Ontwikkeling en karakterisatie van een nieuwe generatie probiotica tegen vulvovaginale candidiasis, op basis van gistcellen en ascosporen

Vulvovaginal candidiasis (VVC), predominantly caused by Candida albicans and to a lesser extent Nakaseomyces glabratus, affects up to 75% of women during their lifetime, with 7-9% experiencing recurrent episodes (RVVC). Current antifungal therapies face limitations, including resistance, high recurrence, and disruption of the vaginal microbiota. This highlights the need for alternative approaches. Probiotics represent a promising option, yet many commercial formulations are not tailored to the vaginal environment and lack long-term stability. Yeast-based probiotics remain largely confined to Saccharomyces var. boulardii for gastrointestinal use, and no yeast spore-based probiotic ("sporebiotic") has been developed for any health indication. This dissertation explores the potential of Saccharomyces cerevisiae as a next-generation probiotic for VVC and RVVC, structured around four aims: (i) characterisation of vaginal C. albicans isolates to assess strain-level heterogeneity, (ii) screening of genetically and ecologically diverse S. cerevisiae strains for activity against C. albicans and N. glabratus under vaginal-simulative conditions, (iii) functional assessment of selected candidates in host-relevant assays and a murine VVC model, and (iv) evaluation of yeast spores as a delivery format with improved gastric survival. Analysis of vaginal C. albicans isolates revealed pronounced variability in growth, filamentation, adhesion, haemolysis, secretion of secreted aspartyl proteinases (Saps), and epithelial cytotoxicity. Although different Candida species exhibit distinct biological behaviours, strain-level variability within a single species is often underestimated, especially among isolates from the same niche. To account for this diversity, two vaginal C. albicans strains (I49 and Ca20A), two laboratory strains (SC5314 and Ca3153), and one vaginal N. glabratus strain (BG2) were selected as representative pathogens for probiotic screening, ensuring that candidate probiotics are tested against multiple relevant backgrounds. Screening of 104 genetically and ecologically diverse S. cerevisiae isolates showed that antagonistic activity against C. albicans and N. glabratus is widespread but highly strain-specific. Many isolates surpassed the reference strain CNCM I-3856 (QUATREFLORA™) in inhibiting growth, hyphal transition, adhesion, and cytotoxicity. These results highlight the value of broad screening across multiple isolates rather than reliance on a single benchmark. Selected strains demonstrated additional functional properties. Several strains enhanced neutrophil antifungal responses, increasing reactive oxygen species production and release of myeloperoxidase and neutrophil elastase, thereby counteracting neutrophil dysfunction. Together, these data support a role for S. cerevisiae in modulating both pathogen traits and host responses. Sporulation emerged as a particularly relevant trait. While sporulation and germination capacity varied across isolates, spores consistently survived simulated gastric conditions far better than vegetative cells. This suggests that spore-based formulations could offer increased stability and resilience compared to conventional yeast probiotics. For in vivo evaluation, three strains (SB001, SB002, SB003) were tested in a murine VVC model. Of these, SB001 displayed the most interesting probiotic profile: compared to untreated infection, it was associated with a reduced C. albicans load on day 2 (BLI), a tendency toward lower C. albicans CFU recovery at day 14, slightly higher immune cell recruitment, and a trend toward less activated neutrophils and eosinophils. Interestingly, treatment appeared to shift neutrophil responses toward a less hyperactivated state while preserving antifungal potential, suggesting a recalibration of inflammation rather than suppression. This dual effect, limiting tissue-damaging dysregulated inflammation while supporting effective clearance, aligns closely with the therapeutic goal of (R)VVC management. In conclusion, this dissertation provides a proof of concept for S. cerevisiae spores as a novel probiotic platform for vaginal health. By integrating pathogen characterisation, probiotic screening, mechanistic assays, and preliminary in vivo evaluation, the work advances both fundamental understanding of yeast-pathogen-host interactions and translational development of yeast-based sporebiotics. The findings highlight that antifungal activity is widely distributed yet strain-specific within S. cerevisiae, that sporulation provides a unique stability advantage, and that selected strains can modulate immune responses in potentially beneficial ways. Beyond addressing an unmet need in (R)VVC, this research positions yeast sporebiotics as an innovative and versatile probiotic strategy.