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WO2018182515A1 - Procédés de modification de champignons et utilisations correspondantes - Google Patents

Procédés de modification de champignons et utilisations correspondantes Download PDF

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Publication number
WO2018182515A1
WO2018182515A1 PCT/SG2018/050142 SG2018050142W WO2018182515A1 WO 2018182515 A1 WO2018182515 A1 WO 2018182515A1 SG 2018050142 W SG2018050142 W SG 2018050142W WO 2018182515 A1 WO2018182515 A1 WO 2018182515A1
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weeks
candida
host organism
fungus
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Norman Xaver PAVELKA
Hoi Wan Gloria TSO
Xiao Hui SEM
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Agency for Science Technology and Research Singapore
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/725—Candida albicans

Definitions

  • the present invention relates to mycology, in particular methods of modifying fungal strains, and more particularly methods of modifying fungal strains of the Candida
  • Candidiasis (caused by Candida species) is the most common opportunistic fungal infection and the fourth most common nosocomial bloodstream infection.
  • Candida species a sign of invasive or systemic candidiasis
  • 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%.
  • antifungal drugs such as azoles, echinocandins, polyenes, allylamines or nucleoside analogues
  • 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.
  • strategies for the development of an antifungal vaccine have been proposed throughout the years.
  • no antifungal vaccine on the market.
  • 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.
  • the virulence of the fungus should be reduced, and/or the competitive fitness of the fungus in the host environment should be increased.
  • 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.
  • 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 with improved competitive fitness in one or more host environment.
  • a vaccine comprising a fungal strain obtained using the method of the first aspect.
  • a method of preventing or treating a disease in a subject in need thereof comprising administering an effective amount of the fungal strain of the second aspect, or an effective amount of the vaccine of the third aspect.
  • 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.
  • FIG. 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.
  • LDH Lactate dehydrogenase
  • FIG. 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.
  • LDH Lactate dehydrogenase
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • FIG. 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 ⁇ ; red scale bar: 20 ⁇ ). The results demonstrate that gut-evolved C. albicans strains are defective in hyphal formation in response to in vitro stimuli.
  • 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.
  • FIG. 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 ⁇ ; red scale bar: 20 ⁇ .
  • Figure 9c are line graphs showing C.
  • 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 10c is a line graph showing the survival rate of WT mice primed with the P10 C.
  • FIG. 1 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).
  • FIG. 1 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.
  • 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).
  • 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 ⁇ ; red scale bar: 20 ⁇ .
  • Figure 12c shows colonization levels of WT pups with SC5314, W2N and R24 strains in the absence of antibiotics treatment (n > 20 mice/group).
  • 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).
  • 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.
  • 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.
  • IgG immunoglobulin G
  • 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.
  • the results demonstrate that the WT mice intramuscularly primed with gut evolved strain are significantly protected from systemic challenge.
  • 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 with improved competitive fitness in one or more host environment.
  • the method further comprises isolating the fungus from the gastrointestinal tract discharge collected, so as to obtain an isolated fungal strain.
  • 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.
  • 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.
  • a species or strain of an organism may be non-pathogenic towards a healthy host organism, but is pathogenic towards an immunocompromised host organism.
  • different strains of the same species may have different level of virulence towards the same host organism.
  • Various methods and/or parameters can be used to indicate the virulence of an organism to a particular host organism.
  • 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.
  • 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.
  • LD50 median lethal dose
  • organs generally spleen or liver
  • CFUs colony forming units
  • Attenuated refers to the weakening or decreasing of the virulence of the wild-type organism.
  • avirulent 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.
  • fit refers to the reproductive success of an organism in a given environment, for example, in a given host environment.
  • competitive fitness refers to the reproductive success of an organism relative to another organism in the same environment.
  • Second test strain 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.
  • the competitive fitness of an organism can be represented by a competitive fitness coefficient.
  • reduce or “reduction” or grammatical variants thereof refer to a decrease in the specified parameter as compared to the same parameter of a reference.
  • reduce 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.
  • 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.
  • the term "fungal strain of reduced virulence" or its grammatical variants can also be defined qualitatively.
  • the fungal strain that the host organism has been exposed to can be considered as a fungal strain of reduced virulence.
  • 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.
  • the term "improve” or grammatical variants thereof refer to an increase in the specified parameter as compared to the same parameter of a reference.
  • “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.
  • 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.
  • 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.
  • microbiota refers to commensal and symbiotic microorganisms found in a host organism.
  • 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.
  • the gut microbiota to be partially or completely removed from the host organism includes bacteria, or fungi, or a mixture of 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.
  • 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.
  • the removal of bacteria is only carried out during step (i) of the method.
  • 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.
  • the removal of bacteria is carried out through steps (i) to (iii) of the method as described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • antibiotic treatment may be useful in steering the evolution of strains in the direction required (for example towards attenuation of virulence).
  • antibiotic agent refers to a substance that may either kill or inhibit the growth of bacteria.
  • 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-phenoxazine- 1,9- dicarboxamide), amikacin (IUPAC name: (2S)-4-Amino-N-[(2S,3S,4R,5S)-5-amino-2- [(2S,3R,4S)
  • 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- (amino)ethyl]oxan-2-yl]oxy ⁇ -2-hydroxycyclohexyl]oxy ⁇ -5-methyl-4- (methylamino)oxane-3,5-diol), imipe
  • the one or more antibiotic agent has activity against anaerobic intestinal bacteria.
  • the antibiotic agent is a mixture of penicillin and streptomycin, as shown in protocols A to H presented in Table 1.
  • the penicillin is penicillin G.
  • the antibiotic agent is a mixture of ampicillin and gentamycin, as shown in protocols I.l and 1.2 presented in Table 1.
  • the antibiotic agent is a mixture of metronidazole and tetracyclin, as shown in protocols J.l and J.2 presented in Table 1.
  • antifungal refers to a substance that may either kill or inhibit the growth of fungi.
  • 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
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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 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 include but are not limited to, for example, a germ-free or a neonatal, pre-weaned or juvenile host organism.
  • 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.
  • 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.
  • 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).
  • 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
  • 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).
  • the time interval between each passage should be such as to sufficiently allow the fungus to replicate between passages.
  • 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.
  • 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
  • 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,
  • 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).
  • 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).
  • 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).
  • 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
  • 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.
  • 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 g
  • capsulatum Histoplasma capsulatum var. duboisii, Histoplasma spp., Hortaea wasneckii, 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 pul
  • 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, Sta
  • 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.
  • the fungus is Candida albicans or Candida tropicalis.
  • 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.
  • 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.
  • 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.
  • the host organism is a non-human host organism, and/or the host environment is a human host environment.
  • the host organism is a non-human host organism, and/or the host environment is a non-human host environment.
  • 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.
  • the non-human mammalian host organism is a mouse (Mus musculus).
  • the non-human mammalian host organism is an adult mouse.
  • 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.
  • the adult mouse may be at least 6 to 8 weeks of age, that is about 42 to 56 days.
  • 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.
  • the host organism 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.
  • the host organism is an immunocompromised host organism.
  • the immunocompromised host organism lacks functioning T and/or B cells.
  • the immunocompromised host organism is a Rag 1 -deficient mouse.
  • the host environment is the host environment of an immunocompromised host.
  • the immunocompromised host lacks functioning T and/or B cells.
  • the immunocompromised host is a Ragl- deficient mouse.
  • the host organism is an immunocompromised host organism
  • the host environment is the environment of a normal host (i.e. not an
  • 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.
  • the host organism is a normal host organism, and the host environment is the environment of a normal host.
  • the host organism is an immunocompromised host organism, and the host environment is the environment of an immunocompromised host.
  • 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.
  • 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
  • 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.
  • 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.
  • the non-human host environment is the gastrointestinal tract of a mouse.
  • 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.
  • 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.
  • 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.
  • the gastrointestinal tract discharge of the host organism comprises solid and/or liquid discharge, or a mixture thereof.
  • the method as disclosed herein can also be used for the generation of fungal strains for biotransformation or for the manufacture of vaccines.
  • a fungal strain obtained using the method of the first aspect is provided.
  • 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).
  • the fungal strain obtained is an isolated fungal strain.
  • 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.
  • the fungal strains obtained using the method as disclosed herein grow as ovoid, yeast form cells, as shown in Example 7.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term "vaccine” may also be used to encompass immunity that is conferred via trained innate immunity.
  • 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.
  • IL-6 i.e. interleukin 6
  • TNF-alpha i.e. Tumor Necrosis Factor-alpha
  • the "vaccines" of the present disclosure can provide systemic acute protection via trained innate immunity
  • 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).
  • 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).
  • 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.
  • 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.
  • 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 heredit
  • 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.
  • autoimmune disease such as Felty's syndrome, rheumatoid arthritis, or systemic lupus erythematosis
  • idiosyncratic reactions to drugs that, in an idiosyncratic way, act as haptens at the surface of neutrophils, initiating immune destruction of neutrophils.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • the vaccine is administered by intramuscular or subcutaneous routes.
  • 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.
  • 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.
  • the vaccine may result in a lessening of the physical symptoms associated with the disease.
  • 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.
  • 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.
  • the vaccine is prepared from freshly produced fungal strains that are obtained using methods of the first aspect.
  • 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.
  • the vaccine prepared may be desiccated, for example, by freeze drying for storage purposes or for subsequent formulation into liquid vaccines.
  • 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.
  • 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.
  • a method of preventing or treating a disease in a subject in need thereof 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.
  • 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.
  • the disease to be prevented or treated is a disease caused by one or more fungal species different from the fungal species being administered.
  • 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.
  • the disease is caused by microorganisms such as, but not limited to, bacteria, archaea, virus, yeast, fungi, protozoa, algae, and the like.
  • 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).
  • the disease is caused by gram-positive bacteria or gram- negative bacteria.
  • 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, Kleb
  • 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,
  • 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, Arthro
  • capsulatum Histoplasma capsulatum var. duboisii, Histoplasma spp., Hortaea wasneckii, 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 pul
  • 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, Sta
  • the fungus is Aspergillus or Candida species.
  • 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.
  • the fungus is Candida albicans or Candida tropicalis.
  • the fungus is Candida species, Aspergillus species, Cryptococcus species, and Histoplasma species
  • the disease is caused by parasites (such as, but not limited to, protozoan, heminthic, and the like).
  • 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.
  • 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.
  • the disease is malaria.
  • 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.
  • 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.
  • 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.
  • the disease is caused by virus such as, influenza virus, dengue virus, zika virus, and chikungunya virus.
  • polymicrobial disease is diseases (or infections) that are caused by multiple infectious agents.
  • polymicrobial diseases may include polyviral diseases, polybacterial diseases, viral and bacterial infections, fungal infections, infections resulting from microbe-induced immunosuppression, and the like.
  • 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.
  • 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.
  • 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.
  • the immune response to be induced is an immune response to one or more fungal species different from the fungal species being administered.
  • the fungal strain of interest is C. albicans
  • the wildtype and clinical strain used may be SC5314.
  • the fungal strain can also be administered as a vaccine, which is provided under the third aspect of the present invention.
  • 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.
  • 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.
  • the Candidiasis to be prevented or treated is caused by Candida albicans or Candida tropicalis.
  • 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.
  • the Candida strain to be administered is Candida albicans or Candida tropicalis strains obtained using the method as disclosed herein.
  • 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.
  • 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.
  • 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 such as, but not limited to,
  • 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.
  • the attenuated strains as obtained by the methods as described herein may be used for various biotechnologicai applications known in the art.
  • 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
  • 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.
  • 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.
  • PBS phosphate -buffered saline
  • 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.
  • 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 ⁇ ddH 2 0.
  • RAGl _/ ⁇ mice protocol C
  • C57BL/6J mice protocol D
  • mice 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.
  • single evolved C. albicans or C. tropicalis colonies were harvested and stored as described above.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • MOI multiplicity of infection
  • 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.
  • PBS phosphate -buffered saline
  • wild-type mice C57BL/6J
  • RAGl _/ ⁇ Jackson Laboratories
  • Example 2 Serial passaging of C. albicans through the gastrointestinal tract of mice
  • Wild-type laboratory mice 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.
  • 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).
  • 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.
  • a similar faecal transplant was performed on a third mouse, and so on, until a total of 8 or 10 serial passages.
  • passages ); passages;
  • 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.
  • Example 3 Increased competitive fitness of evolved C. albicans strains in the mouse gastrointestinal tract
  • 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).
  • test strain either an evolved or an unevolved C. albicans strain
  • reference strain consisting of a strain of C. albicans engineered to express a red fluorescent protein (dTomato).
  • Example 4 Reduced in vitro cytotoxicity of evolved Candida strains
  • 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.
  • Example 5 Reduced in vivo virulence of evolved Candida strains in WT mice
  • 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).
  • C. albicans strain XI which was obtained by protocol A, i.e. after only a single week-long passage through the mouse gut
  • results resulted in 70% animal survival for up to 28 days ( Figure 4 and Table 3).
  • 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.
  • Example 6 Reduced in vivo virulence of evolved C. albicans strains in immunocompromised mice
  • 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.
  • 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.
  • mice 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
  • 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).
  • 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.
  • 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).
  • Example 8 Effect of evolved C. albicans strains against systemic challenge with virulent C. albicans
  • 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).
  • Example 9 Effect of evolved C. albicans in innate immune system
  • 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).

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Abstract

La présente invention concerne un procédé de réduction de la virulence d'un champignon et/ou d'amélioration de l'état physique compétitif d'un champignon dans un ou plusieurs environnements hôtes, consistant à (i) soumettre un organisme hôte à un traitement pour éliminer partiellement ou complètement le microbiote intestinal ou obtenir un organisme hôte exempt de microbiote intestinal, (ii) inoculer un champignon dans le système digestif de l'organisme hôte pour permettre au champignon de coloniser le tractus gastro-intestinal de l'organisme hôte et (iii) récupérer le rejet du tractus gastro-intestinal de l'organisme hôte pour obtenir un champignon présentant une virulence réduite et/ou un état physique compétitif amélioré dans un ou plusieurs environnements hôtes. En particulier, le traitement pour éliminer le microbiote intestinal implique le traitement avec un ou plusieurs agents antibiotiques et un ou plusieurs agents antifongiques et le champignon est Candida albicans. L'invention concerne également un vaccin comprenant le champignon obtenu à l'aide dudit procédé et des utilisations correspondantes.
PCT/SG2018/050142 2017-03-27 2018-03-27 Procédés de modification de champignons et utilisations correspondantes Ceased WO2018182515A1 (fr)

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