Abstract
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.
Highlights
Since its first observation in Europe in the 1970s, porcine epidemic diarrhea virus (PEDV) has spread worldwide due to its highly contagious nature and has been causing significant economic losses [1, 2] by causing wateryMasuda et al Vet Res (2021) 52:102 various receptors, such as porcine aminopeptidase N: APN (Transmissible gastroenteritis virus), angiotensinconverting enzyme 2: angiotensin-converting enzyme 2 (ACE2) (Human coronavirus NL63), carcinoembryonic antigen-cell adhesion molecule: CEACAM1, or dipeptidyl peptidase: DDP4 (MERSCoV), transferrin receptor 1 (PEDV), on host cells and their S2 domain to fuse with the cell membrane [5,6,7,8,9]
The highly glycosylated S protein, consisting of S1 and S2 domains, forms a homotrimer and plays an important role in facilitating host cell attachment and entry [4]. Coronaviruses use their S1 domain to bind to Masuda et al Vet Res (2021) 52:102 various receptors, such as porcine aminopeptidase N: APN (Transmissible gastroenteritis virus), angiotensinconverting enzyme 2: ACE2 (Human coronavirus NL63), carcinoembryonic antigen-cell adhesion molecule: CEACAM1, or dipeptidyl peptidase: DDP4 (MERSCoV), transferrin receptor 1 (PEDV), on host cells and their S2 domain to fuse with the cell membrane [5,6,7,8,9]
Two constructs were designed based on the prediction and generated the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) expressing the candidates for S ectodomain, PEDV/S(1–1332) and PEDV/S(1–1320) as described under the Materials and Methods (Figure 1B)
Summary
Since its first observation in Europe in the 1970s, porcine epidemic diarrhea virus (PEDV) has spread worldwide due to its highly contagious nature and has been causing significant economic losses [1, 2] by causing wateryMasuda et al Vet Res (2021) 52:102 various receptors, such as porcine aminopeptidase N: APN (Transmissible gastroenteritis virus), angiotensinconverting enzyme 2: ACE2 (Human coronavirus NL63), carcinoembryonic antigen-cell adhesion molecule: CEACAM1, or dipeptidyl peptidase: DDP4 (MERSCoV), transferrin receptor 1 (PEDV), on host cells and their S2 domain to fuse with the cell membrane [5,6,7,8,9]. The N-terminal domain of the S protein, which is unique to the genus Alphacoronavirus, has been reported to play an important role in PEDV infection in the intestine, and this second candidate region is thought to have the ability to bind to a type of sialic acids unique to the intestine [15]. An immunological study has shown that the administration of recombinant S1 protein produced by cultured porcine cells to pregnant sows can protect newborns from PEDV infection through passive immunity [17]. Enveloped virus-like particles with S, M, and E proteins produced in cultured insect cells were able to induce neutralizing antibodies equivalent to those of inactivated PEDV vaccines, their production efficiency was lower [18]. By trimer formation, the recombinant S protein is expected to have a structure similar to that on the PEDV envelope, and a selective neutralizing antibody will be inducted
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