Immunogenicity of an S1D epitope from porcine epidemic diarrhea virus and cholera toxin B subunit fusion protein transiently expressed in infiltrated Nicotiana benthamiana leaves

Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636-789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into Agrobacterium tumefaciens and subsequently infiltrated into Nicotiana benthamiana leaves. The highest expression level of S1D was found at 2 days post infiltration (dpi), reached 0.04% of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB-S1D fusion protein were confirmed by Western blot and G_M1-ELISA. The highest expression level of CTB-S1D fusion protein was 0.07% of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB-S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB-S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB-S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV.