The silkworm (Bombyx mori) and its pupae have been used for decades as nutritional additives and applied on the

production of high-quality recombinant proteins via the baculovirus expression vector (BEV) system. The bio-capsule,

the fat-rich body, and some body components of the silkworm pupae, which deliver antigens passing through the

harsh environment of digestive tract and reaching the intestine, have been used as a vehicle for oral vaccines. In the

present study, to develop a novel oral vaccine against porcine epidemic diarrhea virus (PEDV), the PEDV spike (S)

protein was expressed in silkworm pupae and BmN cells using the BEV system. After three doses of oral administrations

with 2-week intervals in pigs, neither PEDV S protein-specific humoral nor mucosal immune responses can be

detected. The failure of eliciting the PEDV-specific immune response suggested that the BEV system using BmN cells

or silkworm pupae as oral immunogen-expression vehicles was not able to overcome the immunological unresponsiveness,

which was possibly due to gastrointestinal specific barriers and oral tolerance. Better strategies to enhance

the delivery and immunogenicity of oral vaccines should be further investigated. Nevertheless, the PEDV S protein

generated in the BmN cells and silkworm pupae herein provides an efficient tool to produce the recombinant antigen

for future applications.