Design and immunogenicity of a recombinant Saccharomyces boulardii secreting the P2-VP8 subunit rotavirus vaccine.

Abstract

Rotavirus is the leading cause of infectious diarrhea in children, and vaccination has significantly reduced the mortality rate. However, the current vaccines have not shown optimal effectiveness in developing countries. Regarding the treatment field, rotavirus vaccine candidates, such as probiotics, virus-like particle, and subunit vaccines, have been developed worldwide, even though their efficacy and safety remain to be improved. We designed and examined the Saccharomyces boulardii secreting P2-VP8 fusion protein as a rotavirus vaccine candidate. The immunological and physicochemical properties of the P2-VP8 fusion protein were evaluated the in silico analyses. A 3D structure of P2-VP8 was generated, refined, and validated. The immune simulation of codon-optimized P2-VP8 fusion protein was investigated by immunoinformatics servers. Then, it was confirmed that the Saccharomyces boulardii was secreting the P2-VP8 fusion protein. C57BL/6 mice were studied for immune response analyses after oral administration of the recombinant Saccharomyces boulardii. IFN-γ and IL-4 were measured to evaluate the activity of Th-1 and Th-2 lymphocytes, respectively. The CFSE [Carboxyfluorescein succinimidyl ester] assay was used for the assessment of lymphocyte proliferation. In silico results revealed that the P2-VP8 fusion protein was immunogenic and non-allergenic. Also, dynamic simulation showed the strong and stable interaction between the P2-VP8 fusion protein and immune cell receptor. Secretion of the P2-VP8 from Saccharomyces boulardii was confirmed by SDS-PAGE and western blotting assays. In vivo results denoted that recombinant Saccharomyces boulardii elicited significant differences in levels of IFN-γ and IL-4 responses compared to control groups. Furthermore, cell proliferation differences were not statistically significant. In this study, probable immunogenicity and stable binding of P2-VP8 to the immune receptor were predicted by in silico study. Recombinant Saccharomyces boulardii could secrete the P2-VP8 subunit rotavirus vaccine. However, our in vivo results demonstrated poor immunogenicity following vaccination and can be improved in future studies.

Supplementary Information: The online version contains supplementary material available at 10.1038/s41598-026-37374-5.

Keywords: Codon optimization; Fusion protein; Immune responses; Immunoinformatics; Rotavirus.