Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice

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.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants

“…We have successfully expressed S proteins of several coronaviruses using silkworm-BEVS ( 21 ), and in this study, we attempted to produce S proteins of SARS-CoV-2 more efficiently and at a lower cost. The schematic diagram of recombinant protein production in silkworm-BEVS is briefly shown in Figure 1A .…”

“…The schematic diagram of recombinant protein production in silkworm-BEVS is briefly shown in Figure 1A . Using this method and based on our previous report of porcine epidemic diarrhea virus S protein expression in silkworm using CMP as a trimerization domain ( 21 ), we generated a construct named SARS2/SNFPP+CMP+TEV-H8STREPH6 to express the S protein of SARS-CoV-2 efficiently. In this construct, three amino acid substitutions were made in the furin recognition site (residues 682-685) of the ectodomain (1-1208aa) of the SARS-CoV-2 S protein to make it resistant to cleavage, and two proline mutations (residues 986 and 987) were introduced to stabilize the conformation.…”

“…Therefore, SDS-PAGE was performed under reducing and non-reducing conditions, and only the band of the S protein fused with CMP was shifted upward in the non-reducing condition compared with that fused with T4fibritin and GCN4, suggesting stable oligomer formation by disulfide bonds ( Figure 3H ). From these results, for further characterization and immunization experiments, we decided to use a construct using CMP trimerization domain, which had been shown to have a high ability to induce neutralizing antibodies in vaccine production against PEDV ( 21 ).…”

“…Using the silkworm-baculovirus expression vector system (silkworm-BEVS), we have succeeded in the mass-production of many recombinant proteins that are difficult to be expressed and secreted by other expression systems ( 21 , 37 ). In addition, when BEVS is used, the silkworm host system often shows higher expression levels of recombinant proteins than the insect cultured cell host system.…”

“…We have previously reported the successful production of recombinant S protein of SARS-CoV-2 in the trimeric state using this BmNPV-silkworm expression system ( 20 ). Our recent study also shows that fusion of coiled-coil derived from chicken cartilage matrix protein (CMP) is effective in stabilizing the ectodomain of porcine epidemic diarrhea virus (PEDV) S protein in the trimeric state and improving its secretion in silkworms ( 21 ). Based on the results of these studies, we have successfully improved the expression of the secreted product by optimizing the trimerization and purification tags.…”