITJXL012 Amaranlhus Class I SEQ ID NO:] 0 ret‘rqflexus HXL013 Glycine max Class I SEQ ID N011] HXL015 Oryza sativa 3 Class I SEQ ID N0112 ‘ HXL032 Triticum aestivum Class I fSEQ ID N0:l3 1 HXL033 Parthenium Class 1 l SEQ ID NO: 14 argenrarum HXL034 Nicoliana Class I 1 SEQ ID N0:15 benthamiana Nle ana Class II SEQ ID NO:16 suaveolens l RECTIFIED SHEET (RULE 91) ISA/AU Type (Class 1, Class II Reference Peptide Source variant) NsD2 Nicotiana Class II SEQ ID NO:l7 suaveolens NaDl Nicotiana alata Class II SEQ ID N0:18 NOD173 Nicotiana Class II SEQ ID NO:l9 occidentalis spp DmAMPl Dahlia merckii Class I SEQ ID N020 HXP4 Artificial t SEQ ID N021 HXP34 Artificial Variant SEQ ID N022 HXP35 Artificial Variant SEQ ID N023 Consensus HXL008 + Ala Artificial Variant SEQ ID N025 HXL035 + Ala Artificial t SEQ ID N026 HXL036 + Ala Artificial Variant SEQ ID N027 HXL001 + Ala Artificial t SEQ ID N028 HXL002 + Ala Artificial Variant SEQ ID N029 HXL003 + Ala Artificial Variant SEQ ID N0:30 HXL004 + Ala Artificial Variant SEQ ID N0:3l HXL005 + Ala Artificial Variant SEQ ID N0:32 HXL009 + Ala Artificial Variant SEQ ID N0:33 HXL012 + Ala Artificial Variant SEQ ID N0:34 HXL013 + Ala Artificial t SEQ ID N0:35 HXL015 + Ala Artificial Variant SEQ ID N0:36 HXL032 + Ala Artificial Variant SEQ ID N0:37 HXL033+ Ala Artificial Variant SEQ ID N0:38 HXL034 + Ala Artificial Variant SEQ ID N0:39 Nle + Ala Artificial Variant SEQ ID N0:40 2015/050294 —16- Type (Class 1, Class II Reference Peptide Source variant) SEQ ID NO:41 SEQ ID NO:42 N0D173 +Ala Variant SEQ ID NO:43 DmAMPl + Artificial Variant SEQ ID NO:44 SEQ ID NO:45 HXP34 + Ala Variant SEQ ID NO:46 HXP35 + Ala Variant SEQ ID NO:47 BRIEF DESCRIPTION OF THE FIGURES s 1(a) through (e) are representations of alignments of amino acids of the various defensins encompassed herein. .

Figures 2(a) through (d) are graphical representations showing the effect of the plant defensins NaDl (dashed line) and HXLOOS (solid line) on the growth of four clinical isolates of Trichophyton rubrum in vitro. Fungal growth was measured by the increase in l density at 595 nm (A595) achieved 72 hours after inoculation of the growth medium and is plotted as a percentage of growth relative to a no—protein control (vertical axis) versus protein concentration , horizontal axis).

Figures 3(a) h (e) are photographic representations of surviving colonies of T. rubrum grown on agar plates after treatment with 100 MM HXLOOS for 72 h. Panels (a) to (d) represent clinical isolates 14—01, 14—02, 14—03 and 13—04 respectively. Surviving colonies are marked with a black rectangle. Panel (e) ents the growth of T. rubrum that has not been d with HXLOOS.

Figure 4 is a representation of an amino acid alignment of HXLOOS, HXL035 and HXL036 and a consensus sequence of these three sequences. Identical amino acids are highlighted in black conserved amino acids are highlighted in grey.

DETAILED DESCRIPTION hout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be tood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any element or integer or method step or group of elements or rs or method steps.

As used in the subject specification, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly es otherwise. Thus, for example, reference to “a defensin” includes a single defensin, as well as two or more defensins; reference to “an WO 91790 —18- agent” es single agent, as well as two or more agents; reference to “the disclosure” includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term “invention”. All such aspects are enabled within the width of the present invention. Any ts and derivatives contemplated herein are encompassed by “forms” of the invention.

Reference to a “defensin” means one or more of the following plant defensins which retains antimicrobial activity. (i) a defensin having the consensus amino acid sequence as set forth in SEQ ID NO:24; (ii) a defensin selected from the group consisting of HXLOOS (SEQ ID NOzl), HXL035 (SEQ ID NO:2) and HXL036 (SEQ ID NO:3); (iii) a defensin selected from the group consisting of HXLOOl (SEQ ID NO:4), HXL002 (SEQ ID NO:5), HXP35 (SEQ ID NO:3), HXL003 (SEQ ID NO:6), HXL004 (SEQ ID NO:7), HXLOOS (SEQ ID NO:8), HXL009 (SEQ ID NO:9), HXLOl2 (SEQ ID NO:lO), HXLOl3 (SEQ ID NO:ll), HXLOlS (SEQ ID NO:l2), HXL032 (SEQ ID NOzl3), HXL033 (SEQ ID , HXL034 (SEQ ID , Nle (SEQ ID NO:l6), NsD2 (SEQ ID NO:l7), NaDl (SEQ ID NO:18), NoDl73 (SEQ ID NO:l9), DmAMPl (SEQ ID NO:20), HXP4 (SEQ ID , HXP34 (SEQ ID NO:22) and HXP35 (SEQ ID NO:23). (iv) a functional naturally occurring or synthetic derivative or variant of any one of SEQ ID NO:1 through 47; (v) a defensin having at least 80% similarity to any one of SEQ ID NO:1 through 47 after optimal alignment; (vi) any one of SEQ ID NO:1 through 47 comprising a N—terminal alanine residue (i.e.

SEQ ID NO:25 through 47); and/or (vii) a defensin having at least 80% similarity to any one of SEQ ID NO:1 through 3 or after optimal ent.

In an embodiment the in is defined by SEQ ID NO: 1 though 3 or contains an N—terminal alanine residue (SEQ ID NO: 25 through 27 respectively). For convenience these defensins are encompassed within the consensus amino acid sequence set forth in SEQ ID No: 24.

Reference hereinafter to a "defensin" or a in "defined herein", means a defensin as listed in paragraphs (i) through (vii) above. The different ins assed by (i) through (vii) represent ent forms of the subject invention.

When the defensin herein is used in combination with a microbicidic agent and that icidic agent is a defensin, then the second defensin may be any in.

A ol is described herein which is used to facilitate management of microbial infection or colonization at ular in vivo anatomical sites or regions in a human or non—human animal subject. The protocol comprises the use of a plant defensin or a functional natural or synthetic derivative or variant thereof to inhibit or otherwise control the growth or colonization of a microorganism on or in in vivo tissue. The present protocol excludes the ent of skin, hair and nails. However, reference to skin does not exclude subcutaneous infection of skin or a surface wound. In an embodiment, the defensin has no adverse activity or medically acceptable minimal activity against mammalian cells. In an optional embodiment, the defensin is used in istic combination with another antimicrobial agent. The latter agent includes a non—defensin peptide, a proteinase inhibitor, another defensin and a proteinaceous or non—proteinaceous (chemical) agent with antimicrobial properties including an antibiotic.

Examples of microbial infection include infection by fungi and bacteria. These include aspergillosis, infection by Candida such as of mucosal membranes (e.g. ), systemic candidiasis, cryptococcosis, subcutaneous infections such as mucorymycosis, bacterial gastroenteritis, respiratory infection, wound infection and an infection leading to a ly transmitted disease.

Enabled herein is a method for inhibiting infection by a microorganism on or in in vivo tissue in a subject, the method comprising contacting the microorganism or tissue comprising the microorganism with an ive amount of a plant defensin or a functional natural or synthetic derivative or variant thereof. In an embodiment, the in vivo tissue is a mucosal or epithelial al surface or tissue or a subcutaneous layer of skin. Hence, taught herein is a method for inhibiting growth or colonization of a microorganism on or in in vivo l or epithelial tissue or a subcutaneous layer, the method sing ting the tissue or microorganism with an effective amount of a plant defensin or a functional natural or synthetic variant or derivative thereof. Generally, the contact is for a time and under conditions sufficient to inhibit growth of the microorganism. The defensins can also be coated on the surface of medical devices such as catheters and implants or condoms or other sheaths.

“Microbial inhibition” includes both iocidic and microbistatic activity, as measured by reduction of microbial biomass (or loss of viability) compared to a control.

Microbial growth can be measured by many different methods known in the art depending on the microorganism. For fungi, for example, a commonly used method of measuring growth of a ntous fungus, entails germinating spores in a suitable growth medium, incubating for a time sufficient to achieve able , and measuring increased optical density in the culture after a specified incubation time. The optical density rises with increased growth. Typically, microbial growth or colonization is necessary for pathogenesis. Therefore, inhibition of pathogen growth provides a suitable indicator for protection from microbial disease, i.e. the greater the inhibition, the more ive the tion. Furthermore, the effectiveness of the microbicidal or microstatic activity can be determined by microscopic examination or culture techniques from a sample or amelioration of e symptoms (e.g. fever, s, tenderness etc.).

Reference to microorganism includes a fungus and a ium.

The treatment protocol includes prophylaxis (i.e. tion) of at risk subjects from infection. A subject “at risk” may be a subject in a particular location or demographic. Hence, “preventing infection” in the present context, means that the human or animal host is treated with the defensin so as to avoid microbial infection or disease symptoms associated therewith or exhibit reduced or minimized or less frequent microbial infection or disease symptoms associate therewith, that are the natural outcome of the host— rganism interactions when compared to the host not exposed to the defensin. That is to say, microbial infection is prevented or reduced from causing disease and/or the associated disease ms. Infection and/or symptoms are reduced by at least about %, 20%, 30%, 40%, 50, 60%, 70% or 80% or greater as ed to a host not so treated with the protocol taught herein. The percentage reduction can be determined by any convenient means appropriate to the host and microorganism. The microorganism may be naturally occurring in the t without being a "pathogen". However, situations can arise where the microorganism multiplies to a level where it becomes pathogenic.

The action of the defensin is to inhibit microbial growth, replication, ion and/or maintenance, amongst other inhibitory ties and/or induce amelioration of ms of microbial infection when the level of rganism or its location in the body (i.e. in the in viva , surface or membrane) results in a pathogenic outcome.

Enabled herein is a method for the treatment or prophylaxis of aspergillosis or a related condition in a subject, the method comprising contacting an infected site on the subject or administering to the subject a plant defensin or a functional natural or synthetic derivative or variant thereof for a time and under conditions sufficient to ameliorate the ms of aspergillosis.

In an embodiment, the microbial infection is infection by a species or strain of Candida.

Enabled herein is a method for the treatment or prophylaxis of a infection of a mucosal membrane in a t, the method comprising contacting an infected site on the subject or administering to the subject a plant defensin or a functional l or synthetic derivative or variant thereof for a time and under conditions sufficient to ameliorate the symptoms of infection.

In an embodiment, the Candida infection leads to thrush.

In an embodiment, the microbial infection is ic candidiasis.

Hence, taught herein is a method for the treatment or prophylaxis of systemic candidiasis in a t, the method comprising administering to the subject a plant defensin or a functional natural or synthetic derivative or variant thereof for a time and under conditions sufficient to rate the symptoms of infection.

In an embodiment, the microbial infection is cryptococcosis.

Hence, taught herein is a method for the ent or prophylaxis of cryptococcosis in a subject comprising contacting an infected site on the subject or administering to the subject a plant in or a functional l or synthetic derivative or variant thereof for a time and under conditions sufficient to ameliorate the symptoms of infection.

In an embodiment, the microbial infection is mucormycosis or a related condition.

According, enabled herein is a method for the treatment or prophylaxis of mucyrmycosis in a subject comprising contacting an infected site on the subject or administering to the t a plant defensin or a functional natural or synthetic derivative or variant thereof for a time and under conditions sufficient to ameliorate the symptoms of infection.

In an embodiment, the microbial infection is a subcutaneous infection. Hence, taught herein is a method for the treatment or prophylaxis of infection of a subcutaneous skin layer in a subject, the method comprising contacting an infected site on the subject or administering to the subject a plant in or a functional natural or synthetic derivative or variant thereof for a time and under conditions sufficient to ameliorate the ms of infection.

In an embodiment, the microbial infection is lasmosis. Hence, taught herein is a method for the ent or prophylaxis of histoplasmosis in a subject comprising contacting an ed site on the subject or administering to the subject a plant defensin or a functional natural or synthetic derivative or variant thereof for a time and under conditions sufficient to ameliorate the symptoms of infection.

In an embodiment, the microbial infection leads to gastroenteritis.

Hence, the present specification is instructional for a method for the treatment or prophylaxis of microbial gastroenterisits in a subject comprising administering to the subject a plant defensin or a functional natural or synthetic tive or variant thereof for a time and under conditions sufficient to ameliorate the symptoms of infection.

In an ment, the ial infection is a respiratory infection. d herein is a method for the treatment or prophylaxis of respiratory infection in a subject, the method comprising administering to the subject a plant defensin or a functional natural or synthetic derivative or t thereof for a time and under conditions sufficient to ameliorate the symptoms of infection.

In an embodiment, the microbial infection is a wound infection.

The subject specification teaches a method for the treatment or prophylaxis of a wound ion in or on a subject, the method comprising contacting the wound on the subject or administering to the subject a plant defensin or a functional natural or tic derivative or variant thereof for a time and under ions sufficient to ameliorate the symptoms of infection.

In an embodiment, the microbial infection leads to a sexually transmitted disease.

Hence, d herein is a method for the treatment or prophylaxis of ly transmitted disease in a subject, the method comprising contacting an infected site on the subject or administering to the subject a plant defensin or a functional l or synthetic derivative or variant thereof for a time and under conditions sufficient to ameliorate the symptoms of infection.

In an embodiment, the in or its functional natural or synthetic derivative or variant is coated on to the surface of a medical device or condom. Examples of medical devices e catheters and implants. Any of the defensins defined herein may be used in these methods such as but not limited to a in defined by the consensus amino acid sequence SEQ ID NO:24 or a onal natural or synthetic derivative or variant thereof which includes a defensin having at least 80% similarity to SEQ ID NO: 24 after optimal alignment or wherein SEQ ID NO:24 has an optional inal alanine residue.

Examples include SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and variants thereof with an N—terminal alanine residue (SEQ ID NO:25 though 27, respectively)..

By “contacting” includes exposure of the rganism or tissue, surface or membrane or site comprising the microorganism to the defensin following administration or application to the human or animal subject. It also includes systemic, local, topical or parenteral administration to the subject or an infected site. Contact may be with a purified plant defensin or formulation comprising same, or a plant extract which comprises the defensin naturally or which has been engineered to produce the defensin. A formulation includes herbal formulations and ceutical formulations. Reference to “contacting” includes the step of administering to a subject or an infected site on a t.

Enabled herein is a formulation comprising a plant defensin or a functional natural or synthetic derivative or variant thereof for use in inhibiting infection by a microrganism on or in in vivo tissue in a human or animal subject.

Further enabled herein a use of a formulation comprising a plant defensin or a functional natural or synthetic derivative or variant thereof in the manufacture of a medicament for inhibiting infection by a rganism on or in in vivo tissue in a human or animal subject.

Also d is a formulation comprising a plant defensin or a functional natural or tic derivative or variant f when used to inhibit infection by a microorganism on or in in vivo tissue in a human or animal subject.

In an embodiment, taught herein is a therapeutic kit comprising a compartment or in tmental form wherein a compartment comprises a plant defensin or a functional natural or synthetic derivative or variant thereof. Second or further compartments may comprise other agents or ents including other antimicrobial agents such as an antibiotic or other microbicidal or microbistatic agent. The contents of each compartment may be d prior to use or used sequentially in any order. Other antimicrobial agents e non—defensin peptides, a proteinase inhibitor, another defensin or a proteinaceous or chemical (non—proteinaceous) antimicrobial agent including an antibiotic.

Reference to a “plant defensin” means those defined herein with reference to Tables 1 and 2. As defined herein, a defensin includes a defensin having at least about 80% similarity to any one of SEQ ID NO:1 h 47. The 80% similarity is determined after optimal alignment and, where necessary, after appropriate spaces are used to optimize the alignment. By “at least 80%” or “at least about 80%” includes 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%. In an embodiment, the defensin is defined by the consensus amino acid sequence SEQ ID NO:24 or a functional natural or synthetic derivative or variant thereof which includes a defensin having at least 80% similarity to SEQ ID NO: 25 after optimal alignment wherein SEQ ID NO:24 has an optional inal alanine residue. Examples include SEQ ID NO;l, SEQ ID NO:2 and SEQ ID NO:3 and their N—terminal alanine variants, SEQ ID NO: 25 through 27, respectively). Some defensins use herein may be referred to as “naturally ing’, “modified”, “variant:9 6‘mutated”, “synthetic derivative or variant” a ric” defensins.

All such defensins retain crobial activity..

Hence, taught herein is a method for inhibiting infection by a microorganism on or in in vivo tissue in a subject, the method comprising contacting the microorganism or tissue sing the microorganism or administering to the subject an effective amount of plant —26- defensin ed from SEQ ID NO:1 h 47 or a functional natural or synthetic derivative or variant thereof or a defensin having at least 80% similarity to any one of SEQ ID NO:1 through 47 after optimal alignment for a time and under conditions sufficient to ameliorate symptoms of the infection. The defensin may have an N—terminal alanine e such as SEQ ID NO:25 through 47.

The term arity" as used herein includes exact identity between compared sequences at the amino acid level. Where there is non—identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In an embodiment, amino acid sequence isons are made at the level of identity rather than similarity.

Terms used to describe sequence relationships between two or more polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", ntage sequence similarity", "percentage sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 amino acid residues in length. Because two polypeptides may each comprise (l) a sequence (i.e. only a portion of the complete amino acid sequence) that is similar between the two polypeptides, and (2) a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing ces of the two polypeptides over a "comparison window" to identify and compare local s of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous es that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the nce sequence (which does not comprise additions or deletions) for optimal alignment of the two ces.

Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, Clustal W2 and TFASTA in the Wisconsin Genetics re Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. ing in the highest tage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. (1997) Nucl. Acids. Res. 25(17).'3389— 3402. A detailed discussion of sequence is can be found in Unit 19.3 of l et al. (1994-1998) In: Current Protocols in Molecular Biology, John Wiley & Sons Inc.

Other alignment software includes BWA (Li and Durbin (2010) Bioinformatics 26: 589- 595) and Bowtie (Langmead et al (2009) Genome Biol 10:R25 and BLAT (Kent (2002) Genome Res 122656—664).

The terms "sequence similarity" and nce identity" as used herein refer to the extent that sequences are identical or functionally or structurally similar on an amino acid— no acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally d sequences over the window of comparison, determining the number of positions at which the cal amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both ces to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence ty" will be understood to mean the "match percentage" calculated by any suitable method or er algorithm using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.

Some defensins used herein may be referred to herein as a “naturally occurring” defensin, a “modified” defensin, a “variant” defensin, a “mutated” defensin or a “chimeric” defensin, depending on its source.

In an embodiment, the defensin is a Class II Solanaceons defensin. In an embodiment, the defensin is modified at the loop region between the first B—strand (B— strand 1) and the oc—helix at the N—terminal end portion of the defensin. In an embodiment, the loop region comprises the 6 amino acids N—terminal of the second invariant cysteine —28- residue or its equivalent. This region is defined as “LooplB”. A Class II Solanaceous defensin is distinguished from other defensins by a relatively conserved inal end n of the mature domain.

Included herein is the use of an artificially created defensin comprising a modified Class II ceous defensin backbone n the loop region between B—strand 1 and the oc—helix on the N—terminal end n is modified by a single or multiple amino acid substitution, addition and/or deletion to te a variant defensin which has anti— pathogen activity. In an embodiment, the loop region is LooplB defined by the 6 amino acid residues N—terminal to the second invariant cysteine residue. Its equivalent region in any defensin is plated herein. In an embodiment, the artificially created defensin comprises a modified Class II defensin.

Examples of suitable defensins are defined by amino acid ce (Table 1) and further characterized in Table 1. These comprise synthetic defensin variants such as HXP4 (SEQ ID NO:21), HXP34 (SEQ ID N022) and HXP35 (SEQ ID NO:23) or the same defensins with an N—terminal alanine residue (SEQ ID NO:45, 46 and, 47, respectively).

Taught herein is a method for inhibiting infection by a microorganism, on or in in viva tissue in or on a subject, the method comprising contacting the microorganism or tissue comprising the microorganism or administering to the subject with an effective amount of a plant in selected from the group consisting of SEQ ID NO:1 h 47 or functional derivative or variant thereof. Generally, the defensin is applied for a time and under conditions sufficient to ameliorate the symptoms of the infection.

The defensin may be provided at a concentration of between 0.1% and 100% w/v, at a frequency of once a day, twice a day, once every two days, once a week, once every two weeks or once a month, for a period of one week, two weeks, three weeks, one month, two months, three months or up to 12 months.

In an optional embodiment, the defensin or its tive or variant is used in combination with another antimicrobial agent. It is proposed that the in and the antimicrobial agent act in synergy. Examples of other agents include a non—defensin antimicrobial peptide, a proteinase tor another defensin or a proteinaceous or non— proteinaceous chemical microbicide including an antibiotic.

Reference to synergy means that the inhibitory effect of a given defensin or other agent alone is greater when both are used er compared to either used alone. Greco er al. (1995) Pharmacol Rev. 47:331—385 define a category of synergy on the basis that the use of two agents in combination has greater ty relative to the additive effects when each is assayed alone. Hence, the definition adopted herein includes all such situations provided that the combined effect of the two agents acting together is greater than the sum of the individual agents acting alone. Furthermore, a combination of agents is deemed istic, as the term is intended herein, if there exists a set of ions, including but not limited to concentrations, where the combined effect of the agents acting together is greater than the sum of the individual components acting alone. Richer (1987) Pestic Sci 19:309—315 describes a mathematical approach to establish proof of y. This approach uses Limpel’s formula for comparing an observed level of inhibition (Io) in the combined ce of two inhibitor agents, X and Y, with an expected additive effect (Ee) resulting from each X or Y acting separately at the same respective concentrations as used to measure their combined effect. Additive percent inhibition, Be, is calculated as X + Y — XY/100 where X and Y are expressed as percent inhibition. Synergism exits where Io > Synergy may be sed as a synergy scale. In an embodiment, a value of up to 14 represents no significant synergy such as 0, l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; a value of from 15 up to 29 represents low synergy such as 15, l6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29; a value of from 30 to 60 represents medium synergy such as , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60; a value greater than 60 represents a high degree of synergy.

By er than 60” includes from 61 to 100 including 61, 70, 80, 90 and 100 and any value in between.

The present method is useful in the treatment or laxis of a t having an infection on or in an in vivo tissue. The defensins defined by SEQ ID NO:1 through 47 are defined in Tables 1 and 2. The present invention encompasses the treatment of any internal e tissue or membrane which is not external skin, hair or nails. Reference to "external skin" does not exclude subcutaneous layers of external skin or a surface wound.

The term ct” includes a human of any age or an animal such as a farm animal (e.g. sheep, pig, horse, cow, donkey, camel, llama, alpaca) or poultry bird (e.g. chicken, duck, turkey, pheasant, peacock), companion animal (e.g. dog or cat), laboratory test animal (e.g. mouse, rat, rabbit, guinea pig or hamster) or captive wild animal (e.g. wild goat).

The microorganism may be a fungus or a bacterium. Reference to a s” includes dermatophytes, yeasts and non—dermatophytic molds (non—dermatophytes).

Dermatophytes include Trichophyton species including Trichophyton rubrum, Trichophyton igitale, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton ense and Trichophyton mentagrophytes, Microsporum fulvum, Epidermophyton floccosum and Microsporum gypseum. Yeasts encompasses Candida species including Candida albicans, Candida glabrata, Candida parasilosis, Candida tropicalis and Candida . Cryptococcus species including coccus neoformans and Cryptococcus gattii, and Malassezia species including Malassezia a, Malassezia furfur, Malassezia dermatis and Malassezia restricta and Penicillium marnefi‘ei. Non— dermatophytic molds include species of Aspergillus including Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger, Rhizopus s including Rhizopus oryzae, Neoscytalidium, Scopulariopsis, Acremonium, Fusarium species including Fusarium solani, Fusarium and oxysporum,, Scytalidium and. Oomycetes include m insidiosum. Reference to a bacterium es Staphylococcus spp, ococcus ssp, ella spp, Proteus spp, E. coli spp, Bacillus spp, Mycobaterium spp, Mycoplasma spp, Bacteroides spp, Fusobacterium spp.

The component or extract is administered with a pharmaceutical carrier, which is non toxic to cells and the individual.

The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous) or systemic. In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like in the case of oral liquid preparations, such as, for example, sions, elixirs and ons; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously ed. If desired, s may be coated by rd aqueous or nonaqueous techniques.

Pharmaceutical compositions of the present invention used for therapy suitable for oral stration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or es or as a solution or a suspension in an aqueous liquid, a non—aqueous liquid, an oil—in—water on or a water—in—oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods e the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the d presentation. For e, a tablet may be prepared by ssion or molding, optionally with one or more ory ingredients.

Compressed tablets may be ed by compressing in a suitable machine, the active ingredient in a free—flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. 2015/050294 The components and/or ts identified of the present invention may be administered orally, parenterally or systemically (including subcutaneous injections, enous, intramuscular, intraperitoneal intrastemal injection, intranasal or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non—toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

When administered by nasal aerosol or tion, these compositions are prepared according to techniques well—known in the art of pharmaceutical formulation and may be prepared as solutions in , employing benzyl alcohol or other le vatives, absorption promoters to enhance bioavailability, arbons, and/or other solubilizing or dispersing agents known in the art.

The defensins of the invention may also be stered in enous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. When administered by injection, the injectable solutions or suspensions may be formulated according to known art, using suitable non—toxic, parenterally—acceptable diluents or solvents, such as mannitol, l,3—butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending , such as sterile, bland, fixed oils, including synthetic mono— or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, these itions may be prepared by mixing the drug with a suitable non—irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.

The ive dosage of the defensins employed in therapy may vary depending on the ular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Thus, the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving tration of drug Within the range that yields efficacy Without toxicity requires a regimen based on the kinetics of the drug's availability to target sites.

This involves a consideration of the distribution, equilibrium, and elimination of a drug.

The defensin may be administered ly to the ed site or provided systemically or by other convenient means generally for a time and under conditions sufficient to ameliorate the symptoms of infection.

Another aspect provides a protocol or method for treating or preventing an animal including a mammalian such as a human subject having in viva tissue ed with a microorganism, the protocol or method comprising stering to the subject or site of infection an antimicrobial effective amount of a composition comprising the plant defensin for a time and under conditions sufficient to treat the infection.

The present defensin may be manufactured based on its amino acid sequence using standard stepwise addition of one or more amino acid residues using, for example, a peptide or protein synthesizer. atively, the defensin is made by recombinant means.

A recombinant defensin may include an additional alanine e at its N—terminus.

Hence, ins contemplated herein may contain the N—terminus alanine residue.

In addition, the defensin may be subject to chemical modification to render the defensin a chemical analog. Such defensin , may exhibit greater ity or longer half life at the point of t with the tissue.

Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, ptide or protein sis and the use of crosslinkers and other methods which impose conformational constraints on the defensin molecule. This term also does not exclude modifications of the defensin, for example, ylations, acetylations, phosphorylations and the like. Included within the definition are, for example, defensins containing one or more analogs of an amino acid (including, for e, unnatural amino acids) or defensins with substituted linkages. Such analogs may have enhanced stability and/or penetrability.

Examples of side chain modifications contemplated by the present invention e modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; ion with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6—trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and xylation of lysine with pyridoxal—5—phosphate followed by reduction with NaBH4.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation ts with ts such as 2,3—butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O—acylisourea ion followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with etic acid or etamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic ide or other substituted ide; formation of mercurial derivatives using 4— chloromercuribenzoate, 4—chloromercuriphenylsulphonic acid, phenylmercury chloride, 2— chloromercuri—4—nitrophenol and other mercurials; carbamoylation with cyanate at alkaline Tryptophan residues may be modified by, for example, oxidation with N— bromosuccinimide or alkylation of the indole ring with 2—hydroxy—5—nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with itromethane to form a 3—nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N—carbethoxylation with diethylpyrocarbonate. es of incorporating ral amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4—amino c acid, 4—amino— 3—hydroxy—5—phenylpentanoic acid, 6—aminohexanoic acid, t—butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4—amino—3—hydroxy—6—methylheptanoic acid, 2—thienyl alanine and/or D—isomers of amino acids. A tional inkers such as the bifunctional imido esters having (CH2)n spacer groups with n = l to n = 6, glutaraldehyde, N—hydroxysuccinimide esters and hetero—bifunctional reagents which usually contain an amino—reactive moiety such as N—hydroxysuccinimide and another group specific—reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide . In addition, peptides can be conformationally constrained by, for example, incorporation of COL and N 0t—methylamino acids, introduction of double bonds between COL and CB atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

Mimetics are another useful group of defensin analog. The term is intended to refer to a substance which has some chemical similarity to the defensin and mimics its anifungal activity. A peptide mimetic may be a peptide—containing molecule that mimics elements of protein secondary structure (Johnson et al., Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al., Eds., Chapman and Hall, New York, 1993). The underlying rationale behind the use of peptide cs is that the e backbone of ns exists chiefly to orient amino acid side chains in such a way as to facilitate activity actions.

As used herein, “comprising” is synonymous with “including,:9 encontaining,” or cterized by,” and is inclusive or nded and does not exclude additional, unrecited ts or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term ising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and s consisting essentially of and consisting of the recited ents or elements. The present disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed .

When a Markush group or other grouping is used herein, all individual members of the group and all combinations and sub—combinations possible of the group are intended to be individually included in the disclosure.

When a range is recited herein, it is intended that all sub—ranges Within the stated range, and all integer values Within the stated range, are intended, as if each nge and integer value was recited.

EXAMPLES Aspects disclosed and enabled herein are now described in the following non— limiting Example.

Methods Purification of ins from Pichia Pastoris A single pPINK—defensin P. pastoris PichiaPink (Trademark) strain 1 colony is used to inoculate 25 mL of BMG medium (described in the Invitrogen Pichia sion Manual) in a 250 mL flask and that is incubated over for 2—3 days in a 30°C shaking incubator (140 rpm). The culture is used to inoculate 200 mL of BMG in a 1 L baffled flask which is placed in a 30°C shaking incubator (140 rpm) overnight. The cells are harvested by centrifugation (1,500 x g, 10 min, 4°C) and resuspended into 1 L of BMM medium in a 5 L baffled flask and incubated in a 28°C shaking incubator for 3 days (2 days for NaDl). The cultures are induced at t=24 and 48h. The expression medium is separated from cells by centrifugation (6000 rpm, 20 min, 4 °C). The medium is adjusted to pH 3.0 before it is applied to an SP Sepharose column (1 cm x 1 cm, Amersham Biosciences) pre— equilibrated with 100mM potassium ate buffer, pH 6.0. The column is then washed with 100 mL of 100mM potassium phosphate buffer, pH 6.0 (wash x 2 for HXL004).

Bound protein is eluted in 10x10 mL of 100mM potassium ate buffer containing 500 mM NaCl. A dot blot is performed to identify factions with the t concentration of eluted protein and those fractions are concentrated down to 1 mL using a centrifugal column and washed 5x using sterile milli Q ultrapure water. The n concentration of Pichia—expressed defensin is determined using the bicinchoninic acid (BCA) protein assay (Pierce Chemical Co.) with bovine serum albumin (BSA) as the protein standard.

The defensins contemplated for use herein are listed in Tables 1 and 2 and include defensins having at least 80% similarity to any one of the listed ins after l alignment. —38- s 1(a) through (e) provide an alignment showing the similarity of amino acid sequences between a number of defensins. Figure 4 provides an alignment of amino acids sequences for HXLOO8 (SEQ ID No: 1), HXL035 (SEQ ID No: 2), and HXL036 (SEQ ID No: 3). This alignment issued to generate a consensus amino acid sequence set for the in SEQ ID NO: 24. Any of these sequences may contain an optional N-terminal alanine residue.

EXAMPLE 1 Inhibition ofthe growth of Trichophyton rubrum and Microsporumfulvum in the presence ofa plant defensin Plant ins include a ceous Class II defensin (NaDl), a modified LooplB variant (HXP4) and Class I ins (HXLOOI, HXLOOZ, HXL004. HXLOOS, HXLOOS, HXL009, HXL012. HXL013, HXL015).

The inhibitory effects of the plant defensins on the growth of Trichophyton rubrum.

T. interdigitale, Micrmporum flrlvum and C. albicans (all obtained from the National Mycology Reference Centre, South Australia Pathology at the Women’s and Children's Hospital, Adelaide, lia ) are measured essentially as described by Broekaert et a]. (1990) FEMS Microbiol Lett 69:55-59.

Spores of T. rubrum, T. interdigitale and M fitlvum are ed from sporulating fungus growing on 1/2 strength Sabouraud se agar. Spores were removed from the plates by the addition of 1/2 strength potato dextrose broth (PDB). C. albicans cells are grown in Yeast Peptone Broth (YPD) for 16 h. Spore and cell concentrations are measured using a tometer.

Antifungal assays are ted in 96 well microtitre plates essentially as herein described. Wells are loaded with 20 u L of filter sterilized (0.22 u m syringe filter.

Millipore) defensin (10x stock for each final concentration) or water and 80 u L of 5 X 104 spores/mL (T. , T, interdigitale, M. fulvum) or 5,000 cells/mL (C. albicans) in 1/2 strength PDB. The plates are incubated at 300C. Fungal growth is assayed by measuring optical y at 595nm (A595) using a microtitre plate reader (SpectraMaX Pro MZ; RECTIFIED SHEET (RULE 91) ISA/AU Molecular Devices. Growth is allowed to proceed until the optical density (OD) of the fungus in the absence of any test defensin reached an OD of 0.2. Each test is performed in duplicate.

After incubation for 72 h, the media from wells containing clinical isolates of T. rubrum incubated with 100 g/mL HXLOOS was plated onto fresh Sabouraud dextrose agar.

Plates were incubated at 30°C for 5 days to allow colonies to develop before being photographed.

The results of the inhibition assays are shown in Table 3. HXLOOS, HXLOOS and HXL035 are the most effective plant defensins across the range of fungal pathogens.

HXLOOl and HXL009 did not y any ty at the concentrations tested. HXL002 and NaD2 are very poor inhibitors of M. fulvum and C. albicans. HXL004, HXLOl2, HXLOl3 and HXLOlS display intermediate activity across the range of pathogens.

The results of inhibition of clinical isolates of T. rubrum by HXLOOS (solid line) and NaDl (dashed line) are shown in Figures 2(a) to 2(d). Both peptides inhibited fungal growth at low concentrations with ICSOs of below 20 ug/mL against four clinical es.

However, in all cases HXLOOS ted growth at a lower concentration than NaDl.

The results of cell survival assays for clinical isolates of T. rubrum are shown in Figures 3(a) to 3(e). Plates that had been ated with cells that had not been incubated with a plant defensin were almost completely covered in . In st, plates that were inoculated with cells had been incubated in the presence of HXLOOS for 72 h only had l—3 colonies indicating that almost all the cells were dead.

TABLE 3 1C50 “.350 {C(53) 153-50 Baffin sin against '3“. againsi M. against 5’ against (.7, min‘zmz uf’zti‘mm inmrdigimie albicazrs ( 3,3, gh'rfl...) ggigin'11_3)( 331 gin'11_,) (53 ghnL) E-EXMK) 1 >MM :> 100 35 HXLGGZ 53’) 35 HX [.003 > 1 {)3} >103) HXUKM i2, 1‘) ‘26:) Lu 1!! 2?; HXLOGS Lu L33 n HXLGG?) 3 333.3 HXL‘Z} 1 2 42 HKB}? 3 :23; HXLO 1 5 3mm(EN U4! ‘Ji HX3,935 1%.} i. 8 NaDZ 4'3 N233} as E0 29 EXAMPLE 2 Inhibition ofthe growth al pathogens in the presence ofa plant defensin Plant defensins include a ceons Class II defensin (NaDl) and Class I defensins (HXL001, HXL002, HXL003, HXL004, HXL005, HXL008, HXL009, HXL012, , HXL015, HXL035, NaD2).

The tory effects of the plant defensins on the growth of Candida albicans, Aspergillus fumigatus (obtained from the National Mycology Reference Centre, South Australia ogy at the Women’s and en's Hospital, Adelaide, lia ), C. glabrata, C. tropicalis, A. flavns, A. niger, A. fumigatns, Cryptococcus neoformans and C. gattii (obtained from Dee Carter, University of Sydney, New South Wales, Australia) are measured essentially as described by Broekaert et al. (1990) FEMS Microbiol Lett 69:55— Spores of A. flavns, A. niger and A. fumigatns are isolated from sporulating fungus g on 1/2 strength Sabouraud dextrose agar. Spores were removed from the plates by the addition of 1/2 th potato dextrose broth (PDB). C. albicans, C. glabrata, C. alis, C. neoformans and C. gattii cells are grown in Yeast Peptone Broth (YPD) for 16 h. Spore and cell concentrations are measured using a hemocytometer.

Antifungal assays are conducted in 96 well microtitre plates essentially as herein described. Wells are loaded with 20 u L of filter sterilized (0.22 u m syringe filter, Millipore) defensin (10X stock for each final concentration) or water and 80 u L of 5 X 104 spores/mL (A. flavns, A. niger, A. fumigatns), 5,000 cells/mL (C. albicans, C. glabrata, C. tropicalis) or 1 X 106 cells/mL (C. neoformans, C. gattii) in 1/2 strength PDB. The plates are incubated at 30°C. Fungal growth is assayed by measuring optical density at 595nm (A595) using a microtitre plate reader (SpectraMaX Pro M2; Molecular Devices. Growth is allowed to proceed until the optical density (OD) of the fungus in the absence of any test defensin reached an OD of 0.2. Each test is performed in duplicate.

The results from the antifungal assay are presented in Table 4.

TABLE 4 1650 1*”«5r:- {£50 , “~50., laww {Cs-:1 16:50 Defens 11 against {7. 231111351: C. against C. against: A. against: A. ages 11131 A. against C. againsi (.7. afbimfis 31%?)mm impica 3:13: flaws n igu r fidfi’iigai 11.3; neqz‘mmmw gt; 1 2’5 i (1: gx'mL) 1’1: gz’mL) ( 11 315/111le 1’_p E/mL’i (11 gme) {11 131mm HXIJXN .351 6 > C-(C! >1 {)0 > 1(be- 1 1:) 41> H); {.002 GE? 2!} EB \Ia “106 >101 :> i {)0 SL > 01".: 4-3 21‘) HXIJikfi-S HKLOGe‘é H}; {.009 (I: l—DLLU 1 2 L‘x’! HXLG 1 3. 12 T» 1 {JC- 1.5 113411015 HXL035 10 NeaDZ r} .-‘ 2;3 4>Lh 20 N3D 1 14 Lu 2015/050294 EXAMPLE 3 Inhibition ofthe growth ofEscherichia coli and Bacillus subtilis in the presence ofplant defensins A single E. coli or B. subtilis colony was used to inoculate 5 ml of Luria—Bertani media and grown overnight at 37°C. The following day, the optical y of the culture was measured and the bacteria diluted to an l y at 600 nm (OD600) of 0.01 in Mueller— Hinton Broth. Diluted E. coli or B. subtilis were added to 96—well plates with defensins at concentrations of 20 uM, 10 uM, 5 uM, 2.5 uM, l.25 uM, 0.625 uM or 0.3125 uM. Plates were read at OD595 to obtain time zero data points. Plates were incubated at 37°C for 18 hours before reading again at OD595 to assess the amount of bacterial growth.

The results of inhibition of E. coli and B. subtilis are shown in Table 5. The plant in HXL004 inhibited the growth of both E. coli and B. subtilis with IC50s of 2.5 and 2.6 uM tively. This activity is similar to the LL37 control for E. coli. HXL012 and HXL013 inhibited growth of B. subtilis with IC50s of 20 and 10 uM respectively. The defensins HXLOOl, HXL002, HXL003, HXL005, HXL008 and NaDl did not inhibit the growth of E. coli or B. subtilis at the concentrations tested.

Table 5 IC50 against IC50 against B.

E. coli ( M) subtilis( M) HXLOOI >20 >20 HXL002 >20 >20 HXL003 >20 >20 HXL004 2.5 2.6 HXL005 >20 >20 HXL008 >20 HXL009 >20 >20 HXL012 >20 20 HXL013 >20 10 NaDl >20 LL37 2.5 Those skilled in the art Will iate that the disclosure described herein is susceptible to variations and modifications other than those ically described. It is to be understood that the disclosure contemplates all such variations and modifications. The disclosure also enables all of the steps, features, compositions and compounds ed to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features or compositions or compounds.

BIBLIOGRAPHY ul et al. (1997) Nucl. Acids. Res. 25(17).'3389—3402 Ausubel et al. (1994—1998) In: Current Protocols in Molecular Biology, John Wiley & Sons Inc Bloch and Richardson (1991) FEBS Lett :101—104 Broekaert et al. (1990) FEMS Microbiol Lett 69:55—59 Cabral et al., (2003) Protein Expres Purif3](]):115—122 Colilla et al. (1990) FEBS Lett 270(1-2):191—194 Fisher et al. (1981) The American Journal ofMedicine 71 (4):571—7 Gomes et al. (2011) Clinical Microbiology Reviews 24(2):411—45 Greco et al. (1995) col Rew —385 Janssen et al. (2003) Biochemistry 42(27):8214—8222 Johnson et al., Peptide Turn Mimetics in Biotechnology and Pharmacy,Pezzuto et al., Eds., Chapman and Hall, New York, 1993 Kent (2002) Genome Res 12: 656—664 Langmead et al (2009) Genome Biol 10:R25 Li and Durbin (2010) Bionformatics 26:589—595 —46- Mitchell & Perfect (1995) al Microbiology Reviews 8(4):515—48) Patterson et al. (2000) Medicine 79(4):250—60 Richer (1987) Pestic Sci 19:309-315 Sagaram et al., (2011) PLoS One 6.4: e18550 Singh et al. (2014) Journal of Oral and Maxillofacial ogy I8(Supp]):SSl—SS5 Supparatpinyo et al. (1996) Lancet 344(8915): 1 10—3 Thianprasit et al. (1996) Current Topics in l Mycology 7(1):43—54 Thomas (2003) Eye (London, d) I 7(8)2853—62 US Patent No. 6,911,577 (Pioneer Hi—Bred International, Inc. and BI. DuPont DeNemours and Company) Wanachiwanawin et al. (2004) Vaccine 22(27—28):3613—21 van der Weerden et al. (2013) Cell Mol Life Sci 70(19) 3545—3570 van der Weerden et al. (2013) Fungal Biol Rev 26: 121—131 Vibhagool et al. (2003) Clinical Infectious Diseases 36(10):1329—31

Claims (18)

CLAIMS :

1. A method for inhibiting infection of a microorganism on or in in vivo tissue in or on a non-human animal subject, said method comprising contacting the microorganism or tissue comprising the microorganism or administering to the subject an effective amount of plant defensin selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 or a functional natural or synthetic derivative or t f or a defensin having at least 80% similarity to any one of SEQ ID NO:1,SEQ ID NO:2 and SEQ ID NO:3, after l alignment for a time and under conditions sufficient to ameliorate symptoms of the infection, wherein the microorganism is Candida spp., and wherein the in vivo tissue is mucosal or epithelial internal .

2. The method of Claim 1, wherein the infection of the mucosal or epithelial al tissue or a subcutaneous layer of skin is oral or vaginal thrush. .

3. The method of Claim 1 or Claim 2, wherein the defensin is defined by the consensus amino acid sequence SEQ ID NO:24.

4. The method of any one of Claims 1 to 3, wherein the defensin comprises an N- terminal e.

5. The method of Claim 4, n a defensin with an N-terminal alanine is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

6. The method of any one of Claims 1 to 5, wherein the defensin is used in combination with an antimicrobial agent.

7. The method of any one of Claims 1 to 6, wherein the antimicrobial agent is a peptide of from about 0.4 to about 12 kD or a proteinase inhibitor.

8. A l coating for a medical device or condom comprising a plant defensin selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, or a functional natural or synthetic derivative or variant thereof or a defensin having at least 80% rity to any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 after optimal alignment.

9. The topical coating of Claim 8, wherein the defensin is defined by the consensus amino acid sequence SEQ ID NO:24.

10. The topical coating of Claim 8 or Claim 9, n the defensin comprises an N-terminal e.

11. The topical coating of Claim 10, wherein a defensin is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

12. Use of a plant defensin ed from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, or a functional natural or synthetic derivative or variant thereof or a defensin having at least 80% similarity to any of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 after optimal ent in the manufacture of a medicament for the treatment or prophylaxis of microbial infection on or in an in vivo tissue in a human subject, wherein the microbial infection is a Candida spp. infection and wherein the in vivo tissue is l or epithelial internal tissue.

13. The use of Claim 12, wherein the defensin is defined by the consensus amino acid sequence SEQ ID NO:24.

14. The use of Claim 12 or Claim 13, wherein the defensin comprises an N- terminal alanine.

15. The use of Claim 14, wherein a defensin is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

16. The use of any one of Claims 12 to 15, wherein the defensin is formulated for administration in combination with an antimicrobial agent.

17. The use of any one of Claims 12 to 16, wherein the antimicrobial agent is a peptide of from about 0.4 to about 12 kD or a proteinase inhibitor.

18. The use of any one of Claims 13 to 17, wherein the defensin is formulated for g onto a medical device or condom.