In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.

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, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkwormproduced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

1. We studied toluene metabolism in dog liver microsomes and the major metabolite was benzyl alcohol with o- and p-cresol as minor metabolites. 2. The enzyme kinetics of toluene biotransformation were examined by means of Lineweaver-Burk analyses. The Michaelis-Menten values differed among the three pathways, the order being; Km, o-cresol > p-cresol > benzyl alcohol; Vmax, benzyl alcohol > o-cresol > p-cresol; and Cl(int), benzyl alcohol > p-cresol > o-cresol. 3. The formation of benzyl alcohol, o- and p-cresol from toluene was substantially inhibited by the P4502E inhibitors such as DDC (diethyldithiocarbamate) and 4-methylpyrazole in all pathways, with IC50's in the range of 0.02-0.59 mM. The P4502B inhibitors, metyrapone and secobarbital also inhibited benzyl alcohol and p-cresol formation, whereas o-cresol was not inhibited by these latter compounds. 4. Anti-rat P4502E1 antibodies inhibited benzyl alcohol, o- and p-cresol formation from 26 to 30% 0.2 ml serum/mg microsomal protein. Furthermore, anti-rat P4502B1/2 antibody inhibited benzyl alcohol and p-cresol formation (47 and 44% respectively), but not that of o-cresol. Anti-rat P4502C11/6 antibody also inhibited benzyl alcohol and p-cresol formation 31 and 24% respectively in a similar manner to that by the anti-rat P4502B1/2 antibody. 5. These results suggested that the P4502B, 2C and 2E isozymes in dog liver contribute to the formation of benzyl alcohol and p-cresol from toluene, and 2E isozyme preferentially contributes to the formation of o-cresol.

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.