SACCHAROMYCES CEREVISIAE YEAST STRAIN FOR THE TREATMENT AND/OR PREVENTION OF OROPHARYNGEAL CANDIDIASIS

Abstract

The yeast strain Saccharomyces cerevisiae number CNCM I-3856 for the treatment and/or prevention of oropharyngeal infections by Candida. This strain has shown a strong ability to reduce living fungi in the oral cavity and to prevent the propagation of the infection by Candida to the esophagus, the stomach and the small intestine.

Claims

1-13. (canceled)

14. A method for preventing and/or treating oropharyngeal candidiasis in a subject, said method comprising a set of administering to said subject a Saccharomyces cerevisiae yeast strain deposited with the CNCM on Oct. 17, 2007 under Accession Number I-3856.

15. The method according to claim 14, wherein the Saccharomyces cerevisiae yeast strain is in the live form or in the inactive form.

16. The method according to claim 14, wherein the Saccharomyces cerevisiae yeast strain is in the form of dry yeast.

17. The method according to claim 16, wherein the Saccharomyces cerevisiae yeast strain in the form of dry yeast is in the form of active dry yeast.

18. The method according to claim 14, wherein the Saccharomyces cerevisiae yeast strain is in a fractionated form.

19. The method according to claim 18, wherein the fractionated form is selected from the group consisting of cell walls of said yeast, β-glucans from said cell walls of said yeast, wall mannoproteins of said yeast, extracts from said yeast, and combination thereof.

20. The method according to claim 14, wherein the effective amount of the Saccharomyces cerevisiae yeast strain number I-3856 is comprised in a pharmaceutical composition, which further comprises at least one physiologically acceptable excipient.

21. The method according to claim 20, wherein the pharmaceutical composition is intended for topical administration or for administration by the oral route.

22. The method according to claim 20, wherein the pharmaceutical composition further comprises at least one additional active pharmaceutical ingredient having a soothing, anti-irritant, analgesic, anti-inflammatory, wound-healing, antibiotic, antipyretic, or antifungal activity.

23. The method according to claim 22, wherein the pharmaceutical composition is intended for topical administration or for administration by the oral route.

24. The method according to claim 21, wherein the pharmaceutical composition further comprises at least one additional active pharmaceutical ingredient having a soothing, anti-irritant, analgesic, anti-inflammatory, wound-healing, antibiotic, antipyretic, or antifungal activity.

25. The method according to claim 20, wherein the pharmaceutical composition is in the form of a toothpaste, a mouthwash, an oral spray, a cream or an oral gel, an orodispersible stick, a stick to be diluted in water, or a vial with a push-button stopper.

26. The method according to claim 21, wherein the pharmaceutical composition is in the form of a toothpaste, a mouthwash, an oral spray, a cream or an oral gel, an orodispersible stick, a stick to be diluted in water, or a vial with a push-button stopper.

27. The method according to claim 22, wherein the pharmaceutical composition is in the form of a toothpaste, a mouthwash, an oral spray, a cream or an oral gel, an orodispersible stick, a stick to be diluted in water, or a vial with a push-button stopper.

27. The method according to claim 23, wherein the pharmaceutical composition is in the form of a toothpaste, a mouthwash, an oral spray, a cream or an oral gel, an orodispersible stick, a stick to be diluted in water, or a vial with a push-button stopper.

28. The method according to claim 14, wherein the oropharyngeal candidiasis is a side effect of a medical treatment, or the oropharyngeal candidiasis is present or likely to develop in a patient in an immunocompromised state.

29. The method according to claim 14, wherein the oropharyngeal candidiasis is present in an infant or an elderly person.

30. The method according to claim 14, wherein administering the Saccharomyces cerevisiae yeast strain further prevents or inhibits the spread of Candida infection to the esophagus, the stomach or the small intestine in the subject with oropharyngeal candidiasis.

31. A method for preventing or inhibiting the spread of Candida infection to the esophagus, the stomach or the small intestine in a patient with oropharyngeal candidiasis, the method comprising a step of administering to said patient a Saccharomyces cerevisiae yeast strain deposited with the CNCM on Oct. 17, 2007 under Accession Number I-3856.

Description

LEGEND OF THE FIGURES

[0080] FIG. 1: Quantification of the total photon flux emission from the oral cavity of mice infected by BLI Candida albicans and treated with different compounds, measured on days +1, +3 and +6 after infection. The results give the mean value±SEM of 5 mice per group in 3 different experiments. ≠, p<0.05 (infected mice treated with FLU, GI or IY vs. infected mice treated with saline solution).

[0081] FIG. 2: Imaging of mice in vivo infected by BLI Candida albicans and treated with different compounds, carried out on day +6 after infection.

[0082] FIG. 3: CFU count on the tongue of mice infected by Candida albicans and treated with different compounds, carried out on days +3 and +6 after infection. The results give the mean value±SEM of 3 mice per group in 3 different experiments. ≠, p<0.05 (infected mice treated with FLU, GI or IY vs. infected mice treated with saline solution).

[0083] FIG. 4: Histological examination on day +8 (A) carried out on uninfected mice treated with different compounds and (B) carried out on mice infected by BLI Candida albicans and treated with different compounds.

[0084] FIG. 5: CFU count (A) in the esophagus, (B) in the stomach, and (C) in the duodenum, of mice infected by BLI Candida albicans and treated with different compounds, carried out on day +6 after infection. The results give the mean value±SEM of 3 mice per group in 3 different experiments. ≠, p<0.05 (infected mice treated with FLU, GI or IY vs. infected mice treated with saline solution).

[0085] FIG. 6: Ex vivo imaging of the esophagus and stomach of mice infected by BLI Candida albicans and treated with different compounds, carried out (A) on day +6 and (B) on day +8 after infection. The results give the mean value±SEM of 5 mice per group in 3 different experiments. ≠, p<0.05 (infected mice treated with FLU, GI or IY vs. infected mice treated with saline solution).

[0086] FIG. 7: CFU counts on the tongue. The fungal burden of the tongue of the infected mice treated with 10 μl of saline solution, or of fluconazole (FLZ—4 mg/ml), or of IY, GI or WG (all at 100 mg/ml on days +1, +2 and +3 post-infection, was evaluated by a CFU assay on days +1, +3 and +6 after infection of the tongue. The results give the mean value ±SEM of 4 to 6 mice in 2 different experiments. #, p<0.05 (infected mice treated with FLZ, IY, GI or WG vs. infected mice treated with saline solution).

[0087] FIG. 8: Effects of IY, GI and WG on expression of SAP2, SAP6, ALS3 and HWP1 during oropharyngeal candidiasis. The expression of the genes (A) SAP2, (B) SAP6, (C) ALS3 and (D) HWP1 was analyzed in cellular fractions of homogenates of tongues of infected mice treated with 10 μl of saline solution, or of fluconazole (FLZ—4 mg/ml), or of IY, GI or WG (all at 100 mg/ml) on days +1, +2 and +3 post-infection. On days 3+ and +6 post-infection, the tongue homogenates were centrifuged, then the cellular fractions were lysed and the total RNA was extracted and reverse-transcribed into cDNA. The genes SAP2, SAP6, ALS3 and HWP1 of Candida albicans were detected by real-time PCR and the amounts of cDNA were reported in 2.sup.−ΔΔCT relative to the transcripts of the Candida albicans inoculum. The results give the mean value±SEM of samples in triplicate from 4 to 6 mice in 2 different experiments. #, p<0.05 (infected mice treated with FLZ, IY, GI or WG vs. infected mice treated with saline solution).

[0088] FIG. 9: Destructive Activity of the Peritoneal Neutrophils. The destructive activity of the peritoneal neutrophils of uninfected mice or of infected mice treated with 10 μl of saline solution, or of fluconazole (FLZ—4 mg/ml), or of IY, GI or WG (all at 100 mg/ml) on days +1, +2 and +3 post-infection, was evaluated on days 3+ and +6 post-infection. The peritoneal neutrophils (1×10.sup.6/ml) were incubated in the presence of Candida albicans (CA-6) (1×10.sup.5/ml) for 2 hours. The results give the mean value±SEM for 4 to 6 mice from each group in 2 different experiments. #, p<0.05 (infected mice treated with FLZ, IY, GI or WG vs. infected mice treated with saline solution).

[0089] FIG. 10: Production of IL-1α, TNF-α and IL-6. The supernatants of tongue homogenates of uninfected mice or of infected mice treated with 10 μl of saline solution, or of fluconazole (FLZ—4 mg/ml), or of IY, GI or WG (all at 100 mg/ml) on days +1, +2 and +3 post-infection were tested, by ELISA, for the presence (A) of IL-1β, (B) of TNF-α and (C) of IL-6, on days +1, +3 and +6 post-infection. The results give the mean value±SEM for 4 to 6 mice from each group in 2 different experiments. *, p<0.05 (infected mice treated with saline solution vs uninfected mice). #, p<0.05 (infected mice treated with FLZ, IY, GI or WG vs. infected mice treated with saline solution).

[0090] FIG. 11: Production of IL-17A/F, IL-22 and IL-23. The supernatants of tongue homogenates of uninfected mice or of infected mice treated with 10 μl of saline solution, or of fluconazole (FLZ—4 mg/ml), or of IY, GI or WG (all at 100 mg/ml) on days +1, +2 and +3 post-infection were tested, by ELISA, for the presence (A) of IL-17A/F, (B) of IL-22 and (C) of IL-23, on days +1, +3 and +6 post-infection. The results give the mean value±SEM for 4 to 6 mice from each group in 2 different experiments. *, p<0.05 (infected mice treated with saline solution vs uninfected mice). #, p<0.05 (infected mice treated with FLZ, IY, GI or WG vs. infected mice treated with saline solution).

[0091] FIG. 12: Production of IFN-α. The supernatants of tongue homogenates of uninfected mice or of infected mice treated with 10 μl of saline solution, or of fluconazole (FLZ—4 mg/ml), or of IY, GI or WG (all at 100 mg/ml) on days +1, +2 and +3 post-infection were tested, by ELISA, for the presence of IFN-α on days +1, +3 and +6 post-infection. The results give the mean value±SEM for 4 to 6 mice from each group in 2 different experiments. *, p<0.05 (infected mice treated with saline solution vs uninfected mice). #, p<0.05 (infected mice treated with FLZ, IY, GI or WG vs. infected mice treated with saline solution).

Example 1

Evaluation of the Activity of Different Yeast Products in an Animal Model of Oropharyngeal Candidiasis

A. Materials and Methods

[0092] Animal model of oropharyngeal candidiasis. As Candida albicans is not a commensal species in laboratory mice, the procedure developed by Solis and Filler (Nature Protoc., 2012, 7(4): 637-642) for obtaining a reproducible infection that mimics pseusomembranous oropharyngeal candidiasis in humans was used on wild-type C57BL/6 mice. The procedure comprises the injection of cortisone acetate, which makes mice susceptible to oral infection by Candida and infection by Candida albicans.

[0093] More specifically, female C57BL/6 mice (Charles River, Calco, Italy), aged from 6 to 8 weeks, were kept in the animal house of Perugia University (Italy). The mice were treated with 225 mg/kg of cortisone acetate (Sigma-Aldrich) every other day beginning one day before infection and were then infected with a suspension of 1×10.sup.6/ml BLI Candida albicans as described previously (Solis and Filler, Nature Protoc., 20125, 7: 637-642) under anesthesia with subcutaneous injection of a mixture of Tiletamine/Zolazepam-Xylazine (50 mg/kg/5 mg/kg) (Mosci et al., Virulence, 2013, 4: 250-254). The oral cavity was tested immediately before infection to confirm prior absence of Candida species (by taking a buccal sample, which was spread on YPD agar with chloramphenicol (50 μg/ml) (both from Sigma-Aldrich)).

[0094] The mice were used in conditions free from specific pathogens—conditions that were checked with tests of sensitivity to undesirable infections. According to the standards of the Federation of European Laboratory Animal Science Associations, no infection was detected. The procedures involving the animals and their care were conducted in accordance with national and international laws and standards. All the experiments on the animals were conducted in compliance with European Directive 2010/63, the European Convention on the Protection of Vertebrates used for Experimental Purposes or for other Scientific Purposes and the national law 116/92. The protocol was approved by the ethics committee of Perugia University for the care and use of animals. All the animals were kept in the animal house of Perugia University. The mice were acclimated for 1 week before beginning the experiments. Each cage contained at most 5 mice, which received food and water ad libitum.

[0095] Products tested. The Saccharomyces cerevisiae strain I-3856 in the live form (GI) and in the inactivated form (IY) were tested in this example. After infection, the mice received an oral injection (10 μl) of saline solution (0.9% NaCl, negative control), of fluconazole (FLZ—reference antifungal used as positive control) (4 mg/ml) or GI and IY yeast products (both at 100 mg/ml) on days +1, +2, +3 and +6.

[0096] Candida. The strain of Candida albicans CA1398 bearing the fusion product ACT1p-gLUC59 (gLUC59) was used. Culture of C. albicans was maintained over several passages on YPD agar (Y: yeast extract, P: peptone and D: anhydrous dextrose—all from Sigma-Aldrich). The fungal cells were collected by suspending a single colony of Candida albicans in a saline solution, washed twice, counted using a hemocytometer, and adjusted to the required concentrations.

[0097] Evaluation of Infection by C. albicans in the Oral Cavity.

[0098] a. CFU assay. The number of colonies of Candida albicans adhering to the oral cavity was evaluated on days +3 and +6 after infection with Candida albicans by spreading dilutions of tongue homogenates on CHROMAgar™ plates (growth medium specific and selective for Candida). Then the viable colonies of Candida albicans were counted after two days of culture at 30° C. The results were expressed in Log CFU/g tissue (or Log UFC/g in French).

[0099] b. Imaging of BLI Candida in the oral cavity. On days +1, +3 and +6 after infection, the mice received 10 μl (0.5 mg/ml in a 1/10 methanol/H.sub.2O mixture) of coelenterazine (Synchem, OHM), and were then imaged using an IVIS-200™ system (Xenogen Inc.) under anesthesia with 2.5% of isoflurane. On the images, the total emission of photon flux from the oral cavity (Region of Interest, ROI) of each mouse was quantified using Living ImageR software. No luminescence was observed for the uninfected mice that received 10 μl of coelenterazine (data not shown).

[0100] c. Histological Examination. Histological examination of the tongue of the mice was carried out on day +8 after infection according to the protocol described by Mosci et al., Virulence, 2013, 4(3): 250-254.

[0101] Spread of Infection to the Esophagus, Stomach and Intestine.

[0102] a. CFU assay. The number of colonies of Candida albicans that developed, after spread of the infection, in the esophagus, stomach and duodenum, was evaluated on day +6 after infection with Candida albicans by spreading dilutions of homogenates of the tissues/organs on CHROMAgar™ plates. The results were expressed in Log CFU/g tissue (or Log UFC/g in French).

[0103] b. Imaging of BLI Candida. On days +6 and +8 after infection, the gastric tracts were excised and 10 μl (0.5 mg/ml in a 1/10 methanol/H.sub.2O mixture) of coelenteratine (Synchem, OHM) was injected via the pharynx into the lumen of the esophagus. The esophagus and the stomach of the mice were then imaged ex vivo using an IVIS-200™ system (Xenogen Inc.). On the images, the total emission of photon flux (Region of Interest, ROI) for each esophagus/stomach system of each mouse was quantified using Living ImageR software.

[0104] Statistical analyses. The differences between the infected mice treated with FLU, GI or IY and the infected mice treated with saline solution were evaluated with the Mann-Whitney U test. A value of p<0.05 was regarded as significant.

[0105] B. Results

[0106] Evaluation of infection by Candida albicans in the oral cavity. FIG. 1 and FIG. 2 show the results of the in vivo imaging of the infected mice treated with the various compounds. These results show that the Saccharomyces cerevisiae strain CNCM I-3856 both in its live form (GI) and in its dead form (IY) is capable of reducing the fungal burden considerably. The beneficial effect is similar to that obtained using fluconazole (FLU—positive control).

[0107] The number of colonies of Candida albicans adhering to the tongue of the mice evaluated on days +3 and +6 after infection with Candida albicans confirms that the Saccharomyces cerevisiae strain CNCM I-3856 both in its live form (GI) and in its dead form (IY) is capable of reducing the fungal burden significantly. The beneficial effect is similar to that obtained using fluconazole (FLU—positive control). (FIG. 3).

[0108] Histological examination carried out on uninfected mice treated with the two yeast products (FIG. 4(A)) shows that the tongue of the uninfected mice treated with GI and IY does not have any lesion and that no recruitment of neutrophils was observed. Histological examination carried out on infected mice treated with the various compounds (FIG. 4(B)) demonstrated that the tongue of the infected mice treated with the Saccharomyces cerevisiae strain CNCM I-3856 both in its live form (GI) and in its dead form (IY) does not have any lesion induced by Candida albicans. The beneficial effect is similar to that obtained using fluconazole (FLU—positive control).

[0109] Spread of infection to the esophagus, stomach and duodenum. Determination of the number of colonies of Candida albicans that developed, after spread of the infection, in the esophagus (FIG. 5(A)), in the stomach (FIG. 5(B)) and in the duodenum (FIG. 5(C)) was evaluated on day +6 after infection with Candida albicans. The results obtained show that the Saccharomyces cerevisiae strain CNCM I-3856 in its live form (GI) is capable of inhibiting the fungal load significantly whereas its dead form (IY) shows a tendency to reduce the fungal load in the esophagus and the stomach. The beneficial effect of GI is similar to that obtained using fluconazole (FLU—positive control). Ex vivo imaging of the esophagus and stomach on days +6 (FIG. 6A)) and +8 (FIG. 6(B)) after infection confirms that the Saccharomyces cerevisiae strain CNCM I-3856 in its live form (GI) and to a lesser degree in its dead form (IY) prevents spread of the infection in the digestive tract.

[0110] C. Conclusions

[0111] Considered collectively, the results obtained in Example 1 show that the Saccharomyces cerevisiae strain CNCM I-3856 both in its live form (GI) and in its dead form (IY) is capable of reducing the fungal burden considerably, thus preventing its spread to the esophagus and stomach. The beneficial effects are similar to those obtained using fluconazole (positive control).

Example 2

Effects of the Saccharomyces Cerevisiae Strain I-3856 on the Immune Response to Oropharyngeal Infection by Candida albicans

[0112] Saliva is one of the innate defense mechanisms against oral infection by Candida. Saliva forms a film on the teeth and the oral epithelium. The main components of this film are mucins and immunoglobulin A (IgA), which may aggregate Candida albicans, which is removed by the action of swallowing. Moreover, saliva contains bioactive agents, such as histatin-5, lysozyme, lactoferrin and calprotectin having fungicidal properties. Although these agents are present in the saliva at low concentrations, their combined effects are either additive or synergistic. Furthermore, the combination of the salivary defense agents and the dynamic effects of the flow of saliva limit the colonization, proliferation and invasion of the oral epithelium by Candida albicans, which leads to innate oral resistance to Candida albicans (Feller et al., J. Oral. Pathol. Med., 2014, 43: 563-569).

[0113] The neutrophils and the T cells play an important role in anti-Candida mucosal immunity. The neutrophils phagocytize and kill the Candida cells by oxidative mechanisms (Moyes et al., Clin. Dev. Immunol., 2011, 346307). The T cells that are mainly involved in the oral response to Candida are the helper T lymphocytes (helper T cells, Th) Th1 and Th17. The Th1 cells produce IFN-γ, which is an effective activator of the neutrophils (Gattoni et al., Clin. Ter., 2006, 157: 457-468). In the presence of IL-6, IL-1β and TGF-β, the T cells differentiate into Th17 and undergo maturation by stimulation with IL-23. The Th17 cells produce IL-17A, IL-17F and IL-22. IL-17A and IL-17F stimulate the epithelial cells to produce antimicrobial peptides and promote recruitment and activation of the neutrophils, thus allowing fungal elimination. IL-22 has effects similar to those of IL-17 with respect to epithelial cells and limits fungal growth (Hebecker et al., Expert. Rev. Anti Infect. Ther., 2014, 12: 867-879; Moyes et al., Clin. Dev. Immunol., 2011, 346307).

[0114] It was demonstrated in Example 1 that the Saccharomyces cerevisiae strain number I-3856, whether in the live form (GI) or in the inactivated form (IY), is able to reduce the oral fungal burden, preventing spread of Candida infection to the esophagus and the stomach. In the present example, the ability of WG (cell walls of the Saccharomyces cerevisiae strain CNCM I-3856) to reduce the oral fungal burden was first evaluated, and then it was determined whether oral administration of IY, GI and WG is able to influence the fungal virulence factors and the inflammatory response in oropharyngeal candidiasis.

[0115] A. Materials and Methods

[0116] Strain of Candida albicans and Culture Conditions. The highly virulent Candida albicans strain (CA-6) was used (Bistoni et al., Infect. Immun., 1986, 51: 6668-674). The Candida albicans culture was maintained by successive passages on YPD agar (Y: yeast extract, P: peptone and D: anhydrous dextrose—all from Sigma-Aldrich). The fungal cells were collected by suspending a single colony of Candida albicans in a saline solution, washed twice, counted using a hemocytometer and adjusted to the required concentrations.

[0117] Animal model of Oropharyngeal candidiasis. Female C57BL/6 mice (Charles River, Calco, Italy), aged from 6 to 8 weeks, were kept in the animal house of Perugia University (Italy). The mice were treated with 225 mg/kg of cortisone acetate (Sigma-Aldrich) every other day beginning one day before infection and were then infected with a suspension of 1×10.sup.6/ml Candida albicans (CA-6) as described previously (Solis and Filler, Nature Protoc., 20125, 7: 637-642) under anesthesia with subcutaneous injection of a mixture of Tiletamine/Zolazepam-Xylazine (50 mg/kg/5 mg/kg) (Mosci et al., Virulence, 2013, 4: 250-254). The oral cavity was tested immediately before infection to confirm prior absence of Candida species (by taking a buccal sample, which was spread on YPD agar with chloramphenicol (50 μg/ml) (both from Sigma-Aldrich). See Example 1 for the ethical declarations.

[0118] Products tested. In this example, the Saccharomyces cerevisiae strain I-3856 was tested in the live form (GI) and in the inactivated form (IY) but also in the form of cell walls (WG). After infection, the mice received an oral injection (10 μl) of saline solution (0.9% NaCl, negative control), of fluconazole (FLZ, 4 mg/ml, positive control) and of IY, GI or WG (all at 100 mg/ml) on days +1, +2 and +3 post-infection.

[0119] CFU assay. See Example 1 for the operating conditions. The fungal burden was determined on days +1, +3 and +6 post-infection.

[0120] Quantitative Analysis of Expression of the SAP2, SAP6, ALS3 and HWP1 genes. Homogenates of tongues from mice with oral infection by Candida albicans and treated as described above with saline solution, or fluconazole, or with IY, GI or WG, were obtained on days +1, +3 and +6 post-infection. The mouse tongue homogenates were centrifuged at 3000 rev/min for 5 minutes, and then the cellular fractions were lysed with Trizol (Life Technology).

[0121] The total RNA was extracted and reverse-transcribed using the reverse transcriptase reaction of the Moloney murine leukemia virus (M-MLV RT) according to the manufacturer's instructions. The concentration of complementary DNA (cDNA) was determined with a spectrophotometer. The SAP2, SAP6, ALS3 and HWP1 genes of Candida albicans were detected using known primers (Naglik et al., J. Med. Microbiol., 2006, 55: 1323-1327; Naglik et al., Microbiology, 2008, 154: 3266-3280; Roudbarmohammadi et al., Adv. Biomed. Res., 2016, 5: 105). The real-time PCR reactions were carried out in 96-well PCR plates with SYBR green (BioRad). For the real-time PCR reactions, 200 ng of cDNA was used. All the samples were measured in triplicate. The relative expression levels of the Candida genes at different times post-infection were reported in 2.sup.−ΔΔCT relative to the transcripts of the Candida albicans inoculum (Pericolini et al., Virulence, 2017, 8: 74-90)). The amplification conditions used are the same for SAP2, SAP6, ALS3 and HWP1: 3 minutes at 95° C., 40 cycles of 10 seconds at 95° C. and 30 seconds at the specific hybridization temperature of the primer. The experiments were carried out with an Eppendorf Mastercycler.

[0122] Tests of Candidacidal Power. On days +3 and +6 post-infection, the peritoneal neutrophils of uninfected mice and of mice infected and treated as described above, were collected 18 hours after an intraperitoneal injection of 0.5 ml of solution of 10% thioglycolate without endotoxin (Difco).

[0123] The neutrophil destruction activity was determined by a CFU inhibition assay. Briefly, neutrophils (10.sup.5 cells) in a suspension of 0.1 ml per well were incubated in a flat-bottomed 96-well tissue culture plate with 10.sup.4 cells of Candida albicans (CA-6) in 0.1 ml of RPMI containing 5% of FBS and were incubated for 2 hours at 37° C., in the presence of 5% CO.sub.2. After incubation, the plates were agitated vigorously and the cells were lysed by adding Triton X-100 (0.1% in distilled water, final concentration of 0.01% in each well). Serial dilutions were prepared from each well with distilled water. The samples were spread on Sabouraud dextrose agar with chloramphenicol (50 mg/ml) in triplicate and the CFU values were evaluated after incubation for 24 hours at 37° C. The so-called control cultures consisted of Candida albicans (CA-6) incubated with RPMI-1640 containing 5% of FCS without effector cells.

[0124] Production of Cytokines. Tongue homogenates, from uninfected mice or from mice orally infected and treated as described above, were obtained on days +1, +3 and +6 post-infection. The tongue homogenates were centrifuged at 3000 rev/min for 5 minutes and the supernatants were collected and the levels of IL-1β, TNF-α, IL-6, IL-17A/F, IL-22, IL-23 and IFN-γ were measured by ELISA assays (eBioscience).

[0125] Statistical analyses. The results are the mean values±SEM of samples in duplicate or triplicate from 4 to 6 mice for each group in two different experiments. The differences between the infected mice treated with saline solution and the uninfected mice or the infected mice treated with FLU, GI, IY or WG and the infected mice treated with saline solution were evaluated with the Mann-Whitney U test. A value of p<0.05 was regarded as significant.

[0126] B. Results and Discussion

[0127] The capacity of WG (cell walls of the Saccharomyces cerevisiae strain CNCM I-3856) was first evaluated for its influence on the burden of Candida albicans in the oral cavity. For this purpose, the mice were treated with IY, GI or WG (all at 100 mg/ml) on days +1, +2 and +3 after infection with Candida albicans (10.sup.6/ml). The CFU values of the tongues of the mice thus treated were evaluated on days +1, +3 and +6 after infection. The results obtained, which are presented in FIG. 7, show that all the compounds tested, including WG, are capable of significantly inhibiting the Candida albicans burden, and the beneficial effect is similar to that obtained using fluconazole (FLZ).

[0128] Next, the present inventors determined whether inhibition of the fungal burden was linked to inhibition of certain virulence factors of Candida albicans. Therefore they determined the expression of aspartic proteases (Sap), which are involved in invasion of the tissues by Candida. Determination of the SAP2 and SAP6 genes was carried out on days +1, +3 and +6 post-infection. The results, presented in FIGS. 8(A) and (B), show that there is significant inhibition of the expression of SAP2 and SAP6 after treatment with GI but not with IY or with WG—the effect being observed 6 days after infection.

[0129] Candida albicans expresses the invasin Als3, which binds to the epithelial cells, resulting in endocytosis of the fungus by invasion and active penetration of the epithelial cells producing cell damage and release of pro-inflammatory cytokines (Naglik et al., Microbes Infect., 2011, 13: 963-976). The HWP1 (hyphal wall protein 1) gene of Candida albicans codes for a protein of the fungal cell wall that is necessary for hyphal growth and for adhesion of the fungus to the epithelial cells (Orsi et al., Microb. Pathog., 2014, 69-70: 20-27). Expression of the two genes ASL3 and HWP1 was found to be reduced significantly after treatment with GI but not with IY or with WG (see FIGS. 8(C) and (D)). These results suggest that the inhibition of CFU by GI could be due to inhibition of adhesion of Candida to the epithelial cells as well as to inhibition of the yeast-hyphal transition with reduced consequent cell damage. Since IY and WG do not affect gene expression in Candida, there are other mechanisms of action responsible for the reduction of CFU observed with these two compounds.

[0130] The neutrophils are major effector immune cells in fungal destruction (Gazendam et al., Immunol. Rev., 2016, 273: 299-311). Therefore the destructive activity of the neutrophils was examined. The results obtained are presented in FIG. 9. They show that the destructive activity of the neutrophils, which is weakened during infection with Candida albicans relative to the uninfected mice, is restored by treatment with the yeast products tested. In particular, 6 days after infection, a large increase in the destructive activity of the neutrophils was observed in the infected mice treated with GI, IY and WG compared to the untreated infected mice.

[0131] In the course of oral candidiasis, by releasing pro-inflammatory cytokines the epithelial cells induce recruitment of neutrophils, which limit the extent of the damage to the epithelial cells by accelerating elimination of Candida (Trautwein-Weidner et al., Mucosal. Immunol., 2015, 8: 221-231). Production of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 was tested in the experimental system used here. The results obtained are presented in FIG. 10. These results show that one day after infection, no change in the production of the pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6, was observed in the infected mice, whether or not treated with the various compounds, compared to the uninfected mice. However, 3 days after infection, the infected mice display a significant increase in production of IL-1β and TNF-α, and treatment with the yeast products or with FLZ results in considerable inhibition of these cytokines. At this moment, the production of IL-6 remains unchanged compared to the untreated mice with the exception of FLZ. When analysis of the pro-inflammatory cytokines was carried out 6 days post-infection, a large increase was observed in the supernatants of tongues of infected mice compared to the supernatants of tongues of uninfected mice, and treatment with GI, WG and FLZ led to a significant decrease in the production of all the cytokines, whereas IY was only capable of significantly inhibiting the production of TNF-α (FIG. 10).

[0132] It is known that IL-17, IL-22 and IL-23 play a role in the elimination of the fungi during oral candidiasis (Hebecker et al., Expert. Rev. Anti Infect. Ther., 2014, 12: 867-879; Moyes et al., Clin. Dev. Immunol., 2011, 346307). Therefore the presence of the cytokines IL-17, IL-22 and IL-23 of the T cells was also determined in the present system. The results obtained (FIG. 11) show that, 6 days after infection, the presence of all these cytokines is greater in the supernatants of tongues of the infected mice than in the supernatants of tongues of the uninfected mice. Treatment with GI and WG resulted in significant inhibition of all the cytokines tested, whereas IY only induced a significant decrease in the case of the production of IL-23.

[0133] An increase in IFN-γ, a cytokine typical of the T cells, was also observed in the supernatants of tongues of infected mice, 3 days and 6 days post-infection, and treatment with the test compounds led to a large reduction in the production of IFN-γ (see FIG. 12).

[0134] Considered together, these results suggest that the inhibition of the pro-inflammatory cytokines observed after treatment with the test compounds could be attributed to a decrease in the fungal burden. It is conceivable that by inhibiting several virulence factors, such as the aspartic proteases and the adhesins of Candida, GI succeeds in inhibiting the Candida burden and the inflammatory response. In the experiments presented here, IY and WG apparently do not have an effect on the virulence factors of Candida, but they induce a large reduction in the fungal burden. It has previously been shown that IY is capable of producing strong aggregation with Candida (Pericolini et al., Virulence, 2017, 8: 74-90). It is conceivable that WG is effective by a similar mechanism. Moreover, the increase in the destructive activity of the neutrophils observed after treatment with all the compounds tested could also explain the large decrease in the fungal burden.

[0135] C. Conclusions

[0136] The results obtained demonstrate that all the yeast products tested (GI, IY and WG) are capable of inhibiting fungal growth of Candida in the oral cavity. The beneficial effect of GI is at least partly due to inhibition of the adhesion of Candida to the epithelial cells through inhibition of the adhesins of Candida and inhibition of the hyphal transition. The results suggest that the effect of WG and of IY is in particular due to a mechanical effect through production of strong aggregation of Candida, which prevents adhesion of Candida to the epithelial cells. All these effects induce a considerable acceleration of the elimination of Candida, which leads to a low or even absent inflammatory response. It is interesting to note that, in all the determinations carried out, the fungal burden obtained after treatment with the yeast products tested (GI, IY, and WG) was comparable to that obtained with fluconazole, the standard antifungal used in the treatment of oropharyngeal candidiasis.