Highlights Influenza A virus-like particles (VLPs) were produced using recombinant insect cells. VLPs were produced using insect cells as host cells without using a baculovirus. A secretory form of VLPs consists of hemagglutinin and matrix protein 1. The VLP productivity is comparable to that of the baculovirus–insect cell system.

Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence.Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.

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Influenza vaccines have long been manufactured in embryonated chicken eggs. This method has some problems such as a long production period (about 6 months) and use of large amounts of infectious pathogens. Recently, the production of recombinant subunit vaccines using the baculovirus–insect cell system has been extensively investigated. In this system, viral immunodominant components can be produced more rapidly and in a larger scale than in the conventional egg-based process. However, continuous production is virtually impossible because infection of recombinant baculovirus results in the death of host insect cells. In the present study, we established stably transformed insect cells that secreted influenza virus-like particles (VLPs) consisting of hemagglutinin (HA), the major protective antigen of influenza A virus, and matrix protein 1 (M1), another structural protein of the virus. Hemagglutination assay and transmission electron microscopy (TEM) suggested that HA produced by recombinant insect cells kept the hemagglutination activity and the morphology of the VLPs was similar to that of wild type influenza virus particles.