Abstract
Background:
Pleurotus ostreatus is an edible mushroom with known immunomodulatory compounds, such as saponins, with potential for vaccine adjuvant development.

Aim:
This study investigated the immunogenicity of an ISCOMATRIX-based adjuvant made with Pleurotus-derived saponins and delivered via mucosal routes to elicit protective immune responses against Streptococcus pneumoniae infection.

Methods:
Saponins were extracted from P. ostreatus using aqueous extraction, defatted with ethyl acetate, and purified using vacuum liquid chromatography. The ISCOMATRIX formulation was prepared by lipid film hydration and characterized accordingly. Mice were immunized through intranasal, oral, or rectal routes and evaluated for delayed-type hypersensitivity, bacterial load, neutrophil recruitment, antibody titers (IgA, IgG), and cytokine levels (IFN-γ, IL-4) using standard assays and enzyme-linked immunosorbent assay.

Results:
Mice immunized intranasally and rectally showed significantly reduced inflammation and bacterial counts compared with controls (P < 0.05). Neutrophil recruitment was notably increased in the nasal and oral groups (P < 0.05), whereas rectal administration was not statistically significant (P > 0.05). All mucosal routes induced significantly elevated IgA and IgG titers, particularly via the intranasal route (P < 0.001). Elevated measures of Interferon-γ and Interleukin-4 were observed in splenocyte cultures from all immunized groups, indicating both Th1 and Th2 activation.

Conclusion:
Mucosal delivery of Pleurotus-derived saponin ISCOMATRIX formulations can induce robust immune responses against S. pneumoniae in animal models. Intranasal administration showed the most consistent immunogenicity among the tested routes. This platform holds potential as a non-invasive alternative to injectable pneumococcal vaccines, warranting further preclinical development.

Key words: Adjuvant; Iscomatrix; Mucous organs; Mushroom; Streptococcus.