Evaluation of mammalian codon usage of fimH in DNA vaccine design

Uropathogenic Escherichia coli (UPEC) bacteria are the principal cause of urinary tract infections (UTI). Because these bacteria propagate intracellularly, the cellular immune response is an important factor in UTIs. Therefore, we designed a genetic construct to induce a cellular immune response. In order to develop a genetic construct that induces strong cellular immunity against this pathogen, we used the fimH synthetic gene according to mammalian codon usage, and the gene expression was compared with wild type codon usage. Initially, we designed two constructs, pVAX/fimH mam and pVAX/fimH wt, which contain mammalian and wild type codon usage, respectively. The Cos-7 cell line was transfected separately with a complex of pVAX/fimH mam-ExGene 500 poly cationic polymer and pVAX/fimH wt-ExGene 500 poly cationic polymer. Expression of the fimH gene in both constructs in COS7 cells was confirmed by RT-PCR, SDS-PAGE, and Western blotting. Both of the pVAX/fimH cassettes expressed inserted fimH genes (mam and wt) in Cos-7 cells. Our results suggest that codon optimization successfully expressed the fimH gene because the fimH gene with mammalian codon usage is compatible with the eukaryotic expression system. Therefore, mammalian codon usage could be appropriate in a pVAX/fimH construct as a DNA vaccine

Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice

Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins