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.

The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy.

Vaccine efficacy of conventional influenza vaccines depend on the antigenic similarity between the selected vaccine strain and annual epidemic strain. Since the influenza virus evolves yearly, a vaccine which is independent from viral antigenic mutation is desired. We have developed chimeric cytokine (CC) and hemagglutinin (HA) incorporated virus-like particle (CCHA-VLP) as a universal influenza vaccine candidate. Using mouse models, it was shown that the vaccine provided broad-based protective activity against several types of human and avian influenza A viruses. In this report, nasal immunization and mixture form (CC- and HA-VLP) were tested to improve usability of this vaccine. Immunogenicity was evaluated by induction of IgG, IgA, and IFN-γ secreting cells. Protective activity was measured as mouse survival rate against lethal challenge with H1N1 and H5N1 viruses and against H3N2 virus by lung viral titer. Nasal immunization showed low immunogenicity and low protective efficacy, but the addition of a sesame oil adjuvant improved vaccine efficacy. Mixture form of CC- and HA-VLP showed comparable or higher vaccine efficacy when compared to the incorporated form, CCHA-VLP. These results contribute to improved usability, such as needle-less administration and easy HA subtypes alteration.