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Title:
METHODS OF MODIFYING FUNGI AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2018/182515
Kind Code:
A1
Abstract:
The present disclosure provides a method for reducing the virulence of a fungus and/or improving the competitive fitness of a fungus in one or more host environment, comprising (i) subjecting a host organism to a treatment for partially or completely removing gut microbiota or providing a host organism free of gut microbiota, (ii) inoculating a fungus into the digestive system of the host organism to allow the fungus to colonize the gastrointestinal tract of the host organism, and (iii) collecting the gastrointestinal tract discharge of the host organism to obtain a fungus with reduced virulence and/or improved competitive fitness in one or more host environment. In particular, the treatment for removing gut microbiota involves treatment with one or more antibiotic agents and one or more antifungal agents, and the fungus is Candida albicans. Also disclosed are a vaccine comprising the fungus obtained using said method, and uses thereof.

Inventors:
PAVELKA NORMAN XAVER (SG)
TSO HOI WAN GLORIA (SG)
SEM XIAO HUI (SG)
Application Number:
PCT/SG2018/050142
Publication Date:
October 04, 2018
Filing Date:
March 27, 2018
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
AGENCY SCIENCE TECH & RES (SG)
International Classes:
C12N1/36; A61K36/06
Other References:
PRIETO D. AND PLA J.: "Distinct stages during colonization of the mouse gastrointestinal tract by Candida albicans", FRONT MICROBIOL, vol. 6, 5 August 2015 (2015-08-05), pages 792.1 - 792.10, XP055547618, [retrieved on 20180518]
PANDE K. ET AL.: "Passage through the mammalian gut triggers a phenotypic switch that promotes Candida albicans commensalism", NAT GENET, vol. 45, no. 9, September 2013 (2013-09-01), pages 1088 - 1091, XP055547622, Retrieved from the Internet [retrieved on 20180518]
CHEN C. ET AL.: "An iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis", CELL HOST MICROBE, vol. 10, no. 2, 17 August 2011 (2011-08-17), pages 118 - 135, XP028264964, [retrieved on 20180518]
VAUTIER S. ET AL.: "Candida albicans colonization and dissemination from the murine gastrointestinal tract: the influence of morphology and Th17 immunity", CELL MICROBIOL, vol. 17, no. 4, April 2015 (2015-04-01), pages 445 - 450, XP055547636, [retrieved on 20180518]
PRIETO D. ET AL.: "Adaptation of Candida albicans to commensalism in the gut", FUTURE MICROBIOL, vol. 11, no. 4, 12 April 2016 (2016-04-12), pages 567 - 583, [retrieved on 20180518]
WANG X.-J. ET AL.: "Vaccines in the treatment of invasive candidiasis", VIRULENCE, vol. 6, no. 4, 6 January 2015 (2015-01-06), pages 309 - 315, XP055415422, [retrieved on 20180518]
BOHM L. ET AL.: "The yeast form of the fungus Candida albicans promotes persistence in the gut of gnotobiotic mice", PLOS PATHOG, vol. 13, no. 10, 25 October 2017 (2017-10-25), pages e1006699.1 - e1006699.26, XP055547654, [retrieved on 20180518]
Attorney, Agent or Firm:
SPRUSON & FERGUSON (ASIA) PTE LTD (SG)
Download PDF:
Claims:
Claims

1. A method for reducing the virulence of a fungus, and/or improving the competitive fitness of a fungus in one or more host environment, the method comprising:

(i) subjecting a host organism to a treatment for partially or completely removing gut microbiota or providing a host organism free of gut microbiota;

(ii) inoculating a fungus into the digestive system of the host organism to allow the fungus to colonize the gastrointestinal tract of the host organism;

(iii) collecting the gastrointestinal tract discharge of the host organism to obtain a fungus with reduced virulence and/or with improved competitive fitness in one or more host environment.

2. The method of claim 1, wherein the gut microbiota comprises bacteria, or bacteria and fungi.

3. The method of claim 2, wherein in case the gut microbiota comprises bacteria and fungi, the method comprises removing fungi and removing bacteria before step (ii), and the method does not comprise removing fungi in steps (ii) and (iii).

4. The method of claim 3, wherein removing fungi and removing bacteria are carried out simultaneously or in sequence.

5. The method of any one of claims 1 to 4, wherein in case the gut microbiota comprises bacteria, the treatment for removing gut microbiota comprises treatment with one or more antibiotic agents; and in case the gut microbiota comprises bacteria and fungi, the treatment for removing gut microbiota comprises treatment with one or more antibiotic agents and one or more antifungal agents.

6. The method of claim 5, wherein the one or more antibiotic agent is selected from the group consisting of Actinomycin D, Amikacin, Amoxicillin, Amoxicillin-clavulanate, Ampicillin, Azithromycin, Aztreonam, Bacitracin, Carbenicillin, Cefepime, Cefixime, Cefoperazone, Cefotaxime, Ceftazidime, Ceftibuten, Ceftriaxone, Chloramphenicol, Ciprofloxacin, Clarithromycin, Clindamycin, Erythromycin, Fosmidomycin,

Gentamicin, Imipenem-cilastatin, Kanamycin, Levofloxacin, Metronidazole,

Moxifloxacin, Neomycin, Novobiocin, Pefloxacin, Penicillin, Piperacillin, Polymyxin B, Prulifloxacin, Roxithromycin, Streptomycin, Sulfamethoxazole-trimethoprim, Tazobactam, Tetracycline Ticarcillin, Ticarcillin-clavulanic acid, Vancomycin, and a mixture thereof.

7. The method of claim 6, wherein the antibiotic agent is a mixture of Penicillin and Streptomycin, or a mixture of ampicillin and gentamycin, or a mixture of

metronidazole and tetracycline.

8. The method of claim 5, wherein the one or more antifungal agent is selected from the group consisting of Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, Econazole,

Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole,

Propiconazole, Ravuconazole, Terconazole, Voziconazole, Abafungin, Amorolfin, Butenafine, Naftifine, Terbinafine, Anidulafungin, Caspofungin, Flucytosine and Micafungin.

9. The method of any one of claims 1 to 8, wherein the fungus to be inoculated into the digestive system of the host organism is pathogenic to the host organism.

10. The method of any one of claims 1 to 8, wherein the fungus to be inoculated into the digestive system of the host organism is non-pathogenic to the host organism.

11. The method of any one of claims 1 to 10, wherein (i) to (iii) are carried out once, twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 18 times, or 20 times, or at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 time, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times, or more.

12. The method of claim 11, wherein when (i) to (iii) are carried out once, the time between (ii) and (iii) is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, or 20 weeks, or at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, or at least 20 weeks, or more.

13. The method of claim 11, wherein when (i) to (iii) are carried out twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 18 times, or 20 times, or at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 time, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times, or more, the time between (ii) and (iii) is 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks, or at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks, or more.

14. The method of any one of claims 1 to 11 and 13, wherein when (i) to (iii) are carried out more than once, a new host organism of the same species is used for each repeat, and (ii) inoculating a fungus into the digestive system of the host organism comprises administering the sample from (iii) of the previous repeat.

15. The method of any one of claims 1 to 14, wherein the fungus is selected from the group consisting of Candida species, Aspergillus species, Cryptococcus species, Histoplasma species, Pneumocystis species, Rhizopus species, Blastomyces species, Coccidioides species, Paracoccidioides species, Penicillum species and Stachybotrys species.

16. The method of claim 15, wherein the fungus is of the genus Candida.

17. The method of claim 16, wherein the fungus is Candida albicans.

18. The method of any one of claims 1 to 17, wherein the host organism is a non-human host organism, and/or wherein the host environment is a non-human host environment.

19. The method of any one of claims 1 to 17, wherein the host organism is a non-human host organism, and/or wherein the host environment is a human host environment.

20. The method of claim 19, wherein the non-human mammalian host organism is selected from the group consisting of mouse, rat, guinea pig, hamster, rabbit, non- human primates, cat, dog and pig.

21. The method of claim 20, wherein the non-human mammalian host organism is a mouse (Mus musculus).

22. The method of any one of claims 1 to 21, wherein the host environment is selected from the group consisting of gastrointestinal tract, oral cavity, vaginal mucosa and skin.

23. The method of claim 22, wherein the host environment is the gastrointestinal tract.

24. The method of claim 22, wherein the host environment is the vaginal mucosa.

25. The method of claim 22, wherein the host environment is the oral cavity.

26. The method of claim 22, wherein the host environment is the skin.

27. The method of any one of claims 1 to 26, wherein the host organism is an immunocompromised host organism.

28. The method of claim 27, wherein the immunocompromised host organism lacks functioning T and/or B cells.

29. The method of claim 28, wherein the immunocompromised host organism is a Ragl- deficient mouse. 30. The method of any one of claims 1 to 29, wherein inoculating the fungus into the digestive system of the host organism in (ii) comprises administering the fungus orally or directly inoculating it into the gastrointestinal tract of the host organism.

31. The method of any one of claims 1 to 30, wherein the gastrointestinal tract discharge of the host organism comprises solid and/or liquid discharge.

32. The method of any one of claims 1 to 31, for the generation of fungal strains for biotransformation and for the manufacture of vaccines. 33. The method of claim 1, comprising:

(i) subjecting a mouse to a treatment with one or more antibiotic agents for removing commensal bacteria from the gut;

(ii) inoculating a Candida albicans strain into the digestive system of the mouse to allow the Candida albicans strain to colonize the gastrointestinal tract of the mouse;

(iii) collecting the gastrointestinal tract discharge of the mouse to obtain a Candida albicans with reduced virulence and/or with improved competitive fitness in the gastrointestinal environment. 34. A fungal strain obtained using the method of any one of claims 1-33.

35. A vaccine comprising a fungal strain of claim 34.

36. A method of preventing or treating a disease in a subject in need thereof, wherein the method comprises administering an effective amount of the fungal strain of claim 34, or an effective amount of the vaccine of claim 35.

37. The method of claim 36, wherein the disease is Candidiasis, and wherein the fungal strain being administered is a Candida strain, or the vaccine being administered comprises a Candida strain. 39. The method of claim 36, wherein the disease is one selected from the group consisting of a bacterial infection, a fungal infection, a parasitic infection, a viral infection, and a polymicrobial infection.

40. The method of claim 38, wherein the fungal infection is caused by a fungal genus selected from the group consisting of Absidia, Ajellomyces, Arthroderma,

Aspergillus, Blastomyces, Candida, Cladophialophora, Coccidioides, Cryptococcus, Cunninghamella, Epidermophyton, Exophiala, Filobasidiella, Fonsecaea, Fusarium, Geotrichum, Histoplasma, Hortaea, Issatschenkia, Madurella, Malassezia, Microsporum, Microsporidia, Mucor, Nectria, Paecilomyces, Paracoccidioides, Penicillium, Pichia, Pneumocystis, Pseudallescheria, Rhizopus, Rhodotorula,

Scedosporium, Schizophyllum, Sporothrix, Trichophyton, and Trichosporon. Absidia corymbifera, Absidia spp., Acremonium falciforme, Acremonium kiliense, Acremonium recifei, Acremonium spp., Ajellomyces capsulatus, Ajellomyces dermatitidis, Ajellomyces spp., Allescheria boydii, Alternaria alternata, Alternaria chartarum, Alternaria dianthicola, Alternaria geophilia, Alternaria infectoria,

Alternaria spp., Alternaria stemphyloides, Alternaria teunissima, Anthopsis deltoidea, Aphanomyces spp., Apophysomyces elegans, Armillaria spp., Arnium leoporinum, Arthroderma benhainiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii, Arthrographis cuboidea, Arthrographis kalrae, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus spp., Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Aureobasidium pullulans, Basisdiobolus ranarum, Beauveria bassiana, Bipolaris australiensis, Bipolaris hawaiiensis, Bipolaris spicifera, Bipolaris spp., Blastomyces dermatitidis, Blastoschizomyces capitatus, Botrytis spp., Candida albicans, Candida auris, Candida ciferrii, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr,

Candida krusei, Candida lambica, Candida lipolytica, Candida lusitaniae, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida spp., Candida tropicalis, Candida viswanathii, Candida zeylanoides, Centrospora spp., Cephalosporium spp., Ceratocystis spp., Chaetoconidium spp., Chaetomium atrobrunneum, Chaetomium spp., Chlamydia trachomatis, Chrysosporium inops,

Chrysosporium keratinophilum, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium parvum , Chrysosporium queenslandicum, Chrysosporium spp., Chrysosporium tropicum, Chrysosporium zonatum, Cladophialophora carrionii, Cladophialophora spp., Cladosporium cladosporioides, Cladosporium elatum, Cladosporium herbarum, Cladosporium sphaerospermum, Cladosporium spp.,

Coccidioides immitis, Coccidioides posadasii, Coccidioides spp., Colletotrichium spp., Conidiobolus coronatus, Conidiobolus incongruus, Conidiobolus lamprauges, Conidiobolus spp., Cryptococcus neoformans, Cryptococcus spp., Cryptoporiopsis spp., Cunninghamella bertholletiae, Cunninghamella spp., Curvularia brachyspora, Curvularia clavata, Curvularia geniculata, Curvularia lunata, Curvularia pallescens,

Curvularia senegalensis, Curvularia spp., Curvularia verruculosa, Cylindrocladium spp., Dactylaria spp., Debaryomyces hansenii, Diplodia spp., Emmonsia parva, Emmonsia parva var. crescens, Emmonsia parva var. parva, Emmonsia pasteuriana, Epidermophyton floccosum, Epidermophyton spp., Exophiala castellanii, Exophiala dermatitidis, Exophiala jeansehnei var. heteromorpha , Exophiala jeanselmei var. lecanii-corni, Exophiala moniliae, Exophiala salmonis, Exophiala spinifera, Exophiala spp., Exophila pisciphila, Exserophilium spp., Filobasidiella neoformans, Fonsecaea compacta, Fonsecaea pedrosoi, Fonsecaea spp., Fulvia spp., Fusarium chlamydosporum, Fusarium oxysporum, Fusarium solani, Fusarium spp., Geotrichum candidum, Geotrichum clavatum, Geotrichum fid, Geotrichum spp., Guignardia spp.,

Helminthosporium spp., Histoplasma capsulatum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma spp., Hortaea werneckii, Issatschenkia orientalis, Kluyveromyces lactis, Lacazia loboi, Lasiodiplodia spp., Lecythophora spp., Leptosphaeria australiensis, Leptosphaeria senegalensis, Leptosphaeria spp., Macrophomina spp., Madurella grisae, Madurella mycetomatis, Madurella spp., Magnaporthe grisea, Magnaporthe spp., Malassezia furfur, Malassezia globosa, Malassezia obtuse, Malassezia pachydermatis,

Malassezia restricta, Malassezia sloojfiae, Malassezia sympodialis, Malbranchea pulchella, Malbranchea sclerotica, Malbranchea spp., Microsporum audouinii, Microsporum canis, Microsporum cookei, Microsporum distortum, Microsporum eguinum, Microsporum ferrugineum, Microsporum fulvum, Microsporum gallinae, Microsporum gypseum, Microsporum nanum, Microsporum spp., Microsporum vanbreusegh, Monilinia spp., Mucor circinelloides, Mucor spp., Mycocentrospora acerina, Nectria haematococca, Nectria spp., Neotestudina rosatii, Neotestudina spp., Neurospora crassa, Nigrospora sphaerica, Nigrospora spp., Nocardia asteroides, Nocardia brasiliensis, Nocardia otitidiscaviarum, Nocardia spp., Ochrononis spp., Onychocola canadensis , Onychocola spp., Oospora spp., Ophiobolus spp.,

Paecilomyces lilacinus, Paecilomyces spp., Paecilomyces variotii, Paracoccidioides brasiliensis, Paracoccidioides spp., Penicillium marneffei, Penicillium spp., Penicillium verrucosum, Phaeoannellomyces spp., Phaeosclera dematioides, Phialemonium obovatum, Phialophora europaea, Phialophora spp., Phialophora verruceosa, Phlyctaena spp., Phoma spp., Phomopsis spp., Phymatotrichum spp.,

Phytophthora spp., Pichia anomala, Pichia guilliermondii, Pichia ohmeri, Pichia spp., Piedraia hortai, Piedraia spp., Pneumocystis carinii, Pneumocystis jiroveci, Pneumocystis spp., Pseudallescheria boydii, Pseudallescheria spp., Puccinia spp., Pyrenochaeta romeroi, Pyrenochaeta spp., Pyrenochaeta unguis -hominis, Pythium insidiosum, Pythium spp., Rhinocladiella aquaspersa, Rhizoctonia spp., Rhizomucor pusillus, Rhizomucor spp., Rhizomucor variabilis, Rhizopus microsporus var. rhizopodiformis, Rhizopus oryzae, Rhizopus spp., Rhodotorula rubra, Rhodotorula spp., Saccharomyces cerevisiae, Saccharomyces spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Scedosporium apiospermum, Scedosporium prolificans, Scedosporium spp., Scerotium spp., Schizophyllum commune,

Schizosaccharomyces pombe, Sclerotinia spp., Scopulariopsis brevicaulis, Scopulariopsis spp., scytalidium spp., Sphaerotheca spp., Sporobolomyces salmonicolor, Sporobolomyces spp., Sporothrix schenckii, Stachybotrys chartarum, Stachybotjys sp., Stemphylium macrosporoideum, Syncephalastrum racemosum, Taeniolella boppii, Taphrina spp., Thielaviopsis spp., Torulopsis spp., Trichoderma spp., Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton spp., Trichophyton verrucosum, Trichophyton violaceum, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides, Trichosporon spp., Ulocladium botrytis, Ulocladium chartarum, Ustilago maydis, Ustilago spp., Venturia spp., Verticillium spp., Wangiella dertnatitidis, Wangiella spp., Whetxelinia spp., Xylohypha spp., and Yarrowia lipolytica.

41. The method of claim 38, wherein the bacterial infection is caused by a genus of bacteria selected from the group consisting of Acetobacter, Acinetobacter, Actinomyces, Agrobacterium spp., Azorhizobium, Azotobacter, Anaplasma spp., Bacillus spp., Bacteroides spp., Bartonella spp., Bordetella spp., Borrelia, Brucella spp., Burkholderia spp., Calymmatobacterium, Campylobacter, Chlamydia spp., Chlamydophila spp., Clostridium spp., Corynebacterium spp., Coxiella, Ehrlichia, Enterobacter, Enterococcus spp., Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus spp., Helicobacter, Klebsiella, Lactobacillus spp., Lactococcus, Legionella, Listeria, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium spp., Mycoplasma spp., Neisseria spp., Pasteurella spp., Peptostreptococcus, Porphyromonas, Pseudomonas, Rhizobium, Rickettsia spp., Rochalimaea spp., Rothia, Salmonella spp., Serratia, Shigella, Staphylococcus spp., Stenotrophomonas, Streptococcus spp., Treponema spp., Vibrio spp., Wolbachia, and Yersinia spp.

42. The method of claim 38, wherein the viral infection is caused by one viral family selected from the group consisting of adenoviruses, herpes viruses, poxviruses, parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses, paramyxoviruses, papillomaviruses, retroviruses, and hepadnaviruses.

43. The method of claim 38, wherein the parasitic infection is caused by an infectious parasite selected from the group consisting of a protozoan parasite and a helminths parasite.

Description:
METHODS OF MODIFYING FUNGI AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore application No. 10201702472T, filed on 27 March 2017, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates to mycology, in particular methods of modifying fungal strains, and more particularly methods of modifying fungal strains of the Candida

BACKGROUND OF THE INVENTION

[0003] Over the past century there has been a rising population of immunocompromised patients, which are at an elevated risk to suffer from opportunistic infections. While hospital- acquired fungal infections are less frequent than bacterial infections, they disproportionately account for higher mortality rates, longer hospitalization times and increased healthcare costs. Risk factors for fungal infections are very broad and nonspecific, and they include chronic respiratory disease, cancer, HIV infection, organ transplantation, neutropenia, presence of central venous catheters, prolonged hospital stay, administration of total parenteral nutrition, exposure to invasive procedures, chemotherapy, hemodialysis, gastric acid suppression and use of broad-spectrum antibiotics.

[0004] Candidiasis (caused by Candida species) is the most common opportunistic fungal infection and the fourth most common nosocomial bloodstream infection. Despite the best available standards of care, the incidence of candidemia, a sign of invasive or systemic candidiasis, is on the rise in the US, and mortality rates often exceed 50% despite use of antifungal drugs. This is especially true in intensive care units and in immunocompromised patients, where Candida bloodstream infections are estimated to strike -400,000 patients a year, with an associated mortality of 46-75%.

[0005] A few classes of antifungal drugs, such as azoles, echinocandins, polyenes, allylamines or nucleoside analogues, are available on the market for the treatment of superficial and systemic fungal infections. However, antifungal drug resistance is emerging and increasing in incidence, especially under settings where susceptible patients need to be treated prophylactically or therapeutically for prolonged periods of time. To prevent fungal infections, strategies for the development of an antifungal vaccine have been proposed throughout the years. However, despite the veterinary and medical importance of fungal infections, there currently exists no antifungal vaccine on the market.

[0006] Live attenuated vaccines are known to be very effective against bacterial and viral pathogens. Methods for the generation of fungal strains with reduced virulence would therefore be of great interest as an initial step towards the development of live attenuated antifungal vaccines. Thus, it is an object of the present invention to provide a method to generate live attenuated fungi, in particular those of the Candida genus. Further, in order for a live attenuated fungus to work effectively as a vaccine, the virulence of the fungus should be reduced, and/or the competitive fitness of the fungus in the host environment should be increased. Thus, it is an object of the present invention to provide a method for reducing the virulence of a fungus, and/or improving the competitive fitness of a fungus in one or more host environment.

SUMMARY OF THE INVENTION

[0007] In a first aspect, there is provided a method for reducing the virulence of a fungus, and/or improving the competitive fitness of a fungus in one or more host environment, the method comprising: (i) subjecting a host organism to a treatment for partially or completely removing gut microbiota or providing a host organism free of gut microbiota; (ii) inoculating a fungus into the digestive system of the host organism to allow the fungus to colonize the gastrointestinal tract of the host organism; and (iii) collecting the gastrointestinal tract discharge of the host organism to obtain a fungus with reduced virulence and/or with improved competitive fitness in one or more host environment.

[0008] In a second aspect, there is provided a fungal strain obtained using the method of the first aspect.

[0009] In a third aspect, there is provided a vaccine comprising a fungal strain obtained using the method of the first aspect.

[0010] In a fourth aspect, there is provided a method of preventing or treating a disease in a subject in need thereof, wherein the method comprises administering an effective amount of the fungal strain of the second aspect, or an effective amount of the vaccine of the third aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0012] Figure 1 is a bar chart showing the competitive fitness of different Candida albicans strains in mouse GI tract. C57BL/6J mice were pretreated with a mixture of penicillin and streptomycin in the drinking water for 3-4 days and then orally gavaged with a total of 1x10 C. albicans cells. The C. albicans cells mixture consisted of a 1: 1 ratio of the indicated tested strain (SC5314, wild-type C. albicans, or strains obtained from different serial passaging protocols) and a fluorescently-tagged common competitor strain. Antibiotic treatment continued throughout the experiment. A minimum of 4 mice was used for each strain. The plot displays mean + standard error of the mean (SEM) of relative GI fitness values calculated from each individual competition experiment. The results indicate that all tested evolved strains demonstrated a significantly increased competitive fitness in the mouse GI tract as compared to the unevolved SC5314 wild-type strain.

[0013] Figure 2 is a bar chart showing the results of C. albicans in vitro cytotoxicity assay. Lactate dehydrogenase (LDH) release from J774.1 or HT-29 cells co-cultured for 6 or 24 hours with a set of evolved strains obtained by different serial passaging protocols at a multiplicity of infection (MOI) of 1 or 0.01, respectively (refer to Table 1 for the details of various serial passaging protocols). Values are normalized as a percentage of the positive control (5 mM tert-Butyl hydroperoxide) and presented as the mean + standard deviation (SD) of the different strains tested in each protocol, while for the wild-type strain (SC5314) the value is the mean + SD of three independent experiments. Statistically significant differences relative to the wild-type are indicated (* p < 0.05). The results demonstrate that with the exception of strains obtained with protocol A (a single week-long passage in the mouse gut), all protocols yielded evolved strains that were significantly less cytotoxic in either J774.1 or the HT-29 cells, or both.

[0014] Figure 3 is a bar chart showing the results of C. tropicalis in vitro cytotoxicity assay. Lactate dehydrogenase (LDH) release from J774.1 or HT-29 cells co-cultured for 6 or 24 hours with two C. tropicalis evolved strains obtained by serial passaging protocol D at a multiplicity of infection (MOI) of 1 or 0.01, respectively. Values are normalized as a percentage of the positive control (5 mM tert-Butyl hydroperoxide) and presented as the mean + standard deviation (SD) of the different strains tested in each protocol, while for the wild-type strain (ATCC 13803) the value is the mean + SD of three independent experiments. Statistically significant differences relative to the wild-type are indicated (* p < 0.05). The results demonstrate that both C. tropicalis strains evolved were significantly less cytotoxic in J774.1 and the HT-29 cells.

[0015] Figure 4 is a line graph showing in vivo C. albicans virulence in C57BL/6 mice. C57BL/6J mice were intravenously injected at day 0 with 5xl0 5 cells of a wild-type C. albicans strain (SC5314) or strains obtained from different serial passaging protocols, as indicated. The graph shows the percentage of mice in each group that survived at each day post-injection. The results demonstrate that C. albicans strains obtained through passaging in the mouse GI tract using the protocols as disclosed herein are significantly less virulent in vivo.

[0016] Figure 5 is a line graph showing in vivo C. tropicalis virulence in C57BL/6 mice. C57BL/6J mice were intravenously injected at day 0 with 5x10 cells of a wild-type C. tropicalis strain (ATCC 13803) or one evolved C. tropicalis obtained at week 5 of the evolution. The graph shows the percentage of mice in each group that survived at each day post-injection. The results demonstrate that the evolved C. tropicalis obtained through passaging in the mouse GI tract using the protocol as disclosed herein are significantly less virulent in vivo.

[0017] Figure 6 is a line graph showing in vivo C. albicans virulence in immunocompromised RAG1KO mice. RAGl _/~ mice were intravenously injected at day 0 with 5xl0 5 cells of a wild-type C. albicans strain (SC5314) or strains obtained from different serial passaging protocols, as indicated. The graph shows the percentage of mice in each group that survived at each day post-injection. The results demonstrate that C. albicans strains obtained through passaging in the mouse GI tract using the protocols as disclosed herein are significantly less virulent in immunocompromised mice.

[0018] Figure 7a shows a schematic overview of the different evolution protocols. Figure 7b is a line graph of the numbers of colony forming units per gram of stool for different serial passaging protocols. Data represents mean of 7-8 independent evolution experiments per protocol. Dotted horizontal line represents geometric mean of all week-1 isolates. The results demonstrate that independently of serial passaging frequency and mouse host genotype, evolving C. albicans populations progressively increased their colonization levels over time. Figure 7c is a bar chart showing the competitive fitness of the tested strains in the mouse GI tract. Data are mean + s.e.m. n = 4 tolO mice/group. Mann- Whitney test, *P < 0.0001. The results demonstrate that similar to the efgl ~ mutant, 10- weeks gut-evolved (P10) C. albicans strains achieved increased competitive fitness in the mouse GI tract, as compared to WT (SC5314) and 1-week gut-evolved (PI) strains (Wl and Rl). Figure 7d shows the percentage of smooth colonies (i.e. non-filamentous isolates) in the samples obtained using different serial passaging protocols. Circles: medians, boxes: 1st to 3rd quartile, violin shapes: density estimates. The results demonstrate that percentages of non-filamentous isolates are higher at the end (i.e. week 8 or 10) than after 1 week of the evolution experiment. Figure 7e shows pictures of cellular morphologies of various C. albicans strains (Black scale bar: 200 μιτι; red scale bar: 20 μιη). The results demonstrate that gut-evolved C. albicans strains are defective in hyphal formation in response to in vitro stimuli.

[0019] Figure 8a is a heat map showing clustering of gut-evolved C. albicans strains based on mutational pattern across 87 verified open reading frames (ORFs) carrying de novo, non-synonymous substitutions (NSSs). List of non-synonymous substitutions (NSSs) can be found both in Figure 8a and Table 4). The genes analyzed are ordered based on chromosomal location. The result reveals that recurrent mutations are present in FL08 and other transcription factors required for filamentous growth. Figure 8b demonstrates convergent acquisition of specific mutations in the FL08 gene across multiple independent evolution experiments, clustering around closely located amino acid positions. Figure 8c shows pictures of cellular morphologies of C. albicans strains with and without the FL08 gene (Black scale bar: 200 μιη; red scale bar: 20 μιη). The result demonstrates that flo8 ~ ' ~ mutant is unable to form filament in hyphal-inducing media. Figure 8d is a bar chart showing the competitive fitness of C. albicans strains with and without the FL08 gene in the mouse GI tract. Data are mean + s.e.m. n = 5 mice/group. Mann-Whitney test, *P < 1 0.0001. The result demonstrates that the flo8—/— mutant C. albicans strains is hyper-fit in the mouse GI tract.

[0020] Figure 9a is a bar chart showing the results of C. albicans in vitro cytotoxicity assay. Data are mean + s.e.m. n = 3 independent experiments. Mann-Whitney test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results show that similar to the efgl ~ ' ~ mutant, P10 C. albicans strains with (filled circles) or without (empty circles) FL08- inactivating mutations and the flo8 ~ ' ~ mutant have reduced in vitro cytotoxicity against J774A.1 mouse macrophages and HT-29 human gut epithelial cells, as compared to WT (SC5314 or FL08IFL08) and PI strains (Wl and Rl). Figure 9b shows representative periodic acid-Schiff- stained kidney sections of mice infected with WT (SC5314) or gut- evolved C. albicans strains (W2N, R24). Black scale bar: 200 μιη; red scale bar: 20 μιη. Figure 9c are line graphs showing C. albicans virulence in WT and Ragl-/- adult mice, n = 5-10 mice/group. The results show that similar to the efgl ~ mutant, P10 C. albicans strains with (filled circles) or without (empty circles) LOS-inactivating mutations are less virulent in WT and Ragl _/~ mice, in comparison to WT (SC5314) or PI strains (Wl and Rl). Figure 9c shows that regardless of whether the evolved strains have a mutation in FL08 or not, the evolved strains are still less virulent than wild type. In other words, FL08-inactivating mutations are sufficient but not required for virulence attenuation, meaning that other mutations can also do the job. This result clearly shows that (1) evolution in the gut inevitably lead to virulence attenuation, and (2) that deleting FL08 may be sufficient to generate hyper-fit strains in the mouse gut.

[0021] Figure 10a is a line graph showing the survival rate of WT mice systemically primed with gut-evolved C. albicans strains against C. albicans systemic challenge, n = 10 mice/group. The results show that WT mice systemically primed with P10 C. albicans strains are significantly protected from systemic candidiasis. Survival is significantly higher than mice primed with efgl ~ mutant or with a sub-lethal dose of WT C. albicans cells. Figure 10 a shows evolved strins R24, R2N, and W2N protect both wild type and Ragl "7" mice significantly better than efgl against systemic candidiasis. Figure 10b is a line graph showing the survival rate of Ragl _/~ mice systemically primed with gut-evolved C. albicans strains against C. albicans systemic challenge, n = 5-10 mice/group. Filled circles represent P10 strains harbored LOS-inactivating mutations, while empty circles represent P10 strains that did not harbor LOS-inactivating mutations. The results show that Ragl _/~ mice systemically primed with P10 C. albicans strains are significantly protected from systemic candidiasis. In most cases, survival is significantly higher than that of mice primed with the efgl ~ mutant. Figure 10c is a line graph showing the survival rate of WT mice primed with the P10 C. albicans strain R24 for different durations against systemic C. albicans challenge. n=10 mice/group. The results show that the R24-primed WT mice are significantly protected from systemic candidiasis as early as 1 day post-priming. Figure lOd and lOe are bar charts showing serum (d) and kidney (e) IL-6 levels of WT (n = 11-18 mice/group) and Ragl _/~ mice (n = 4-7 mice/group) infected with live P10 strains (W2N or R24) strains. Kidney cytokine levels were normalized based on organ weight. The results demonstrate that serum (d) and kidney (e) IL-6 levels are elevated at 7 (d) and 28 (e) days post-infection, as compared to mice infected with live or heat-killed (HK) WT (SC5314). Figure lOf and lOg are bar charts showing IL-6 and TNF-a levels in splenocytes extracted from WT mice 28 days after infection with a P10 strain (W2N or R24). Cytokines levels were measured 48 hours post-stimulation, n = 6-10 mice/group. Data are mean + s.e.m. Significant differences are shown only for comparisons against mock- and SC5314-primed mice. The results demonstrate that the splenocytes produce higher levels of IL-6 (f) and TNF-a (g) upon ex vivo stimulation with HK C. albicans. In contrast, the efgl ~ mutant fails to significantly train splenocytes. Figure lOh to lOj are line graphs showing survival rate of WT mice primed with P10 strains (W2N or R24) from systemic challenge with A. fumigatus (h), S. aureus (i) or P. aeruginosa (j). n = 10-11 mice/group. Log-rank test (a-c, h-j) or Mann- Whitney test (d-g). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (a-j). The results demonstrate that the WT mice primed with P10 strains are significantly protected from systemic challenge ih A. fumigatus, S. aureus or P. aeruginosa. In many cases, survival is significantly higher than that of mice primed with the efgl _/~ mutant. Figure lOi shows evolved strains W2N and R2N protect significantly better than efgl against systemic aspergillosis, but not S. aureus challenge. Figure lOj shows evolved strain R24 protects significantly better than efgl against P. aeruginosa.

[0022] Figure 11 shows line graphs of percentage of weight change of WT (a) and Ragl _/~ mice (b) infected systemically with P10 evolved C. albicans strains or the efgl ~ mutant. Data are mean of n = 5-10 mice/group. The results show that similar to mice infected with efgl ~ ~ C. albicans, mice infected by the P10 strains initially lost 10-20% of weight up until 7 dpi, then gradually regained their initial weight at -21 dpi and all mice survived until 28 dpi.

[0023] Figure 12a shows pictures of cellular morphologies of NA1 and NA2 strains (obtained after 9 weeks of evolution in the gut of non-antibiotic-treated mice) on hyphal- inducing stimuli. Black scale bar: 200 μιη; red scale bar: 20 μιη. Figure 12b shows survival of WT adult mice systemically infected with NA1 and NA2 strains. Data are mean + s.e.m. n = 9 mice/group. Figure 12c shows colonization levels of WT pups with SC5314, W2N and R24 strains in the absence of antibiotics treatment (n > 20 mice/group). Figure 12d shows colonization levels of WT pups (n = 10 mice/group) and adults (n = 2-10 mice/group) with SC5314 and efgl ~ in the absence or presence of antibiotics. Mann-Whitney test, *P 6 < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results show that C. albicans strains obtained after 9 weeks of evolution (W9 strains) all retained the ability to respond to hyphal- inducing stimuli as well as their virulence in the intravenous infection model, when antibiotic was not used during the serial passaging process. Figure 12 also demonstrates that antibiotic treatment is useful in steering the evolution in the direction required (for example towards attenuation of virulence).

[0024] Figure 13a shows a line graph of survival of WT mice primed with a lower dose (10 4 CFUs) of WT (SC5314), efgl ~ ' ~ , W2N or R24 strains, challenged with a lethal dose (5 X 10 5 ) of WT C. albicans 28 days post-priming, n = 10 mice/group. The results show that priming with a sub-lethal dose of WT C. albicans SC5314 delayed host mortality but eventually all animals succumbed to the challenge. Figure 13a shows evolved strains W2N and R24 protect significantly better than the efgl strain against systemic candidiasis, even when used at a lower immunization dose, i.e. 10 4 CFUs as opposed to the regular 5 x 10 5 CFUs. Figure 13b and 13c are survival curves of WT (b) and Ragl _/~ mice (c) primed with the efgl ~ mutant or a P10 gut-evolved C. albicans strain, and challenged with a lethal dose (5 X 10 5 ) of WT C. albicans 3 months post-priming, n = 5-10 mice/group. Log-rank test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results show that at 3 months post- priming Ragl _/~ mice were no longer protected by most P10 strains, while WT mice still retained a partial protection. Figure 13b shows that at three months immunization, evolved strain R24 (but not other evolved strains) still protects Ragl "7" significantly better than the efgl strain. Figure 13 further corroborate the idea that the protection was mediated by trained innate immunity, rather than by a more classical adaptive memory.

[0025] Figure 14 shows results of longitudinal analysis of total serum IgG (a) and anti-C. albicans serum IgG levels (b) in WT mice infected with 10 4 CFUs of live WT (SC5314), or with 5 X 10 5 CFUs of heat-killed WT (HK-SC5314) or P10 C. albicans strains (W2N, R24). Data are mean + s.e.m. n = 3 mice/group. The results demonstrate that the increased protection observed in P10 primed mice over those immunized with WT C. albicans correlated with increased total as well as anti-C. albicans- specific immunoglobulin G (IgG) titers in the serum. Figure 14 shows that, at least in wild type mice that do have T and B cells, in addition to trained innate immunity, classical adaptive immune response can also be triggered by the evolved strains. Without wishing to be bound by theory, it is believed that this result shows that evolved strains both (a) classical anti-Candida specific adaptive immunity; and (b) non-specific trained innate immunity.

[0026] Figure 15 shows survival of Ragl _/~ mice primed with W2N or R24, after challenge with A. fumigatus 28 days post-priming, n = 10 mice/group. Log-rank test, ****p < 0.0001. The results show that cross -protection against systemic aspergillosis of mice primed with gut-evolved C. albicans strains is independent of adaptive immunity.

[0027] Figure 16 shows a line graph of survival of WT mice (a) and Ragl _/~ mice (b) primed intramuscularly with a single dose (10 10 CFUs) of WT (SC5314) or R24 strains, challenged with a lethal dose (5xl0 5 ) of WT C. albicans 28 days post-priming, n = 7-10 mice/group. The results demonstrate that the WT mice intramuscularly primed with gut evolved strain are significantly protected from systemic challenge. Figure 16c shows a line graph of survival of WT mice primed intramuscularly with two doses separated by 14 days of the gut evolved strain R24. n = 10 mice/group. The results show that the use of two doses of the gut evolved strain improve the survival of the mice against the systemic challenge. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0028] It has been surprisingly found by the inventors that passaging a fungus in the gastrointestinal tract of a host can produce fungal strains with reduced virulence and/or improved competitive fitness. Thus, in the first aspect, there is provided a method for reducing the virulence of a fungus, and/or improving the competitive fitness of a fungus in one or more host environment, the method comprising: (i) subjecting a host organism to a treatment for partially or completely removing gut microbiota or providing a host organism free of gut microbiota; (ii) inoculating a fungus into the digestive system of the host organism to allow the fungus to colonize the gastrointestinal tract of the host organism; and (iii) collecting the gastrointestinal tract discharge of the host organism to obtain a fungus with reduced virulence and/or with improved competitive fitness in one or more host environment. In some examples, the method further comprises isolating the fungus from the gastrointestinal tract discharge collected, so as to obtain an isolated fungal strain. In effect, the method of the first aspect comprises passaging (for at least one passage) the virulent, wild-type fungus in the digestive system of a host organism in vivo.

[0029] The term "virulence" as used herein refers to the degree of damage or harm caused by an organism to animals or humans. Such animals or humans are often referred to as the host of the organism. Although the term "virulence" is often used interchangeably with the term "pathogenicity", some authorities distinguish these two terms by defining "pathogenicity" as a qualitative term, and by defining "virulence" as a quantitative term. By this standard, an organism may be considered as pathogenic or non-pathogenic in a particular context (for example, the organism being pathogenic or non-pathogenic to a particular host). In some examples, the same species or strain of an organism may be pathogenic or nonpathogenic towards different host organisms. Specifically, a species or strain of an organism may be non-pathogenic towards a healthy host organism, but is pathogenic towards an immunocompromised host organism. In some other examples, different strains of the same species may have different level of virulence towards the same host organism.

[0030] Various methods and/or parameters can be used to indicate the virulence of an organism to a particular host organism. For example, an in vitro cytotoxic assay can be used to assess the degree of damage that an organism can cause to its host cells. In such tests, the lower the cytotoxicity of the tested organism to its host cells, the lower the virulence of the organism towards its host cells is, and vice versa. In another example, an in vivo survival test using animal models can be used to assess the degree of damage that an organism can cause to its host animals. In such tests, the higher the survival rate of the host animals is, the lower the virulence of the organism is, and vice versa. Virulence can also be quantitated by the median lethal dose (LD50) in experimental animals, the numbers of organs (generally spleen or liver) colonized by the organism, and the colony forming units (CFUs) from the infected organs.

[0031] The term "attenuated" as used herein refers to the weakening or decreasing of the virulence of the wild-type organism.

[0032] The term "avirulent" as used herein refers to previously virulent organism that has been attenuated to a sufficient degree such that the administration of the attenuated organism to the animal host would not cause any detectable or measurable disease. Generally, such avirulence can be shown by a decrease in the LD50, the numbers of colonized organs, or the number of CFUs by a factor of 10, or a factor of 100, or by a factor of 1000 or more. [0033] The term "fitness" as used herein refers to the reproductive success of an organism in a given environment, for example, in a given host environment. The term "competitive fitness" as used herein refers to the reproductive success of an organism relative to another organism in the same environment. Competitive fitness can be measured experimentally by co-inoculating a particular environment with a mixture of two strains, of which one serves as a reference and the other as the test strain. The competitive fitness of the test strain can be then calculated from the rate at which the test strain alters its relative proportion in the environment in respect to the reference strain. If the relative proportion of the test strain increases over time in respect to the reference strain, then it is said to be competitively fitter than the reference strain in the tested environment. Conversely, if the relative proportion of the test strain decreases over time in respect to the reference strain, then the test strain is said to be competitively less fit than the reference strain in the tested environment. If a first test strain increases its relative proportion in respect to the reference strain at a faster rate than a second test strain, then the first test strain is said to have higher competitive fitness than the second test strain, and vice versa.

[0034] The competitive fitness of an organism can be represented by a competitive fitness coefficient. The competitive fitness coefficient of a test strain can be obtained by linear regression according to the following formula: log 2 [R(f)/fl(io)] = sy R (t-t 0 ), where R(t) represents the ratio between the test strain and the reference strain at time t; R(to) represents the ratio between the test strain and the reference strain at time to; s is the selection coefficient; JR is the growth rate of the reference strain expressed as cell divisions per hour; t represents the time points in hours and ¾ the initial time point. If the value of y R is known in the given environment, the equation can be solved for the selection coefficient s. Otherwise, the unknown entity SJR is used as the competitive fitness coefficient.

[0035] The term "reduce" or "reduction" or grammatical variants thereof refer to a decrease in the specified parameter as compared to the same parameter of a reference. For example, in the context of "reducing the virulence of a fungus", "reducing" refers to a decrease in the virulence of the fungus, when compared to the virulence of a reference strain of the fungus, such as a wild-type strain of the fungus. In some examples, the virulence of a fungus obtained using a method as disclosed herein is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% as compared to the virulence of a wild-type strain of the fungus. In some examples, the term "fungal strain of reduced virulence" or its grammatical variants can also be defined qualitatively. For example, when a host organism that has been exposed to a fungal strain does not develop the disease or condition usually caused by the wild-type strain of the same fungus, the fungal strain that the host organism has been exposed to can be considered as a fungal strain of reduced virulence. In some examples, reduced virulence may be used herein if the difference with the wild-type reference strains is (statistically) significant. The calculation whether a reduction is (statistically) significant can be determined through known methods in the art.

[0036] The term "improve" or grammatical variants thereof refer to an increase in the specified parameter as compared to the same parameter of a reference. For example, in the context of "improving the competitive fitness of a fungus", "improving" refers to an increase in the competitive fitness of the fungus, when compared to the competitive fitness of a reference strain of the fungus, such as a wild-type strain of the fungus. In some examples, the competitive fitness of a fungus obtained using a method as disclosed herein is increased or improved by at 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% as compared to the competitive fitness of a wild-type strain of the fungus. In some example, increased fitness may be used herein if the fitness coefficient is (statistically) significantly higher than wild- type strains. The calculation whether an increase may be considered (statistically) significant is to be determined through known methods in the art.

[0037] The term "microbiota" as used herein refers to commensal and symbiotic microorganisms found in a host organism. The term "gut microbiota" or "gastrointestinal microbiota" as used interchangeably refers to a community of microorganisms that live in the digestive tracts of the host animals or humans. In some examples, in step (i) of the method as described herein, the gut microbiota to be partially or completely removed from the host organism includes bacteria, or fungi, or a mixture of bacteria and fungi. In cases where the gut microbiota to be removed includes bacteria and fungi, the method includes removing bacteria and fungi before inoculating a fungus into the digestive system of the host organism in step (ii), but the method does not include removing fungi during steps (ii) inoculating a fungus into the digestive system of the host organism to allow the fungus to colonize the gastrointestinal tract of the host organism; and (iii) collecting the gastrointestinal tract discharge of the host organism. Further, in cases where the gut microbiota to be removed includes bacteria and fungi, the removal of fungi and bacteria can be carried out simultaneously, or can be carried out in sequence. For example, when the removal of fungi and bacteria are carried out in sequence, the removal of fungi can be carried out first, followed by the removal of bacteria. Alternatively, the removal of bacteria can be carried out first, followed by the removal of fungi.

[0038] In some examples, wherein in case the gut microbiota to be removed comprises bacteria, or bacteria and fungi, the removal of bacteria is only carried out during step (i) of the method. In some other examples, the removal of bacteria is carried out through steps (i) subjecting a host organism to a treatment for partially or completely removing gut microbiota to (ii) inoculating a fungus into the digestive system of the host organism to allow the fungus to colonize the gastrointestinal tract of the host organism, but not in step (iii) collecting the gastrointestinal tract discharge of the host organism. In some further examples, the removal of bacteria is carried out through steps (i) to (iii) of the method as described herein.

[0039] In some examples, the host organism is free of gut microbiota or no gut microbiota. Examples of host organisms that are free of gut microbiota are germ-free animals. Thus, if germ-free animals are used, removal of gut microbiota would not be a necessary step of the method. As would be understood by the person skilled in the art, host organism that is free of gut microbiota is provided from artificially maintained environment. Thus, depending on the stringency of the artificially maintained environment, in some examples, the host microorganism are substantially free of gut microbiota.

[0040] On the other hand, if the host organism provided has gut microbiota and it is to be treated for completely removing gut microbiota, it would be understood by the person skilled in the art that the host would be "substantially free".

[0041] As used herein, throughout the disclosure, the term "substantially free" refers to both the gut microbiota of a host organism maintained in germ-free environment and to the gut microbiota after a treatment to completely remove the gut microbiota, which as would be understood by the person skilled in the art to be a considerable amount of microbiota has been removed, though 100% removal may not be practically possible.

[0042] In some examples, wherein in case the gut microbiota to be removed comprises bacteria but not fungi, the treatment for removing gut microbiota comprises treatment with one or more antibiotic agents. In some other examples, wherein in case the gut microbiota to be removed comprises bacteria and fungi, the treatment for removing gut microbiota comprises treatment with one or more antibiotic agents and one or more antifungal agents.

[0043] As illustrated in Figure 12, antibiotic treatment may be useful in steering the evolution of strains in the direction required (for example towards attenuation of virulence).

[0044] The term "antibiotic" or "antibiotic agent" as used herein refers to a substance that may either kill or inhibit the growth of bacteria. Examples of antibiotic agent include, but are not limited to, actinomycin D (IUPAC name: 2-Amino-N,N'- bis[(6S,9R,10S,13R,18aS)- 6, 13 -diisopropyl-2,5 ,9-trimethyl- 1 ,4,7 , 11 , 14-pentaoxohexadecahydro- 1 H-pyrrolo [2,1- i][ 1,4,7, 10, 13]oxatetraazacyclohexadecin-10-yl]-4,6-dimethyl-3-oxo-3H-ph enoxazine- 1,9- dicarboxamide), amikacin (IUPAC name: (2S)-4-Amino-N-[(2S,3S,4R,5S)-5-amino-2- [(2S,3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6-(hydroxymethyl)oxa n-2-yl]oxy-4- [(2R,3R,4S,5R,6R)-6-(aminomethyl)-3,4,5-trihydroxy-oxan-2-yl ]oxy-3-hydroxy- cyclohexyl]-2-hydroxybutanamide), amoxicillin (IUPAC name: (2S,5R,6R)-6-{ [(2R)-2- Amino-2-(4-hydroxyphenyl)acetyl]amino}-3,3-dimethyl-7-oxo-4- thia-l- azabicyclo[3.2.0]heptane-2-carboxylic acid), amoxicillin-clavulanate (IUPAC name of clavulanic acid: (2R,5R,Z)-3-(2-Hydroxyethylidene)-7-oxo-4-oxa-l-aza- bicyclo[3.2.0]heptane-2-carboxylic acid, ampicillin (IUPAC name: (2S,5R,6R)-6-([(2R)-2- amino-2-phenylacetyl]amino)-3,3-dimethyl-7-oxo-4-thia-l-azab icyclo[3.2.0]heptane-2- carboxylic acid, azithromycin (IUPAC name: 2R,3S,4R,5R,8R,10R,l lR,12S,13S,14R)-2- ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-15-oxo- l l-{ [3,4,6-trideoxy-3- (dimethylamino)-P-D-xylo-hexopyranosyl]oxy}-l-oxa-6-azacyclo pentadec-13-yl 2,6- dideoxy-3C-methyl-3-0-methyl-a-L-ribo-hexopyranoside, aztreonam (IUPAC name: 2- { [(lZ)-l-(2-Amino-l,3-thiazol-4-yl)-2-{ [(2S,3S)-2-methyl-4-oxo-l-sulfoazetidin-3- yl] amino }-2-oxoethylidene] amino }oxy-2-methylpropanoic acid, bacitracin (IUPAC name: (4R)-4-[(2S)-2-({2-[(lS)-l-amino-2-methylbutyl]- 4,5-dihydro-l,3-thiazol-5-yl}formamido)- 4-methylpentanamido]-4-{ [(lS)- 1-{ [(3S,6R,9S,12R,15S,18R,21S)- 18-(3-aminopropyl)-12- benzyl-15-(butan-2-yl)-3-(carbamoylmethyl)- 6-(carboxymethyl)-9-(lH-imidazol-5- ylmethyl)-2,5, 8, 11,14,17,20- heptaoxo- 1,4,7, 10, 13,16, 19-heptaazacyclopentacosan-21- yl] carbamoyl}- 2-methylbutyl]carbamoyl}butanoic acid, carbenicillin (IUPAC name: (2S,5R,6R)-6-{ [carboxy(phenyl)acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-l- azabicyclo[3.2.0] heptane-2-carboxylic acid), cefepime (IUPAC name: (6R,7R,Z)-7-(2-(2- aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((l-methylpy rrolidinium

oxo-5-thia-l-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate), cefixime (IUPAC name: (6R,7R)-7- { [2-(2- Amino- l,3-thiazol-4-yl)-2-(carboxymethoxyimino)acetyl]amino}-3-eth enyl-8-oxo-5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid), cefoperazone (IUPAC name: (6R,7R)- 7-[(2R)-2- { [(4-Ethyl-2,3-dioxopiperazin- 1 -yl)carbonyl] amino } -2-(4- hydroxyphenyl)acetamido]-3-{ [(l-methyl-lH-l,2,3,4-tetrazol-5-yl)sulfanyl]methyl}-8-oxo-

5- thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid), cefotaxime (IUPAC name: (6R,7R,Z)-3-(Acetoxymethyl)-7-(2-(2-aminothiazol-4-yl)-2-(me thoxyimino)acetamido)-8- oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid), ceftazidime (IUPAC name: (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yl oxyimino)acetamido)-8-oxo- 3-(pyridinium-l-ylmethyl)-5-thia-l-aza-bicyclo[4.2.0]oct-2-e ne-2-carboxylate), ceftibuten (IUPAC name: (6R,7R)-7-([(Z)-2-(2-amino-l,3-thiazol-4-yl)-5-hydroxy-5-oxo pent-2- enoyl]amino) -8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid), ceftriaxone (IUPAC name: (6R,7R)-7-{ [(2Z)-2-(2-amino-l,3-thiazol-4-yl)->2- (methoxyimino)acetyl] amino } -3- { [(2-methyl-5,6-dioxo- 1 ,2,5,6-tetrahydro- 1 ,2,4-triazin-3- yl)thio]methyl}-8-oxo-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid), chloramphenicol (IUPAC name: 2,2-dichloro-N-[(lR,2R)-l,3-dihydroxy-l-(4- nitrophenyl)propan-2-yl]acetamide), ciprofloxacin (IUPAC name: l-cyclopropyl-6-fluoro-4- oxo-7-(piperazin-l-yl)-quinoline-3-carboxylic acid), clarithromycin (IUPAC name: (3R,4S,5S,6R,7R,9R,l lS,12R,13S,14R)-6-{ [(2S,3R,4S,6R) -4-(dimethylamino)-3-hydroxy-

6- methyloxan-2-yl]oxy} -14-ethyl-12,13-dihydroxy-4-{ [(2R,4R,5S,6S)-5-hydroxy -4- methoxy-4,6-dimethyloxan-2-yl]oxy}-7 -methoxy-3,5,7,9,11,13-hexamethyl -1- oxacyclotetradecane-2,10-dione), clindamycin (IUPAC name: methyl 7-chloro-6,7,8- trideoxy-6-{ [(4R)-l-methyl-4-propyl-L-prolyl]amino}-l-thio-L-threo-a-D-g alacto- octopyranoside), erythromycin (IUPAC name: (3R,4S,5S,6R,7R,9R,l lR,12R,13S,14R)-6- { [(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl ]oxy}-14-ethyl-7,12,13- trihydroxy-4-{ [(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy }- 3,5,7,9,1 l,13-hexamethyl-l-oxacyclotetradecane-2,10-dione), fosmidomycin (IUPAC name: 3-[Formyl(hydroxy)amino]propylphosphonic acid), gentamicin (IUPAC name: (3R,4R,5R)- 2-{ [(lS,2S,3R,4S,6R)-4,6-diamino-3-{ [(2R,3R,6S)-3-amino-6-[(lR)-l-

(methylamino)ethyl]oxan-2-yl]oxy}-2-hydroxycyclohexyl]oxy }-5-methyl-4- (methylamino)oxane-3,5-diol), imipenem-cilastatin (IUPAC name of imipenem: (5R,6S)-6- [( 1 R)- 1 -hydroxyethyl] -3 -( { 2- [(iminomethyl)amino] ethyl } thio)-7-oxo- 1 - azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid; IUPAC name of cilastatin: (Z)-7-[(2R)-2- Amino-3 -hydroxy-3 -oxopropyl] sulf anyl-2- { [( 1 S )-2,2- dimethylcyclopropanecarbonyl] amino }hept-2-enoic acid), kanamycin (IUPAC name: 2- (aminomethyl)- 6-[4,6-diamino-3- [4-amino-3,5-dihydroxy-6-(hydroxymethyl) tetrahydropyran-2-yl]oxy- 2-hydroxy- cyclohexoxy]- tetrahydropyran- 3,4,5-triol), levofloxacin (IUPAC name: (S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methylpiperazin-l-yl )- 7-oxo-7H-pyrido[l,2,3-de]-l,4-benzoxazine-6-carboxylic acid), metronidazole (IUPAC name: 2-(2-Methyl-5-nitro-lH-imidazol-l-yl)ethanol), moxifloxacin (IUPAC name: 1- Cyclopropyl-7-[(lS,6S)-2,8-diazabicyclo[4.3.0]nonan-8-yl]-6- fluoro-8-methoxy-4- oxoquinoline-3-carboxylic acid), neomycin (IUPAC name: (2RS,3S,4S,5R)-5-Amino-2- (aminomethyl)-6-((2R,3S,4R,5S)-5-((lR,2R,5R,6R)-3,5-diamino- 2-((2R,3S,4R,5S)-3-amino-

6- (aminomethyl)-4,5-dihydroxytetrahydro-2H-pyran-2-yloxy)-6-hy droxycyclohexyloxy)-4- hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yloxy)tetrahydro- 2H-pyran-3,4-diol), novobiocin (IUPAC name: 4-Hydroxy-3-[4-hydroxy-3-(3-methylbut-2-enyl)benzamido]-8- methylcoumarin-7-yl 3-0-carbamoyl-5,5-di-C-methyl-a-L-lyxofuranoside), pefloxacin (IUPAC name: l-ethyl-6-fluoro-7-(4-methylpiperazin- l-yl)-4-oxo-quinoline-3-carboxylic acid), penicillin (IUPAC name: (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-(2-phenylacetamido)-4- thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid), piperacillin (IUPAC name: (2S,5R,6R)- 6- { [(2R)-2- [(4-ethyl-2,3 -dioxo-piperazine- 1 -carbonyl)amino] -2-phenyl-acetyl] amino } -3 ,3 - dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxyli c acid), polymyxin B (IUPAC name: N-[4-amino-l-[[l-[[4-amino-l-oxo-l-[[6,9,18 ris(2-aminoethyl)-15-benzyl-3-(l- hydroxyethyl)-12-(2-methylpropyl)-2,5,8,l l,14,17,20-heptaoxo-l,4,7,10,13, 16,19- heptazacyclotricos-21 -yl] amino]butan-2-yl] amino] -3 -hydroxy- 1 -oxobutan-2-yl] amino] - 1 - oxobutan-2-yl]-6-methyloctanamide), prulifloxacin (IUPAC name: (RS)-6-Fluoro-l-methyl-

7- [4-(5-methyl-2-oxo-l,3-dioxolen-4-yl)methyl-l-piperazinyl]-4 -oxo-4H-[l,3]thiazeto[3,2- a]quinoline-3-carboxylic acid), roxithromycin (IUPAC name: (3R,4S,5S,6R,7R,9R,l lS,12R,13S,14R)-6-{ [(2S,3R,4S,6R)-4-(Dimethylamino)-3-hydroxy- 6-methyloxan-2-yl] oxy } - 14-ethyl-7 , 12,13 -trihydroxy-4- { [(2R,4R,5S ,6S )-5-hydroxy-4- methoxy-4,6-dimethyloxan-2-yl]oxy}-3,5,7,9,l l,13-hexamethyl-10-(2,4,7-trioxa-l-azaoctan- l-ylidene)-l-oxacyclotetradecan-2-one), streptomycin (IUPAC name: 5-(2,4-diguanidino- 3,5,6-trihydroxy-cyclohexoxy)- 4-[4,5-dihydroxy-6-(hydroxymethyl)-3-methylamino- tetrahydropyran-2-yl] oxy-3-hydroxy-2-methyl-tetrahydrofuran-3-carbaldehyde), sulfamethoxazole-trimethoprim (IUPAC name of sulfamethoxazole: 4-Amino-N-(5- methylisoxazol-3-yl)-benzenesulfonamide; IUPAC name of trimethoprim: 5-(3,4,5- Trimethoxybenzyl)pyrimidine-2,4-diamine), tazobactam (IUPAC name: (2S,3S,5R)-3- Methyl-7-oxo-3-(lH-l,2,3-triazol-l-ylmethyl)-4-thia-l-azabic yclo[3.2.0]heptane-2- carboxylic acid 4,4-dioxide), tetracycline (IUPAC name: (4S,6S,12aS)-4-(dimethylamino)- l,4,4a,5,5a,6,l l,12a-octahydro-3,6, 10,12, 12a-pentahydroxy-6-methyl-l,l l- dioxonaphthacene-2-carboxamide), ticarcillin (IUPAC name: (2S,5R,6R)-6-{ [(2R)-2- carboxy-2-(3-thienyl)acetyl]amino}-3,3-dimethyl-7-oxo-4-thia -l-azabicyclo[3.2.0]heptane-2- carboxylic acid), ticarcillin-clavulanic acid, vancomycin (IUPAC name: (1S,2R,18R,19R,22S,25R,28R,40S)- 48- { [(2S,3R,4S,5S,6R)- 3- { [(2S,4S,5S,6S)- 4- amino- 5- hydroxy- 4,6- dimethyloxan- 2- yl]oxy}- 4,5- dihydroxy- 6- (hydroxymethyl)oxan- 2- yl]oxy}- 22- (carbamoylmethyl)- 5,15- dichloro- 2,18,32,35,37- pentahydroxy- 19- [(2R)- 4- methyl- 2- (methylamino)pentanamido]- 20,23,26,42,44- pentaoxo- 7,13- dioxa- 21,24,27 ,41,43pentaazaoctacyclo[26.14.2.23,6.214,17.18,12.129,33.010 ,25.034,39]pentacont a- 3,5,8(48),9,11,14,16,29(45),30,32,34,36,38,46,49- pentadecaene- 40- carboxylic acid), and mixture(s) thereof.

[0045] In some examples, the one or more antibiotic agent has activity against anaerobic intestinal bacteria. In one specific example, the antibiotic agent is a mixture of penicillin and streptomycin, as shown in protocols A to H presented in Table 1. In one specific example, the penicillin is penicillin G. In another specific example, the antibiotic agent is a mixture of ampicillin and gentamycin, as shown in protocols I.l and 1.2 presented in Table 1. In yet another specific example, the antibiotic agent is a mixture of metronidazole and tetracyclin, as shown in protocols J.l and J.2 presented in Table 1.

[0046] The term "antifungal" or "antifungal agent" as used herein refers to a substance that may either kill or inhibit the growth of fungi. Examples of antifungal agent include, but are not limited to, amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, Miconazole, miconazole, mmoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, voziconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, flucytosine and micafungin.

[0047] In some examples, the treatment for removing gut microbiota can include one or more of the following combinations of antibiotic agents and/or antifungal agents: antibiotic agents ampicillin and gentamicin and antifungal agent fluconazole; antibiotic agents ampicillin and gentamicin; antibiotic agents metronidazole and tetracycline and antifungal agent fluconazole; antibiotic agents metronidazole and tetracycline; antibiotic agents penicillin and streptomycin.

[0048] In some examples, the treatment for removing gut microbiota in step (i) of the method as described herein is administered starting from at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days prior to inoculating a fungus into the digestive system of the host organism in step (ii) of the method. This is to ensure that the gut microbiota is partially or completely removed from the host organism before inoculating a fungus into the digestive system of the host organism in step (ii). In some examples, the term "partially" means at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 99% of the gut microbiota are removed from the host organism, prior to inoculating a fungus into the digestive system of the host organism in step (ii) of the method.

[0049] In some examples, the treatment for removing gut microbiota from the host organism in step (i) of the method as described herein is administered at least once every three days, at least once every two days, at least once a day, at least twice a day, at least 3 times a day, at least 4 times a day, at least 5 times a day, or at least 6 times a day or continuously throughout the day. In some examples, when the treatment for removing gut microbiota from the host organism in step (i) is administered continuously throughout the day, it is administered through the drinking water of the host organism. [0050] In some examples, instead of subjecting the host organism to a treatment for partially or completely removing gut microbiota, a host organism that is not colonized by any gut microbiota or that is colonized by an incomplete, immature or reduced-complexity microbiota can be used. Examples of such host organism not colonized by any gut microbiota or that is colonized by an incomplete, immature or reduced-complexity microbiota include but are not limited to, for example, a germ-free or a neonatal, pre-weaned or juvenile host organism.

[0051] The term "reduced-complexity" as used herein refers to a microbial complexity that is lower than the mature gut microbiota that is typically present in fully developed adult hosts of the same species. The terms "immature" and "incomplete" are almost synonymous to "reduced-complexity", in the sense that new-born hosts do not yet carry a gut microbiota that is fully developed (hence "immature"), misses some gut microbes that are acquired later on in life (hence "incomplete") and is still not yet as complex as an adult gut microbiota (hence "reduced-complexity"). The "complexity of the gut microbiota" can be measured by the number of different microbial species/taxa and/or by their relative abundance in the gut.

Each of these parameters can be determined using either culture-dependent or culture- independent (e.g. sequencing-based) methods.

[0052] In order to obtain a modified fungal strain with reduced virulence, and/or improved competitive fitness, the in vivo passaging methodas disclosed herein has to be performed a sufficient number of times or for a sufficiently long period of time until the fungal strain has acquired the desired properties. The number of serial passages or length of time needed may vary, depending upon the nature of the starting fungal strain, the type of host organism, and/or the nature of the gut microbiota to be removed, but the number or total length of time will be readily determinable without undue experimentation by persons skilled in the art, given the teachings contained herein.

[0053] In order to reduce the virulence of a fungus, and/or improve the competitive fitness of a fungus in one or more host environment, the passaging technique as disclosed herein may need to be carried out once only, or may need to be carried out multiple times (known as serial passaging). Thus, in some examples, in order to reduce the virulence of a fungus, and/or improve the competitive fitness of a fungus in one or more host environment, steps (i) subjecting a host organism to a treatment for partially or completely removing gut microbiota; (ii) inoculating a fungus into the digestive system of the host organism to allow the fungus to colonize the gastrointestinal tract of the host organism; and (iii) collecting the gastrointestinal tract discharge of the host organism is carried out once, twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 18 times, or 20 times, or at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 time, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times, or more, until the desired fungus or fungal strain is obtained. In some specific examples, steps (i) to (iii) as described above are carried out once (see for example, Table 1, protocol A, C, D), 8 times (see for example, Table 1, protocol B), 10 times (see for example, Table 1, protocol E, F, 1.1, 1.2, J.l, J.2) or 15 times (see for example, Table 1, protocol G and H).

[0054] The time interval between each passage should be such as to sufficiently allow the fungus to replicate between passages.

[0055] In some examples, when the passage is carried out once, the time between steps (ii) inoculating a fungus into the digestive system of the host organism; and (iii) collecting the gastrointestinal tract discharge of the host organism is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, or 20 weeks, or at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, or at least 20 weeks, or more, in order to allow the inoculated fungus to colonize the gastrointestinal tract of the host organism. In some specific examples, the time between steps (ii) and (iii) as described above is 1 week (see for example, Table 1, protocol A) or 10 weeks (see for example, Table 1, protocol C and D).

[0056] In some other examples, when steps (i) to (iii) as described above are carried out twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 18 times, or 20 times, or at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 time, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times, or more, the time between inoculating a fungus into the digestive system of the host organism in step (ii) and collecting the gastrointestinal tract discharge of the host organism in step (iii) is 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks, or at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks, or more. In some specific examples, steps (i) to (iii) as described above are carried out 8 times, and the time between steps (ii) and (iii) is 1 week (see for example, Table 1, protocol B). In some other specific examples, steps (i) to (iii) as described above are carried out 10 times, and the time between steps (ii) and (iii) is 1 week (see for example, Table 1, protocol E, F, 1.1, 1.2, J. l, J.2). In some further specific examples, steps (i) to (iii) as described above are carried out 15 times, of which the time between steps (ii) and (iii) is 1 week for the first 10 times; and 2 weeks for the remaining 5 times (see for example, Table 1, protocol G and H).

[0057] In some other examples, when steps (i) to (iii) as described above are carried out twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 18 times, or 20 times, or at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 time, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times, or more, a new host organism of the same species is used for each repeat, and (ii) inoculating a fungus into the digestive system of the host organism comprises administering the sample obtained from collecting the gastrointestinal tract discharge of the host organism in step (iii) of the previous repeat.

[0058] Fungal species suitable for use in the methods as described herein include, but are not limited to: Absidia corymbifera, Absidia spp., Acremonium falciforme, Acremonium kiliense, Acremonium recifei, Acremonium spp., Ajellomyces capsulatus, Ajellomyces dermatitidis, Ajellomyces spp., Allescheria boydii, Alternaria alternata, Alternaria chartarum, Alternaria dianthicola, Alternaria geophilia, Alternaria infectoria, Alternaria spp., Alternaria stemphyloides, Alternaria teunissima, Anthopsis deltoidea, Aphanomyces spp., Apophysomyces elegans, Armillaria spp., Arnium leoporinum, Arthroderma benhainiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii, Arthrographis cuboidea, Arthrographis kalrae, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus spp., Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Aureobasidium pullulans, Basisdiobolus ranarum, Beauveria bassiana, Bipolaris australiensis, Bipolaris hawaiiensis, Bipolaris spicifera, Bipolaris spp., Blastomyces dermatitidis, Blastoschizomyces capitatus, Botrytis spp., Candida albicans, Candida auris, Candida ciferrii, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida lusitaniae, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida spp., Candida tropicalis, Candida viswanathii, Candida zeylanoides, Centrospora spp., Cephalosporium spp., Ceratocystis spp., Chaetoconidium spp., Chaetomium atrobrunneum, Chaetomium spp., Chlamydia trachomatis, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium parvum Chrysosporium queenslandicum, Chrysosporium spp., Chrysosporium tropicum, Chrysosporium zonatum, Cladophialophora carrionii, Cladophialophora spp., Cladosporium cladosporioides, Cladosporium elatum, Cladosporium herbarum, Cladosporium sphaerospermum, Cladosporium spp., Coccidioides immitis, Coccidioides posadasii, Coccidioides spp., Colletotrichium spp., Conidiobolus coronatus, Conidiobolus incongruus, Conidiobolus lamprauges, Conidiobolus spp., Cryptococcus neoformans, Cryptococcus spp., Cryptoporiopsis spp., Cunninghamella bertholletiae, Cunninghamella spp., Curvularia brachyspora, Curvularia clavata, Curvularia geniculata, Curvularia lunata, Curvularia pallescens, Curvularia senegalensis, Curvularia spp., Curvularia verruculosa, Cylindrocladium spp., Dactylaria spp., Debaryomyces hansenii, Diplodia spp., Emmonsia parva, Emmonsia parva var. crescens, Emmonsia parva var. parva, Emmonsia pasteuriana, Epidermophyton floccosum, Epidermophyton spp., Exophiala castellanii, Exophiala dermatitidis, Exophiala jeansehnei var. heteromorpha , Exophiala jeanselmei var. lecanii- corni, Exophiala moniliae, Exophiala salmonis, Exophiala spinifera, Exophiala spp., Exophila pisciphila, Exserophilium spp., Filobasidiella neoformans, Fonsecaea compacta, Fonsecaea pedrosoi, Fonsecaea spp., Fulvia spp., Fusarium chlamydosporum, Fusarium oxysporum, Fusarium solani, Fusarium spp., Geotrichum candidum, Geotrichum clavatum, Geotrichum fid, Geotrichum spp., Guignardia spp., Helminthosporium spp., Histoplasma capsulatum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma spp., Hortaea werneckii, Issatschenkia orientalis, Kluyveromyces lactis, Lacazia loboi, Lasiodiplodia spp., Lecythophora spp., Leptosphaeria australiensis, Leptosphaeria senegalensis, Leptosphaeria spp., Macrophomina spp., Madurella grisae, Madurella mycetomatis, Madurella spp., Magnaporthe grisea, Magnaporthe spp., Malassezia furfur, Malassezia globosa, Malassezia obtuse, Malassezia pachydermatis, Malassezia restricta, Malassezia slooffiae, Malassezia sympodialis, Malbranchea pulchella, Malbranchea sclerotica, Malbranchea spp., Microsporum audouinii, Microsporum canis, Microsporum cookei, Microsporum distortum, Microsporum eguinum, Microsporum ferrugineum, Microsporum fulvum, Microsporum gallinae, Microsporum gypseum, Microsporum nanum, Microsporum spp., Microsporum vanbreusegh, Monilinia spp., Mucor circinelloides, Mucor spp., Mycocentrospora acerina, Nectria haematococca, Nectria spp., Neotestudina rosatii, Neotestudina spp., Neurospora crassa, Nigrospora sphaerica, Nigrospora spp., Nocardia aster oides, Nocardia brasiliensis, Nocardia otitidiscaviarum, Nocardia spp., Ochrononis spp., Onychocola canadensis , Onychocola spp., Oospora spp., Ophiobolus spp., Paecilomyces lilacinus, Paecilomyces spp., Paecilomyces variotii, Paracoccidioides brasiliensis, Paracoccidioides spp., Penicillium marneffei, Penicillium spp., Penicillium verrucosum, Phaeoannellomyces spp., Phaeosclera dematioides, Phialemonium obovatum, Phialophora europaea, Phialophora spp., Phialophora verruceosa, Phlyctaena spp., Phoma spp., Phomopsis spp., Phymatotrichum spp., Phytophthora spp., Pichia anomala, Pichia guilliermondii, Pichia ohmeri, Pichia spp., Piedraia hortai, Piedraia spp., Pneumocystis carinii, Pneumocystis jiroveci, Pneumocystis spp., Pseudallescheria boydii, Pseudallescheria spp., Puccinia spp., Pyrenochaeta romeroi, Pyrenochaeta spp., Pyrenochaeta unguis-hominis, Pythium insidiosum, Pythium spp., Rhinocladiella aquaspersa, Rhizoctonia spp., Rhizomucor pusillus, Rhizomucor spp., Rhizomucor variabilis, Rhizopus microsporus var. rhizopodiformis, Rhizopus oryzae, Rhizopus spp., Rhodotorula rubra, Rhodotorula spp., Saccharomyces cerevisiae, Saccharomyces spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Scedosporium apiospermum, Scedosporium prolificans, Scedosporium spp., Scerotium spp., Schizophyllum commune, Schizosaccharomyces pombe, Sclerotinia spp., Scopulariopsis brevicaulis, Scopulariopsis spp., scytalidium spp., Sphaerotheca spp., Sporobolomyces salmonicolor, Sporobolomyces spp., Sporothrix schenckii, Stachybotrys chartarum, Stachybotjys sp., Stemphylium macrosporoideum, Syncephalastrum racemosum, Taeniolella boppii, Taphrina spp., Thielaviopsis spp., Torulopsis spp., Trichoderma spp., Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton spp., Trichophyton verrucosum, Trichophyton violaceum, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides, Trichosporon spp., Ulocladium botrytis, Ulocladium chartarum, Ustilago maydis, Ustilago spp., Venturia spp., Verticillium spp., Wangiella dertnatitidis, Wangiella spp., Whetxelinia spp., Xylohypha spp., and Yarrowia lipolytica.

[0059] In some examples, the fungus is a Candida species, such as Candida albicans, Candida ciferrii, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida lusitaniae, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida spp., Candida tropicalis, Candida viswanathii or Candida zeylanoides. In some specific examples, the fungus is Candida albicans or Candida tropicalis.

[0060] The term "host organism" as used herein refers to an organism in which the fungus of interest can be modified in using the method as disclosed herein. It is to be noted that the host organism is used in the method of the first aspect solely as the vehicle to generate the fungus or fungal strain that has the desired property, i.e. reduced virulence, and/or improved competitive fitness in one or more host environment,. It is to be further noted that the method of the first aspect does not confer any medical treatment effect on the host organism in any way. The term "host environment" as used in the context of "improving the competitive fitness of a fungus in one or more host environment" refers to the cell, tissue, or organ of a host, or any substances produced by and/or released from a cell, tissue, or organ of a host. Examples of the host environment include but are not limited to gastrointestinal tract, oral cavity, vaginal mucosa and skin.

[0061] In some examples, the host organism in which the fungus of interest can be modified, and the host providing the host environment in which the competitive fitness of a fungus is improved, are of different species, or are different individuals of the same species. In some examples, the host organism is a non-human host organism, and/or the host environment is a human host environment. In some other examples, the host organism is a non-human host organism, and/or the host environment is a non-human host environment. In some examples, the non-human host organism is a non-human mammalian host organism, including but are not limited to mouse, rat, guinea pig, hamster, rabbit, non-human primates, cat, dog and pig. In one specific example, the non-human mammalian host organism is a mouse (Mus musculus). In some examples, the non-human mammalian host organism is an adult mouse. In general, a mouse that is older than about 40 days, or older than about 50 days, or older than about 60 days, is considered as an adult mouse. In some examples, the adult mouse may be at least 6 to 8 weeks of age, that is about 42 to 56 days. In some other examples, the non-human host environment is a non-human mammalian host environment, including but are not limited to mouse, rat, guinea pig, hamster, rabbit, non-human primates, cat, dog and pig. As an illustrative example, when the host organism is a mouse, and the host environment is the gut of a human, it means the method as described herein can be used to produce a fungal strain that has improved competitive fitness in human gut, while all steps (i) to (iii) of the method are performed in mouse.

[0062] In some examples, the host organism is an immunocompromised host organism. In some examples, the immunocompromised host organism lacks functioning T and/or B cells. In some examples, the immunocompromised host organism is a Rag 1 -deficient mouse.

[0063] Similarly, in some examples, the host environment is the host environment of an immunocompromised host. In some examples, the immunocompromised host lacks functioning T and/or B cells. In some examples, the immunocompromised host is a Ragl- deficient mouse.

[0064] In some examples, the host organism is an immunocompromised host organism, and the host environment is the environment of a normal host (i.e. not an

immunocompromised host). In some other examples, the host organism is a normal host organism (i.e. not an immunocompromised host organism), and the host environment is the environment of an immunocompromised host. In some other examples, the host organism is a normal host organism, and the host environment is the environment of a normal host. In some further examples, the host organism is an immunocompromised host organism, and the host environment is the environment of an immunocompromised host.

[0065] In some specific examples, the method as described herein is a method for reducing the virulence of a Candida strain, and/or improving the competitive fitness of a Candida strain in one or more host environment, the method comprises (i) subjecting a mouse to a treatment with one or more antibiotic agents for removing commensal bacteria from the gut; (ii) inoculating a Candida strain into the digestive system of the mouse to allow the Candida strain to colonize the gastrointestinal tract of the mouse; (iii) collecting the gastrointestinal tract discharge of the mouse to obtain a Candida strain with reduced virulence and/or with improved competitive fitness in the gastrointestinal environment. In some other examples, the method comprises (i) subjecting a rabbit to a treatment with one or more antibiotic agents for removing commensal bacteria from the gut; (ii) inoculating a Candida strain into the digestive system of the rabbit to allow the Candida strain to colonize the gastrointestinal tract of the rabbit; (iii) collecting the gastrointestinal tract discharge of the rabbit to obtain a Candida strain with reduced virulence and/or with improved competitive fitness in the gastrointestinal environment

[0066] In some other specific examples, the method as described herein is a method for reducing the virulence of an Aspergillus strain, and/or improving the competitive fitness of an Aspergillus strain in one or more host environment, the method comprises (i) subjecting a mouse to a treatment with one or more antibiotic agents for removing commensal bacteria from the gut; (ii) inoculating an Aspergillus strain into the digestive system of the mouse to allow the Aspergillus strain to colonize the gastrointestinal tract of the mouse; (iii) collecting the gastrointestinal tract discharge of the mouse to obtain an Aspergillus strain with reduced virulence and/or with improved competitive fitness in the gastrointestinal environment.

[0067] In some examples, the fungus to be inoculated into the digestive system of the host organism is pathogenic to the host organism. In some other examples, the fungus to be inoculated into the digestive system of the host organism is non-pathogenic to the host organism.

[0068] In one specific example, the non-human host environment is the gastrointestinal tract of a mouse.

[0069] In some examples, inoculating the fungus into the digestive system of the host organism in step (ii) of the method disclosed herein comprises administering the fungus orally or directly inoculating it into the gastrointestinal tract of the host organism. When inoculating the fungus into the digestive system of the host organism in step (ii) comprises administering the fungus orally, the fungus is administered through, for example, the drinking water or the food. When inoculating the fungus into the digestive system of the host organism in step (ii) comprises inoculating the fungus directly into the gastrointestinal tract of the host organism, the fungus is administered by, for example, gastric gavage.

[0070] In some examples, the gastrointestinal tract discharge of the host organism comprises solid and/or liquid discharge, or a mixture thereof.

[0071] The method as disclosed herein can also be used for the generation of fungal strains for biotransformation or for the manufacture of vaccines.

[0072] In a second aspect, there is provided a fungal strain obtained using the method of the first aspect. This means, in particular, that the fungal strain obtained has reduced virulence (as shown in Examples 4, 5 and 6, which demonstrate that the Candida strains obtained have reduced in vitro cytotoxicity, and reduced in vivo virulence in both wild-type mice and immunocompromised mice), and/or improved competitive fitness in one or more host environment (as shown in Example 3). In some examples, the fungal strain obtained is an isolated fungal strain.

[0073] In some examples, the fungal strains obtained using the method as disclosed herein have reduced virulence as compared to the wild-type strains of the same species, as shown in Example 4, 5 and 6. In some examples, in contrast to wild-type strains that grow predominantly as filamentous forms in vivo, the fungal strains obtained using the method as disclosed herein grow as ovoid, yeast form cells, as shown in Example 7. In addition, in some examples, the fungal strains obtained using the method as disclosed herein are more resistant to hyphal-inducing stimuli, as compared to the wild-type strains of the same fungal species, as shown in Example 7.

[0074] The fungal strains obtained using the method of the first aspect are expected to have utility as immunogens in antimicrobial vaccines for animals, as shown in Example 8 as well as Figure 16, which demonstrates that the C. albicans strains obtained can protect the mice against systemic challenge with the wild-type, virulent C. albicans. Thus, in a third aspect, there is provided a vaccine comprising a fungal strain obtained using the method as disclosed herein. Such a vaccine would comprise an immunologically effective amount of the fungal strain and a pharmaceutically acceptable carrier.

[0075] The term "vaccine" as used herein is intended to encompass a preventative vaccine, i.e. one that is given to stimulate an immune response so that if the subject subsequently is exposed to the antigen in nature, the pre-formed immune response will increase the subject's ability to fight off the agent or cells carrying the antigen. The term "vaccine" as used herein is also intended to encompass a therapeutic vaccine, i.e. one that is given to a subject who already has a disease associated with an antigen, wherein the vaccine can elicit an immune response or boost the subject's existing immune response to the antigen to provide an increased ability to fight the agent or cells carrying the antigen. In some examples, the term "vaccine" may also be used to encompass immunity that is conferred via trained innate immunity. Without wishing to be bound by theory, it is believed that the "vaccine" of the present disclosure does not only provide protective and therapeutic effect through specific antigenic recognition that typically occurs in adaptive immune system immunity. As illustrated in the examples section (for example Example 9), the "vaccine" of the present disclosure also extends protective and therapeutic effects beyond the specific antigen to additional unrelated pathogens, which demonstrates trained innate immunity. The phrase "trained innate immunity" or "trained immunity" is readily understood in immunology to be immunity that is conferred independently of adaptive immune systems (such as T and B lymphocytes). The "trained innate immunity" are characterized by at least one of (1) protection can also be observed independently of T and B cells (for example as seen in adaptive immune system mouse models such as Rag 1 -knockout mice), (2) significant cross- protection observed towards completely different pathogens, (3) protection correlates with increased innate cytokine responses (for example IL-6 (i.e. interleukin 6) and TNF-alpha (i.e. Tumor Necrosis Factor-alpha)) and (4) the protection is observed as early as one day post- immunization, which is understood by the person skilled in the art to be incompatible with mounting a classical adaptive immune memory that typically requires at least a few weeks.

[0076] Without wishing to be bound by theory, because the "vaccines" of the present disclosure can provide systemic acute protection via trained innate immunity, it is believed that the "vaccines" of the present disclosure can be provided to patients/subjects where infections may occur and fast immunity is required (i.e. less than the typical time adaptive immune system takes to establish). For example, the protection offered by the "vaccines" of the present disclosure and mediated via trained innate immunity may be useful against systemic candidiasis and other (opportunistic) infections (for example which typically only happens in hospitals). In this setting, a subject/patient at high risk of such infections would only need to take the "vaccine" as described herein at the time of admission and it only needs to protect the subject/patient for the duration of his/her hospitalization. In some examples, if hospitalization were to occur again within six months of the first admission and administration of the "vaccines" as described herein, the patient/subject would simply need to take another dose at the time of second admission. This is because, as shown in the experimental data section, protection was observed even as early as 1 day post-immunization.

[0077] In various examples, the vaccine can be administered to subjects at risk for fungal infections. These include subjects with impairment of neutrophil function due to decreased neutrophil production in the bone marrow, increased neutrophil destruction, or qualitative defects in neutrophil function. Factors that can cause a decrease in neutrophil production include, but are not limited to (1) administration of cytotoxic drugs, including alkylating agents such as cyclophosphamide, busulfan, and chlorambucil, and antimetabolites such as methotrexate, 6-mercaptopurine and 5-flurocytosine; (2) administration of other drugs known to inhibit neutrophil production including, but not limited to, certain antibiotics, phenothiazines, diuretics, anti-inflammatory agents, and antithyroid drugs; (3) bacterial sepsis infections, viral infections such as HTV, EBV or hepatitis; typhoid, malaria, brucellosis, and tularemia; (4) primary hematologic diseases resulting in bone marrow failure, as well as both hereditary syndromes and acquired defects; (5) bone marrow failure due to tumor invasion or myelofibrosis; (6) nutritional deficiencies such as deficiency of either vitamin B 12 or folate;

(7) bone marrow destruction due to accidental radiation; and (8) radiotherapy for cancer treatment. Factors that can cause an increase in destruction of neutrophils, thereby rendering an individual susceptible to fungal infections, include, without limitation, the presence of antineutrophil antibodies, autoimmune disease (such as Felty's syndrome, rheumatoid arthritis, or systemic lupus erythematosis), or idiosyncratic reactions to drugs that, in an idiosyncratic way, act as haptens at the surface of neutrophils, initiating immune destruction of neutrophils.

[0078] Qualitative defects in neutrophil function that can lead to increased susceptibility to fungal infections include many disease states, for example, leukocyte adhesion deficiency syndromes, neutrophil chemotactic defects, and neutrophil phagocytic and killing defects. Neutrophil function is also compromised by administration of corticosteroids used in the treatment of a wide variety of diseases. Thus, patients treated with corticosteroids are at increased risk of fungal infections.

[0079] Additional factors increasing individual susceptibility to fungal infections include: (1) treatment with broad spectrum antibiotics, especially in the hospital setting and in Intensive Care settings in particular; (2) application of intravenous catheters, particularly central venous catheters; (3) surgical wounds, particularly those associated with intraabdominal surgeries; (4) tissue, bone marrow or solid organ transplantation; (5) cancer chemotherapy; (6) Acquired Immune Deficiency Syndrome; (7) Intensive Care Unit stay; and

(8) diabetes. In addition, neonates and aged patients are at increased risk. Immunosuppressed patients (e.g., patients with acquired immunosuppression, e.g., due to HIV infection or immunosuppressive medical treatments such as chemotherapy; or with an inherited immunosuppressive disorder) can exhibit increased susceptibility to fungal infections as well. In some specific examples, the vaccines can be used for the immunization of subjects which are difficult to be immunized using other vaccines. Examples of such subjects include but are not limited to immunocompromised subjects. In some examples, the immunocompromised subjects lack functioning T and/or B cells.

[0080] Vaccine compositions can be administered to provide a beneficial effect specific to the strain administered, or which also is beneficial towards one or more additional strains (e.g., by inducing a cross -reactive response). Thus, one may administer vaccines derived from one particular fungal species to induce a beneficial effect in a subject at risk for infection with another fungal species. That is, the present vaccine composition may be used to stimulate trained innate immunity.

[0081] The vaccine can be administered to a subject by a variety of administration routes. Methods of administration of such a vaccine are known in the art, and include but are not limited to, oral, nasal, intraveneous, intradermal, intraperitoneal, intramuscular, intralymphatic and subcutaneous routes of administration. In some preferred examples, the vaccine is administered by intramuscular or subcutaneous routes.

[0082] In some examples, the vaccine is suitable for single-dose administration. In some other examples, the vaccine is suitable for multiple-dose administration, such as two, or three, or four, or five, or six, or more doses.

[0083] A subject to be administered with such vaccines, can be any vertebrate, preferably a mammal, including domestic animals, sport animals, and primates, including humans. The vaccine may be administered as a prophylactic, where the subject is vaccinated in order to be immunized against a particular disease. The vaccine may also be administered as a therapeutic, where the subject having a particular disease is vaccinated in order to improve the immune response to the disease or a disease-related protein. In some examples, the vaccine may result in a lessening of the physical symptoms associated with the disease.

[0084] Vaccine formulations are known in the art. The vaccine can, but need not be administrated with an adjuvant or a carrier. Adjuvants include, (complete or incomplete) Freund's adjuvant; other bacterial cell wall components; aluminum-based salts; calcium- based salts; silica; polynucleotides; toxoids; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; and block copolymer adjuvants. Carriers include polymeric controlled release formulations, biodegradable implants, liposomes, oils, esters, and glycols. Vaccine composition can also include one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient refers to a substance suitable for delivering a fungal composition to a site in vivo or ex vivo. Excipients can maintain a fungal composition in a form that is capable of eliciting an immune response at a target site. Examples of pharmaceutically acceptable excipients are saline, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity. Other auxiliary compounds include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances can also include preservatives, such as thimerosal, m- or o-cresol, formalin and benzol alcohol.

[0085] In some examples, the vaccine is prepared from freshly produced fungal strains that are obtained using methods of the first aspect. In some other examples, the fungal strains obtained using methods of the first aspect are stored under the appropriate storage conditions, and the vaccine is prepared from the stored fungal strains. In some examples, the vaccine prepared may be desiccated, for example, by freeze drying for storage purposes or for subsequent formulation into liquid vaccines.

[0086] An immunologically effective amount is determinable by means known in the art without undue experimentation, given the teachings contained herein. The amount of fungal strain should be sufficient to stimulate an immune response in disease-susceptible animals while still being avirulent. This will depend upon the particular animal, fungal species, and disease involved.

[0087] The efficacy of the vaccines can be evaluated in a subject, for example in mice. A mouse model is recognized as a model for efficacy in humans and is useful in assessing and defining the vaccines as disclosed herein. The mouse model is used to demonstrate the potential for the effectiveness of the vaccines in any individual. Vaccines can be evaluated for their ability to provide either a prophylactic or therapeutic effect against a particular disease. For example, in the case of fungal infection, a population of mice can be vaccinated with a desired amount of the appropriate vaccine. The mice can be subsequently infected with the pathogenic fungus and assessed for protection against infection. The progression of the infectious disease can be observed relative to a control population (either non vaccinated or vaccinated with vehicle only). In some examples, the survival rate of the mice can be used as an indicator of the efficacy of the vaccine being tested.

[0088] In a fourth aspect, there is provided a method of preventing or treating a disease in a subject in need thereof, wherein the method comprises administering an effective amount of fungal strain obtained using the method as disclosed herein, or an affective amount of a composition comprising a fungal strain obtained using the method as disclosed herein. In some examples, the disease to be prevented or treated is a disease caused by a clinical strain (i.e. wild-type) of the same fungal species being administered to the subject. In some other examples, the disease to be prevented or treated is a disease caused by one or more fungal species different from the fungal species being administered.

[0089] In some examples, the disease to be prevented or treated is a disease caused by a different microorganism from the fungal strain obtained using the method described herein. In some examples, the disease is caused by microorganisms such as, but not limited to, bacteria, archaea, virus, yeast, fungi, protozoa, algae, and the like. Thus, in some example, the disease includes, but is not limited to, a bacterial disease (such as bacterial infection), fungal disease (such as fungal infection), a parasitic disease (such as parasite infections), a viral disease (such as a viral infection), a polymicrobial disease (such as a polymicrobial infection).

[0090] In some examples, the disease is caused by gram-positive bacteria or gram- negative bacteria. In some examples, the disease is caused by bacteria from the genus of Acetobacter, Acinetobacter, Actinomyces, Agrobacterium spp., Azorhizobium, Azotobacter, Anaplasma spp., Bacillus spp., Bacteroides spp., Bartonella spp., Bordetella spp., Borrelia, Brucella spp., Burkholderia spp., Calymmatobacterium, Campylobacter, Chlamydia spp., Chlamydophila spp., Clostridium spp., Corynebacterium spp., Coxiella, Ehrlichia, Enterobacter, Enterococcus spp., Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus spp., Helicobacter, Klebsiella, Lactobacillus spp., Lactococcus, Legionella, Listeria, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium spp., Mycoplasma spp., Neisseria spp., Pasteurella spp., Peptostreptococcus, Porphyromonas, Pseudomonas, Rhizobium, Rickettsia spp., Rochalimaea spp., Rothia, Salmonella spp., Serratia, Shigella, Staphylococcus spp., Stenotrophomonas, Streptococcus spp., Treponema spp., Vibrio spp., Wolbachia, and Yersinia spp. In some examples, the bacteria may include, but are not limited to Acetobacter aurantius, Acinetobacter baumannii, Actinomyces Israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Azorhizobium caulinodans, Azotobacter vinelandii, Anaplasma phagocytophilum, Anaplasma marginale, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaminogenicus (Prevotella melaminogenica), Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia complex, Burkholderia cenocepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila. (such as C. pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium diphtheriae, Cory neb acterium fusiforme, Coxiella burnetii, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis Peptostreptococcus, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rhizobium Radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus, avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Wolbachia, Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis . In some example, the bacterial infection may include, but is not limited to, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and the like.

[0091] In some examples, the disease is caused by fungus from the genus of Absidia, Ajellomyces, Arthroderma, Aspergillus, Blastomyces, Candida, Cladophialophora, Coccidioides, Cryptococcus, Cunninghamella, Epidermophyton, Exophiala, Filobasidiella, Fonsecaea, Fusarium, Geotrichum, Histoplasma, Hortaea, Issatschenkia, Madurella, Malassezia, Microsporum, Microsporidia, Mucor, Nectria, Paecilomyces, Paracoccidioides, Penicillium, Pichia, Pneumocystis, Pseudallescheria, Rhizopus, Rhodotorula, Scedosporium, Schizophyllum, Sporothrix, Trichophyton, and Trichosporon. Absidia corymbifera, Absidia spp., Acremonium falciforme, Acremonium kiliense, Acremonium recifei, Acremonium spp., Ajellomyces capsulatus, Ajellomyces dermatitidis, Ajellomyces spp., Allescheria boydii, Alternaria alternata, Alternaria chartarum, Alternaria dianthicola, Alternaria geophilia, Alternaria infectoria, Alternaria spp., Alternaria stemphyloides, Alternaria teunissima, Anthopsis deltoidea, Aphanomyces spp., Apophysomyces elegans, Armillaria spp., Arnium leoporinum, Arthroderma benhainiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii, Arthrographis cuboidea, Arthrographis kalrae, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus spp., Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Aureobasidium pullulans, Basisdiobolus ranarum, Beauveria bassiana, Bipolaris australiensis, Bipolaris hawaiiensis, Bipolaris spicifera, Bipolaris spp., Blastomyces dermatitidis, Blastoschizomyces capitatus, Botrytis spp., Candida albicans, Candida auris, Candida ciferrii, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida lusitaniae, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida spp., Candida tropicalis, Candida viswanathii, Candida zeylanoides, Centrospora spp., Cephalosporium spp., Ceratocystis spp., Chaetoconidium spp., Chaetomium atrobrunneum, Chaetomium spp., Chlamydia trachomatis, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium parvum , Chrysosporium queenslandicum, Chrysosporium spp., Chrysosporium tropicum, Chrysosporium zonatum, Cladophialophora carrionii, Cladophialophora spp., Cladosporium cladosporioides, Cladosporium elatum, Cladosporium herbarum, Cladosporium sphaerospermum, Cladosporium spp., Coccidioides immitis, Coccidioides posadasii, Coccidioides spp., Colletotrichium spp., Conidiobolus coronatus, Conidiobolus incongruus, Conidiobolus lamprauges, Conidiobolus spp., Cryptococcus neoformans, Cryptococcus spp., Cryptoporiopsis spp., Cunninghamella bertholletiae, Cunninghamella spp., Curvularia brachyspora, Curvularia clavata, Curvularia geniculata, Curvularia lunata, Curvularia pallescens, Curvularia senegalensis, Curvularia spp., Curvularia verruculosa, Cylindrocladium spp., Dactylaria spp., Debaryomyces hansenii, Diplodia spp., Emmonsia parva, Emmonsia parva var. crescens, Emmonsia parva var. parva, Emmonsia pasteuriana, Epidermophyton floccosum, Epidermophyton spp., Exophiala castellanii, Exophiala dermatitidis, Exophiala jeansehnei var. heteromorpha , Exophiala jeanselmei var. lecanii- corni, Exophiala moniliae, Exophiala salmonis, Exophiala spinifera, Exophiala spp., Exophila pisciphila, Exserophilium spp., Filobasidiella neoformans, Fonsecaea compacta, Fonsecaea pedrosoi, Fonsecaea spp., Fulvia spp., Fusarium chlamydosporum, Fusarium oxysporum, Fusarium solani, Fusarium spp., Geotrichum candidum, Geotrichum clavatum, Geotrichum fid, Geotrichum spp., Guignardia spp., Helminthosporium spp., Histoplasma capsulatum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma spp., Hortaea werneckii, Issatschenkia orientalis, Kluyveromyces lactis, Lacazia loboi, Lasiodiplodia spp., Lecythophora spp., Leptosphaeria australiensis, Leptosphaeria senegalensis, Leptosphaeria spp., Macrophomina spp., Madurella grisae, Madurella mycetomatis, Madurella spp., Magnaporthe grisea, Magnaporthe spp., Malassezia furfur, Malassezia globosa, Malassezia obtuse, Malassezia pachydermatis, Malassezia restricta, Malassezia slooffiae, Malassezia sympodialis, Malbranchea pulchella, Malbranchea sclerotica, Malbranchea spp., Microsporum audouinii, Microsporum canis, Microsporum cookei, Microsporum distortum, Microsporum eguinum, Microsporum ferrugineum, Microsporum fulvum, Microsporum gallinae, Microsporum gypseum, Microsporum nanum, Microsporum spp., Microsporum vanbreusegh, Monilinia spp., Mucor circinelloides, Mucor spp., Mycocentrospora acerina, Nectria haematococca, Nectria spp., Neotestudina rosatii, Neotestudina spp., Neurospora crassa, Nigrospora sphaerica, Nigrospora spp., Nocardia aster oides, Nocardia brasiliensis, Nocardia otitidiscaviarum, Nocardia spp., Ochrononis spp., Onychocola canadensis , Onychocola spp., Oospora spp., Ophiobolus spp., Paecilomyces lilacinus, Paecilomyces spp., Paecilomyces variotii, Paracoccidioides brasiliensis, Paracoccidioides spp., Penicillium marneffei, Penicillium spp., Penicillium verrucosum, Phaeoannellomyces spp., Phaeosclera dematioides, Phialemonium obovatum, Phialophora europaea, Phialophora spp., Phialophora verruceosa, Phlyctaena spp., Phoma spp., Phomopsis spp., Phymatotrichum spp., Phytophthora spp., Pichia anomala, Pichia guilliermondii, Pichia ohmeri, Pichia spp., Piedraia hortai, Piedraia spp., Pneumocystis carinii, Pneumocystis jiroveci, Pneumocystis spp., Pseudallescheria boydii, Pseudallescheria spp., Puccinia spp., Pyrenochaeta romeroi, Pyrenochaeta spp., Pyrenochaeta unguis-hominis, Pythium insidiosum, Pythium spp., Rhinocladiella aquaspersa, Rhizoctonia spp., Rhizomucor pusillus, Rhizomucor spp., Rhizomucor variabilis, Rhizopus microsporus var. rhizopodiformis, Rhizopus oryzae, Rhizopus spp., Rhodotorula rubra, Rhodotorula spp., Saccharomyces cerevisiae, Saccharomyces spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Scedosporium apiospermum, Scedosporium prolificans, Scedosporium spp., Scerotium spp., Schizophyllum commune, Schizosaccharomyces pombe, Sclerotinia spp., Scopulariopsis brevicaulis, Scopulariopsis spp., scytalidium spp., Sphaerotheca spp., Sporobolomyces salmonicolor, Sporobolomyces spp., Sporothrix schenckii, Stachybotrys chartarum, Stachybotjys sp., Stemphylium macrosporoideum, Syncephalastrum racemosum, Taeniolella boppii, Taphrina spp., Thielaviopsis spp., Torulopsis spp., Trichoderma spp., Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton spp., Trichophyton verrucosum, Trichophyton violaceum, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides, Trichosporon spp., Ulocladium botrytis, Ulocladium chartarum, Ustilago maydis, Ustilago spp., Venturia spp., Verticillium spp., Wangiella dertnatitidis, Wangiella spp., Whetxelinia spp., Xylohypha spp., and Yarrowia lipolytica.

[0092] In some examples, the fungus is Aspergillus or Candida species. In some examples, the fungus is a Candida species, such as Candida albicans, Candida ciferrii, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida lusitaniae, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida spp., Candida tropicalis, Candida viswanathii or Candida zeylanoides. In some specific examples, the fungus is Candida albicans or Candida tropicalis. In some examples, the fungus is Candida species, Aspergillus species, Cryptococcus species, and Histoplasma species

[0093] In some examples, the disease is caused by parasites (such as, but not limited to, protozoan, heminthic, and the like). In some examples, the disease caused by protozoan parasites includes, but not limited to, malaria, African trypanosomiasis, amebiasis, babesiosis, chagas disease, cryptosporidiosis, cyclosporiasis, giardiasis, leishmaniasis, microsporidiosis, toxoplasmosis, and the like. In some example, disease caused by helminths, include, but not limited to, diseases caused by roundworms (such as, but not limited to, filariasis, strongyloidiasis, trichinellosis, toxocariasis, and the like), diseases caused by flukes (such as, but not limited to, paragonimiasis, schistosomiasis, and the like), diseases caused by tapeworms (such as, but not limited to, cysticercosis, echinococcosis, and the like), and the like. In some examples, the disease is malaria.

[0094] In some examples, the disease is caused by virus including, but not limited to adenoviruses, herpes viruses, poxviruses, parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses, paramyxoviruses, papillomaviruses, retroviruses (such as Human Immunodeficiency Virus) and hepadnaviruses. In some examples, the viral infectious disease may include, but is not limited to common cold, influenza, chickenpox, cold sores, Ebola, AIDS, avian influenza, SARS, dengue, herpes, shingles, measles, mumps, rubella, rabies, human papillomavirus, viral hepatitis, coxsackie virus, Epstein Barr virus and the like. In some examples, the disease is caused by virus such as, influenza virus, dengue virus, zika virus, and chikungunya virus.

[0095] In some examples, polymicrobial disease is diseases (or infections) that are caused by multiple infectious agents. In some examples, polymicrobial diseases may include polyviral diseases, polybacterial diseases, viral and bacterial infections, fungal infections, infections resulting from microbe-induced immunosuppression, and the like. In some exmaples, polymicrobial diseases include, but are not limited to, abscesses, AIDS-related opportunistic infections, conjunctivitis, gastroenteritis, hepatitis, multiple sclerosis, otitis media, periodontal diseases, respiratory diseases, and genital infections.

[0096] There is also provided a method of inducing an immune response in a subject in need thereof, wherein the method comprises administering an effective amount of fungal strain obtained using the method as disclosed herein, or an affective amount of a composition comprising a fungal strain obtained using the method as disclosed herein. In some examples, the immune response to be induced is an immune response to a clinical strain (i.e. the wild- type) of the same fungal species being administered to the subject. In some other examples, the immune response to be induced is an immune response to one or more fungal species different from the fungal species being administered. For example, when the fungal strain of interest is C. albicans, the wildtype and clinical strain used may be SC5314.

[0097] The fungal strain can also be administered as a vaccine, which is provided under the third aspect of the present invention.

[0098] Human and animal subjects at risk for (or exposed to) infection by strains of any of the following species may be administered fungal strains and/or vaccine compositions described herein including, without limitation: Aspergillus spp., Candida spp., Cryptococcus spp., Fusarium spp., Histoplasma spp., Pneumocystis spp., Trichophyton spp., Saccharomyces spp., Paracoccidioides spp., and Coccidioides spp.

[0099] In some specific examples, the method of the fourth aspect is a method of preventing or treating Candidiasis, or inducing an immune response to Candidiasis in a subject. Candidiasis is a fungal infection due to any Candida species, such as Candida albicans, Candida auris, Candida ciferrii, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida lusitaniae, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida spp., Candida tropicalis, Candida viswanathii or Candida zeylanoides. In some specific examples, the Candidiasis to be prevented or treated is caused by Candida albicans or Candida tropicalis. In some examples, the method comprises administering an effective amount of a Candida strain obtained using the method as disclosed herein, or an affective amount of a composition comprising a Candida strain obtained using the method as disclosed herein. In some specific examples, the Candida strain to be administered is Candida albicans or Candida tropicalis strains obtained using the method as disclosed herein. [00100] The fungal strains or vaccines described herein can also be administered in combination with one or more additional therapeutic agents, such as anti-fungal agents. Among the agents that can be used in combination therapy are polyenes (such as Amphotericin B, Mepartricin, atamycin, Nystatin, and the like), echinocandins (such as, but not limited to, candins, Caspofungin, Micafungin. aminocandins, anidulafungin, and the like), sordarins, azoies (such as, but not limited to, fluconazole, ketoconazole, itraconazole, posaconazole. clotrimazole, and the like), allylamines, morpholines, pradimicins, and other antifungals. The antifungal agents that are administered or to be administered in combination may act, for example, by blocking ergosterol synthesis (e.g., azoies or allylamines), by interfering with the ceil wall (e.g., echinocandins), by interfering with the ceil membrane (polyenes) or by interfering with protein translation (e.g., sordarins). Fungal strains may also be administered in combination with an antibody (or antigen-binding portion thereof) that specifically binds to a fungal component (e.g., a fungal polypeptide or carbohydrate). The antibody (or antigen-binding portion thereof) can be a monoclonal antibody, such as a human or humanized monoclonal antibody.

[00101] In some examples, the attenuated strains as obtained by the methods as described herein may be used for various biotechnologicai applications known in the art.

[00102] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00103] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00104] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXAMPLES [00105] The following examples illustrate methods by which aspects of the invention may be practiced or materials suitable for practice of certain embodiments of the invention may be prepared.

[00106] Example 1. Materials and Methods

[00107] Fungal strains and growth conditions [00108] Candida albicans (C. albicans) SC5314 (wild-type strain), dTOM-SC5314 (fluorescently labelled wild-type strain), all evolved C. albicans strains and wild-type C. tropicalis (ATCC 13803) and the evolved C. tropicalis strains were grown in yeast extract- peptone-dextrose media supplemented with 2% glucose (YPD), shaken at 150 rpm at 37°C overnight. Aspergillus fumigatus (A. fumigatus) AF293 (ATCC) were grown for 3-5 days on Potato dextrose agar (Sigma) and washed with phosphate -buffered saline (PBS) plus 0.05% Tween-20 to harvest A. fumigatus conidia. Staphylococcus aureus and Pseudomonas aeruginosa were grown in Luria-Bertani (LB) broth shaken at 150 rpm at 37°C overnight.

[00109] Animal work

[00110] C57BL/6J and RAG1 _/" (Jackson Laboratories) female mice (6-9 weeks) were used in all experiments. All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of A*STAR (Biopolis, Singapore) in accordance with the guidelines of the Agri-Food & Veterinary Authority (AVA), the National Advisory Committee for Laboratory Animal Research (NACLAR).

[00111] Gastrointestinal colonization [00112] Overnight cultures of wild-type C. albicans strain SC5314 or of wild-type C. tropicalis strain ATCC 13803 were harvested, washed twice in double-distilled water (ddH 2 0) and counted with a hemocytometer. Mice were pre-treated with an antibiotic cocktail in their drinking water for 3-4 days before starting gastrointestinal colonization (see specific protocol for details). Antibiotics were replenished twice weekly and maintained for the entire duration of the experiments. Mice were intragastrically gavaged with 1x10 live C. albicans cells and housed in separated cages. Stool was collected, weighed, homogenized in ddH 2 0 and plated on YPD-PS agar, i.e. YPD agar plates supplemented with 50U/ml of penicillin and streptomycin (Gibco). Colonization levels were assessed by counting the number of colony-forming units (CFUs) per gram of stool.

[00113] In vivo gastrointestinal evolution (serial passaging protocols A, B, E and F) [00114] Mice (C57BL/6J or RAG _/~ ) were pre-treated with 1 mg/ml of penicillin G sodium and 2 mg/ml streptomycin sulfate (both from Sigma) in their drinking water for 3-4 days and then colonized with 1x10 live SC5314 as described above. After 7 days, about 0.5 g of stool was collected from each mouse. The collected stool was weighed, homogenized in ddH 2 0, centrifuged at 13,000 rpm and resuspended in 300 μΐ ddH 2 0. A new set of naive mice, pre- treated with the same antibiotics as mentioned previously, was gavaged with the homogenized stool from the previous passage. After 7 days of colonization, the procedure was repeated using another set of naive mice as the recipients and serial passaging was performed weekly for a total of 1 (protocol A), 8 (protocol B) or 10 (protocol E and F) passages. Antibiotic treatment in the drinking water was maintained throughout the experiment. At the end of each protocol, fresh stool samples from the colonized mice were weighed, homogenized in ddH 2 0 and plated on YPD-PS agar to isolate single evolved colonies, which were then frozen at -80 °C in 50% glycerol.

[00115] In vivo gastrointestinal evolution (long-term colonization protocols C and D)

[00116] RAGl _/~ mice (protocol C) or C57BL/6J mice (protocol D) were pretreated with penicillin and streptomycin and colonized with 1x10 live C. albicans SC5314 (protocols C and D) or live C. tropicalis ATCC 13803 (protocol D) as described above. Stool was collected every week up to a total of 10 weeks, but no serial passaging between mice was performed. Antibiotic treatment was maintained throughout the experiment. At the end of the experiment, single evolved C. albicans or C. tropicalis colonies were harvested and stored as described above.

[00117] In vivo gastrointestinal evolution (serial passaging protocols G and H) [00118] RAGl _/~ mice (protocol G) or C57BL/6 mice (protocol H) were pretreated with penicillin and streptomycin as described above and colonized with 1x10 live cells of strain R2N, an evolved C. albicans strain obtained after 10 serial passages in RAGl _/~ mice (protocol E). After 14 days, about 0.5 g of stool was collected from each mouse. The collected stool was weighed, homogenized in ddH 2 0, centrifuged at 13,000 rpm and resuspended in 300μ1 ddH 2 0. Naive mice, pre-treated with penicillin and streptomycin as mentioned previously, were gavaged with the homogenized stool. After 14 days of colonization, the procedure was repeated using another naive mouse as the recipient and in vivo passaging was performed bi-weekly up to 5 passages. Antibiotic treatment was maintained throughout the experiment. At the end of the experiment, single evolved C. albicans colonies were harvested and stored as described above.

[00119] In vivo gastrointestinal evolution (serial passaging protocols I and J)

[00120] C57BL/6J mice were first colonized with lxlO 7 live SC5314 essentially as described above, with the only exception that mice were pretreated with 1 mg/ml ampicillin sodium salt and 0.1 mg/ml gentamycin sulfate (protocol I), or with 1 mg/ml metronidazole and 1 mg/ml tetracyclin (protocol J) instead of penicillin and streptomycin. In addition, some mice were also pretreated with 0.25 mg/ml of fluconazole in their drinking water (protocols 1.1 and J. l). Thereafter, the protocol proceeded similarly to protocol F for a total of 10 weekly serial passages. Antibiotic treatment was maintained throughout the experiment, but fluconazole (where applicable) was withdrawn 3 days prior to C. albicans gavage. At the end of the experiment, single evolved C. albicans colonies were harvested and stored as described above.

[00121] In vivo gastrointestinal competition assay

[00122] C57BL/6J mice were pre-treated with penicillin and streptomycin as described above and gavaged with a 1: 1 mixture of competing C. albicans strains (test strain vs. fluorescent dTOM-SC5314 strain) with a total number of 1x10 C. albicans in 300 μΐ of ddH 2 0. Antibiotic treatment was maintained throughout the experiment. Stool was collected at 6, 12, 24, 30, 36 and 48 hours after initiation of competition experiment. Stools were homogenized in ddH 2 0 and plated on YPD-PS agar. Relative abundances of the two competing strains were determined by counting the number of colonies of the test and fluorescent strains under a fluorescent stereomicroscope (Stereoscopes Olympus Fluorescence MVX10). The relative fitness coefficient of the test strain was then obtained by linear regression according to the following formula:

[00123] log 2 [R(f)/*(io)] = s 7R (t-t 0 ),

[00124] where R(i) represents the ratio between the test strain and the reference strain at time i; s is the selection coefficient; y R is the growth rate of the reference strain expressed as cell divisions per hour; t represents the time points in hours and to the initial time point.

[00125] In vitro cytotoxicity (LDH release) assay

[00126] The mouse macrophage cell line J774.1 and the human gut epithelial cell line HT- 29 were used to determine the cytotoxicity of the evolved strains in vitro by the lactate dehydrogenase (LDH) release assay. Briefly, the macrophages or epithelial cells were grown in complete medium (without phenol red) and co-cultured for 6 or 24 hours with one of several C. albicans or C. tropicalis strains (control or evolved) at a multiplicity of infection (MOI) of 1 or 0.01, respectively, or incubated with 5 mM tert -butyl hydroperoxide as a positive control. After incubation, LDH release was measured with the CytoTox 96 ® Non- Radioactive Cytotoxicity Assay (Promega) according to the manufacturer's protocol. The cytotoxicity of the different evolved strains was then calculated as the average of three independent biological replicates and represented as a percentage of the positive control.

[00127] In vivo virulence assay (systemic infection model)

[00128] Control and evolved C. albicans and C. tropicalis strains were harvested from overnight cultures and number of cells was counted with a hemocytometer. Cells were washed twice in phosphate -buffered saline (PBS), and wild-type (C57BL/6J) or RAGl _/~ (Jackson Laboratories) mice were infected intravenously with a lethal dose (5 x 10 5 CFUs) of live SC5314 or one of different evolved C. albicans strains or with a lethal dose (5 x 10 ) C. tropicalis wild-type or the evolved C. tropicalis strains. Mouse survival was monitored and recorded daily.

[00129] Example 2. Serial passaging of C. albicans through the gastrointestinal tract of mice

[00130] Wild-type laboratory mice (strain C57BL/6J), which are normally not colonized by C. albicans, were pre-treated with a cocktail of antibiotics (penicillin and streptomycin, or ampicillin and gentamycin, or metronidazole and tetracyclin, depending on the protocol) in their drinking water for 3-4 days, followed by intragastric gavage of the wild-type C. albicans (strain SC5314). Some mice were additionally pre-treated with the antifungal agent fluconazole in their drinking water to clear their gut from any potential endogenous fungi. As expected, robust and stable colonization of the GI tract, routinely maintained in the range of 10 5-108 colony forming units (CFUs) of C. albicans per gram of stool, was achieved by this method (data not shown). After 7 days of colonization of the first mouse, collected stool samples were collected and a faecal transplant was performed by intragastric gavage to a second mouse, which had also been pre-treated with the same cocktail of antibiotics for 3-4 days prior to the transplant. Robust and stable colonization, i.e. 10 5 -108 CFUs of C. albicans per gram of stool was detected in the recipient mouse (data not shown), indicating that the faecal transplant was successful. After 7 days of colonization of the second mouse, a similar faecal transplant was performed on a third mouse, and so on, until a total of 8 or 10 serial passages. Several variations on this protocol, differing by Candida species, by type of antibiotic agent, by number of passages, by duration of each passage or by mouse host genotype, were also carried out. With regards to the latter, C. albicans cells were passaged either in wild-type (WT) mice or immunocompromised Ragl knock-out (KO) mice, which are developmentally impaired in their ability to produce any functional T and B cells. One of the strains (named 'R2N'), which was obtained after 10 weekly serial passages in Ragl KO mice, was also used for further passaging in either WT or Ragl KO mice for an additional 5 bi-weekly serial passages. A summary of the different combinations of parameters (designated as protocols A through J) that were tested is presented in Table 1. In each and every single protocol, robust and stable colonization in each mouse as well as successful transmission of C. albicans from mouse to mouse could be achieved. At the end of each experiment, the passaged C. albicans strain (which is referred to as an 'evolved strain') is recovered by plating the stool of the last colonized animal on an appropriate selective plate, followed by single-colony isolation of one or more individual evolved clones.

Numbe Total

Antifunga

Protoc Antibiotic r of Passage length evolution Mouse host

1

ol treatment passag (weeks) time genotype treatment

es (weeks)

Penicillin/

A Strep tomyci N/A 1 1 1 WT n

Penicillin/

B N/A 8 1 8 WT Strep tomyci n

Penicillin/

C Strep tomyci N/A 1 10 10 Ragl n

Penicillin/

D Strep tomyci N/A 1 10 10 WT

n

Penicillin/

E Strep tomyci N/A 10 1 10 Ragl n

Penicillin/

F Strep tomyci N/A 10 1 10 WT

n

1 (first 10

Penicillin/

passages);

G Strep tomyci N/A 15 20 Ragl

2 (next 5

n

passages)

1 (first 10 Ragl (first 10

Penicillin/

passages); passages);

H Strep tomyci N/A 15 20

2 (next 5 WT (next 5 n

passages) passages)

Ampicillin/

1.1 N/A 10 1 10 WT

Gentamycin

Ampicillin/

1.2 Fluconazole 10 1 10 WT

Gentamycin

Metronidaz

J.l ole/ N/A 10 1 10 WT

Tetracycline

Metronidaz

J.2 ole/ Fluconazole 10 1 10 WT

Tetracycline

Table 1. Serial passaging protocols. The table summarizes the various implementations of the method that have been used to evolve C. albicans strains in the GI tract of antibiotics- treated mice. The different protocols (A through J) differed by antibiotic treatment, number of passages, length of each passage, and the mouse host genotype. For protocols G and H, different sets of parameters were used in the first 10 passages compared to the next 5 passages. WT: C57BL/6J mice. Ragl: RAG1 _/" mice.

[00131] Example 3. Increased competitive fitness of evolved C. albicans strains in the mouse gastrointestinal tract

[00132] If the mouse GI tract acts as a selective pressure on the C. albicans cells during the serial passaging experiment, the evolved strains recovered at the end of the serial passaging experiment are expected to display an increased competitive fitness in comparison to the 'unevolved' strain. To test this prediction, an in vivo competition assay was carried out. Briefly, WT mice were pre-treated with oral antibiotics for 3-4 days and intragastrically gavaged with a 1: 1 mixture of a test strain (either an evolved or an unevolved C. albicans strain) and a reference strain, consisting of a strain of C. albicans engineered to express a red fluorescent protein (dTomato). At regular intervals, stools were plated on appropriate selective media and the relative frequency of fluorescent and non-fluorescent fungal colonies was assessed. Using a statistical regression technique, the rate of change of fluorescent vs. non-fluorescent colonies over time was then converted into a measure of the competitive fitness of the test strain in comparison to the reference strain. Consistent with the hypothesis, all tested evolved strains, which were obtained using any one of several different variants of the protocol as described here, demonstrated a significantly increased competitive fitness in the mouse GI tract as compared to the unevolved SC5314 wild-type strain (Figure 1).

[00133] Example 4. Reduced in vitro cytotoxicity of evolved Candida strains

[00134] To measure the virulence of the evolved C. albicans and C. tropicalis strains, the evolved strains were subjected to an in vitro cytotoxicity assay as a way to assess their ability to cause host cell damage. Several evolved strains, collectively obtained by one of several different serial passaging protocols, were individually added to monolayer cultures of either a mouse macrophage cell line (J774.1) or a human gut epithelial cell line (HT-29) to test their ability to cause cell damage, which was measured by release of the intracellular enzyme lactate dehydrogenase (LDH). With the exception of strains obtained with protocol A (a single week-long passage in the mouse gut), which yielded strains that tended to be less cytotoxic than the WT unevolved strain (but this difference was not statistically significant), all protocols yielded evolved strains that were significantly less cytotoxic in either J774.1 or the HT-29 cells, or both (Figures 2 and 3). Closer inspection of data from individual evolved strains revealed that C. albicans strain XI, which was obtained by protocol A, displayed reduced cytotoxicity towards both J774.1 and HT-29 cells to a degree that was comparable to evolved strains obtained by one of the other protocols (Table 2). Taken together, these results demonstrate that passaging through the mouse gut not only increases the competitive fitness of a fungal pathogen to the mouse gut environment, but also reduce its ability to cause harm to host cells. Furthermore, this data indicates that even a single week- long passage in the mouse GI tract is sufficient to reduce the virulence of a fungal pathogen, albeit at lower frequency and efficiency as with longer or more passages.

Table 2. In vitro and in vivo virulence data of C. albicans strains. The table presents virulence data for specific evolved C. albicans strains obtained by one of the different serial passaging protocols. LDH release by macrophages or epithelial cells after the indicated time- point and at the indicated multiplicity of infection (MOI) is represented as a percentage against a positive control in each assay (cell damage induced by tert-Butyl hydroperoxide). Survival of mice of the indicated genotype was followed for 28 days after intravenous injection with the indicated strain at the indicated dose. SC3514: wild-type, ancestral C. albicans strain used as a control. Not tested: experiment not performed.

[00135] Example 5. Reduced in vivo virulence of evolved Candida strains in WT mice

[00136] It was further investigated if evolved C. albicans and C. tropicalis strains in addition to displaying reduced in vitro cytotoxicity against host cells were also associated with reduced in vivo virulence towards live animals. To test this hypothesis, a systemic infection model, consisting of intravenous injection of a lethal dose of live C. albicans or C. tropicalis cells in WT mice, followed by recording of the number of days required for each mouse to succumb to the infection, was employed. Whereas injection of the WT C. albicans unevolved strain SC5314 or the WT C. tropicalis strain ATCC 13803 resulted in 100% of the animals to die in <7 days, all tested C. albicans strains, obtained by one of protocols E, F, G, I or J, as well as the gut-evolved C. tropicalis strain CT8, resulted in 100% mouse survival for up to 28 days post-infection (Figures 4 and 5, and Tables 2 and 3). Importantly, also C. albicans strain XI (which was obtained by protocol A, i.e. after only a single week-long passage through the mouse gut) resulted in 70% animal survival for up to 28 days (Figure 4 and Table 3). Together these results demonstrate that serial passaging of the pathogenic fungus C. albicans or C. tropicalis through the mouse GI tract selects not only for variants that are competitively fitter in this host environment but wherein these variants are also significantly less virulent both in vitro and in vivo against the same host species in which they have been passaged.

Table 3: In vitro and in vivo virulence data of C. tropicalis strains. The table presents virulence data for specific evolved C. tropicalis strains obtained by protocol D at different weeks of evolution. LDH release by macrophages or epithelial cells after the indicated time- point and at the indicated multiplicity of infection (MOI) is represented as a percentage against a positive control as described for Table 2. Survival of C57BL/6J mice was followed for 28 days after intravenous injection with the indicated strain. ATCC 13803: wild-type, ancestral C. tropicalis strain used as a control. Not tested: experiment not performed.

[00137] Example 6. Reduced in vivo virulence of evolved C. albicans strains in immunocompromised mice [00138] Since systemic infections by C. albicans most often occur in patients with compromised immunity, it was tested whether the attenuation in pathogenicity observed in the evolved strains described herein extends also to systemic infection in animals lacking a functional immune system. To this end, the above-described systemic infection experiments were repeated in mice genetically disrupted of the Ragl gene (Ragl " ' " mice), which are completely deficient in adaptive immunity. While only 1 out of 9 Ragl " ' " animals survived 28 days after an intravenous challenge with the unevolved SC5314 C. albicans strain, 100% of mice survived systemic infection with either one of three evolved C. albicans strains obtained by protocol E or with one of two evolved strains obtained with protocol F; the other strain obtained by protocol E, termed 'W2N', nevertheless yielded 80% animal survival after 28 days, which corresponded to a significantly reduced virulence as compared to the unevolved SC5314 (Figure 4 and Table 2). Therefore, it was concluded that serial passaging through the mouse gut of a pathogenic C. albicans strain significantly reduces the ability of the fungus to cause harm to either immunocompetent or immunocompromised animals. [00139] Example 7. Cellular morphologies of evolved C. albicans strains and the reduced in vivo virulence of evolved C. albicans strains

[00140] Cellular morphologies of the C. albicans strains were also examined by the inventors. It is noted that P8 and P10 strains were significantly more refractory to hyphal- inducing stimuli than PI or unevolved strains (Figure 7d-e). These results suggested that adaptive evolution in the mouse GI tract yields yeast-locked mutants with increased intra-GI tract fitness.

[00141] In a heat map analsyis of the clustering of gut-evolved C. albicans strains, 87 verified open-reading frames (ORFs) were identified as carrying > 1 denovo mutation in > evolved strain (Figure 8a and Table 4).

[00142] Table 4: Non-synonymous, de novo mutations acquired by gut-evolved C. albicans st rains in verified open reading frames (ORFs)

Evolution Strain ORF ID Gene Amino acid changes

Protocol name

B6/8p XH_Ll-8-4 orfl9.723 BCR1 Gln83fs,Pro84fs

orfl9.1166 CTA3 Pro403_Gln404del

orfl9.1093 FL08 Leu667fs

orfl9.4961 STP2 Glu 172delins ValLy sGln

XH_L4-8-l orfl9.3356 ESP1 Asnl326Ser

orfl9.1093 FL08 Gln659*

orfl9.6877 PNG2 Lys434Glu orfl9.3429 FGR47 Gly787_Glu788insGlyGly,Thr530Asn,Asn3

43_Asn345del

orfl9.1234 FGR6-10 Val82Ile,Arg334His,Ala425Thr,Glu480Asp orfl9.1093 FL08 Gln697*

orfl9.465 IFF9 Gly482Asp,Met487Thr

orfl9.5179 LIP5 Leul67Ser

orfl9.5734 POP2 Glnl48_Glnl49del

orfl9.4208 RAD52 Gln22_Gln24del

orfl9.5124 RBR3 Ser558Asn,Asn557Ser,Pro448fs,Lys447fs,S er446Thr

orfl9.5595 SHE3 Asn443del,Tyr444_Asn446del,Tyr444Asn orfl9.5058 SMI1 Pro560_Glu561del

orfl9.801 TBF1 Glu799fs,Glu799Gln,Gln800fs,Gln801Lys,

Glu803fs,Gln805fs

[00143] As the hyphal morphogenesis program is a key virulence factor of C. albicans, it was hypothesized that gut-evolved strains would be less damaging to host cells and be less virulent during infection. To test this, the evolved C. albicans strains were co-cultured with murine macrophages or human colon epithelial cells and lactate dehydrogenase (LDH) release was quantified as a measure of their cytotoxicity. It was found that P10 strains induced significantly lower cell damage than wild type or PI strains (Figure 9a). The virulence of gut-evolved strains was then tested in a mouse model of hematogenously disseminated candidiasis. As expected, using a lethal dose of WT C. albicans, formation of hyphae was observed in the kidneys of WT mice 2 days post-infection (dpi), as well as severe necrosis, moderate perivascular edema and moderate diffuse pyogranulomatous renal capsulitis in most animals (Figure 9b). Eventually, all mice infected with WT or PI strains succumbed within 3-4 dpi (Figure 9c). In contrast, at the same infection dose, P10 strains remained in the yeast form in kidneys at 2 dpi, and only mild to moderate necrosis was observed, with some animals displaying mild edema or capsulitis (Figure 9b). Mice infected by the P10 strains initially lost 10-20% of weight up until 7 dpi, then gradually regained their initial weight at -21 dpi and all mice survived until 28 dpi (Figure 9c and 11). Similar results were obtained when immunocompromised Ragl 7 mice were infected, where the survival rates of mice infected by P10 strains were significantly higher than those infected by the WT or PI strains (Figure 9c). Hence, the experimental system as described herein reproducibly yields C. albicans strains that are hyper-fit in the mouse GI tract, genetically locked in the yeast form and avirulent. [00144] To reconcile above findings with the fact that, despite the human GI tract being the natural niche of C. albicans, most clinical isolates of C. albicans retain their virulence and their filamentation ability, three independent, long-term evolution experiments were performed in non-antibiotic-treated mice, which harbor a rich GI microbiome like most healthy humans. Gut colonization was achieved by intragastrically inoculating 2-weeks-old pups with the same ancestral C. albicans strain used for the previous evolution experiments and maintained for up to 9 weeks. C. albicans strains obtained after 9 weeks of evolution (W9 strains) all retained the ability to respond to hyphal-inducing stimuli as well as their virulence in the intravenous infection model (Figure 12a-b). Furthermore, P10 strains, while being hyper-fit in an antibiotic-treated gut, actually colonized the GI tract of untreated mice less efficiently than WT cells (Figure 12c-d). Taken together, the results indicate that, treatment to at least partially remove gut microbiota is required to obtain the evolved strains with reduced virulence, and/or improved competitive fitness.

[00145] Example 8. Effect of evolved C. albicans strains against systemic challenge with virulent C. albicans

[00146] To test if gut-evolved C. albicans strains can be used as effective vaccines that protect against infections with virulent C. albicans, WT C57BL/6 mice were primed with the evolved C. albicans strains followed by systemic challenge with a lethal dose of a fully virulent C. albicans strain (SC5314) 28 days later. All mock-vaccinated animals died within 3-4 days post-challenge (dpc) (Figure 10a). Priming with a sub-lethal dose of WT C. albicans SC5314 delayed host mortality but eventually all animals succumbed to the challenge (Figure 13a). In contrast, 60- 80% of mice primed with a full dose of a P10 strain survived the secondary challenge (Figure 10a). Similar to WT mice, Rag l _ ~ mice immunized with P10 strains were also significantly more protected from systemic candidiasis (Figure 10b). In particular, -85% of R24-vaccinated mice survived at least until 32 dpc. It was also observed that significant protection against infection with a fully virulent C. albicans strain was achieved as early as 1 day post-priming with the R24 gut-evolved C. albicans strain (Figure 10c).

[00147] Example 9. Effect of evolved C. albicans in innate immune system

[00148] In view of the correlation observed in the increased protection observed in P10-primed mice over those immunized with wild type C. albicans with the increased total as well as anti-C. albicans-specific immunoglobulin G (IgG) titers in the serum (Figure 14), experiments in Ragl " /_ mice were repeated to test the contribution of adaptive immunity in this protective mechanism. Similar to WT mice, Ragl ^ mice immunized with P10 strains were significantly more protected from systemic candidiasis than non-immunized mice or those immunized with efgl ~h cells (Figure. 10c). In particular, -85% of R24-vaccinated mice survived at least until 32 dpc. This indicated the P10 strains were able to raise protective immune responses independently of B and T cells.

[00149] As understood by the person skilled in the art, while adaptive immunity typically requires a few weeks to mount long-lived memory responses, innate immune responses are characterized by a more rapid onset but shorter lifetimes. Consistent with an innate-like memory mechanism, significant protection against infection with a fully virulent C. albicans strain was achieved as early as 1 day post-priming with the R24 gut-evolved C. albicans strain (Figure 10c); and at three months post-priming Ragl _/~ mice were no longer protected by most P10 strains, while wild type (WT) mice still retained a partial protection probably due to a bystander adaptive immune memory (Figure 13b).

[00150] It is understood that trained innate immunity is characterized by increased cytokine responses that are associated with innate immune response (for example IL-6 and TNF- alpha). Consistent with this hallmark, the increased protection observed at day 28 in mice immunized with P10 strains R24 or W2N correlated with increased IL-6 in the serum and the kidneys and with increased IL-6 and TNF-a production upon ex vivo re- stimulation of splenocytes with heat-killed C. albicans cells (Figure lOd to lOg).

[00151] The non-antigen-specific nature of innate immunity predicts that priming with a P10 strain should confer broad cross -protection against a wide range of pathogens. To test this, in this Example 9, the inventors first intravenously primed naive mice with a P10 strain and then challenged them systemically at 14 dpi with a lethal dose of the unrelated fungal pathogen A. fumigatus, a gram-positive bacterium, Staphylococcus aureus, or a gram- negative bacterium, Pseudomonas aeruginosa. In all cases, animals immunized with a P10 strain were significantly protected from infection in comparison to control mice (Figure 10 h to lOj). Confirming that this cross -protection was independent of adaptive immunity, Ragl _/~ mice showed similar cross -protection to A. fumigatus as wild type (WT) mice (Figure 15). Altogether, gut-evolved P10 strains were able to raise protective immune responses characterized by rapid onset, short lifetime, strong innate cytokine responses, pathogen-aspecificity and independence from T and B cells; thus, this Example 9 clearly shows the gut-evolved strain produced by the method of the present disclosure can provide trained innate immunity (trained immunity).