Interleukin 12-containing influenza virus-like-particle vaccine elevate its protective activity against heterotypic influenza virus infection

To produce monovalent and bivalent influenza vaccines composed of virus-like particles (VLPs) containing hemagglutinin (HA), we generated four recombinant Baculoviruses derived from Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa california nuclear polyhedrosis virus (AcNPV). Monovalent Fukushima (A/tufted duck/Fukushima/16/2011 [H5N1]) (FkH5) and Anhui (A/Anhui/1/2013 [H7N9]) (AnH7) VLP influenza vaccines were produced in silkworm pupae infected with FkH5-BmNPV or AnH7-BmNPV. To produce a bivalent FkH5 and AnH7 vaccine, the pupae were simultaneously inoculated with FkH5-BmNPV and AnH7-BmNPV. Then, interleukin (IL)-containing bivalent vaccines were produced by Eri silkworm pupae following triple infection with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV. Fluorescent antibody tests in Sf9 cells triple-infected with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV showed coexpression of FkH5, AnH7, and IL-12 antigens, suggesting the presence of VLPs containing all three antigens. We then performed competitive hemagglutination inhibition (CHI) tests to calculate the VLP vaccine constituents. Inoculation with two recombinant viruses led to the production of bivalent vaccines containing very similar amounts of the H5 and H7 antigens, suggesting that our dual infection system can be used to produce bivalent VLP vaccines. Immunisation of mice with our developed monovalent and bivalent VLP vaccines induced the production of HI antibody, which protected against a sublethal dose of influenza virus. These IL-12-containing vaccines tended to display increased protection against hetero-subtype influenza viruses.