Development of Influenza Universal Vaccine Materials Using Antigenic M1 and M2 Extracellular Domain (M2e) of Influenza A/Korea/01-2-9/2009 (H1N1) Virus Expressed from Stably Transfected Trichoplusia ni BT1 Tn 5B1-4 Cells

Several influenza subunit vaccine candidates has been developed from four major envelop proteins, matrix protein 1 (M1), matrix protein 2 (M2), hemagglutinin (HA) and neuraminidase (NA). However, there is a limitation to develop subunit vaccines because influenza viruses have numerous variants with antigenic differences in the surface proteins HA and NA. Recently, M2, especially extracellular domain (M2e) of M2, has been suggested to be a promising candidate antigen for developing universal influenza vaccines because M2 remains nearly invariant among different strains. Here, stably transfected lepidopteran insect Trichoplusia ni BT1 Tn 5B1-4 (Tn5) cells expressing M1 and M2e fused with transmembrane domain of HA were generated to develop influenza universal VLP vaccine candidates. Full length M1 and M2 cDNA of influenza A/Korea/01-2-9/2009 (H1N1) virus were generated by RT-PCR. M1 and three tandem repeats of M2e genes (6M2e) C-terminally fused with transmembrane domain of HA were cloned into pIZTx2 to generate pIZTx2/M1-6M2e-HA-TMD. Stably transfected polyclonal Tn5 cell populations were isolated after 5 weeks of transfection with pIZTx2/M1-6M2e-HA-TMD and selection with zeocin. The expression of M1 and 6M2e-HA-TMD were confirmed by Western blot analysis using anti-M1 and M2e antibodies. Furthermore, influenza M1/6M2e-HA-TMD VLPs were concentrated by ultracentrifugation. The morphology of influenza M1/6M2e-HA-TMD VLPs will be examined by electron microscopy. This work was supported by a grant from the Next-Generation BioGreen 21 Program (PJ009526), Rural Development Administration, Republic of Korea.