Test of a new liposomal adjuvant for the commercial influenza vaccine in ferret

1. Test of a new liposomal adjuvant for the commercial influenza vaccine in ferret Martel C. a, Hammer Jensen T. a, Viuff B. a, Nielsen L.P. b, Agger E.M. b, Blixenkrone-Møller M. a, Andersen P. b, Aasted B. a a Copenhagen University, Faculty of Life sciences, Institute of Veterinary Pathobiology, Denmark b Statens Serum Institute, Department of Immunology of Infectious Diseases, Denmark Abstract: Inactivated vaccines against influenza elicit mainly a humoral immune response, and only a small T-cell response. This is one of the main reasons for the necessity to revaccinate yearly. Recently, the Statens Serum Institut has developed a cationic liposome-based adjuvant (dimethyldioctadecylammonium/trehalose 6,6’-dibehenate, DDA/TDB) which induces both humoral and cell-mediated immune responses and thereby could trigger the T-cell response necessary for enhanced protection and possibly cross-protection among influenza subtypes. This adjuvant was tested in ferrets in a model using a recent circulating H1N1 virus. Levels of gamma-interferon in leucocytes were monitored by FACS in ferrets challenged by H1N1. Presence of influenza-specific antibodies was checked by ELISA. Virus production was investigated by quantitative RT-PCR and pathological effects in the respiratory tract were investigated. This study suggested that DDA/TDB might be used to enhance the immunogenicity of the current influenza vaccine. • Material and methods: • 24 ferrets were immunized three times with two-weeks intervals, then challenged after a month of rest with 107 TCID of H1N1 • Vaccine group: 8 ferrets, 3 x commercial vaccine Vaccine + adjuvant group: 8 ferrets, 3 x vaccine + DDA/TDB • Control Adjuvant group: 3 ferrets, 3 x DDA/TDB Control PBS group: 5 ferrets, 3 x PBS • Viral titers in nasal washes were measured by RT-PCR • Influenza-specific antibodies were detected in serum via ELISA using homemade rabbit anti-ferret IgG • PBL positive for intracellular gamma-IFN staining after PMA stimulation were analyzed by flow cytometry Results: Figure 1: Virus titers in nasal washes at day 3 after challenge (peak replication day) measured by quantitative RT-PCR Figure 2: Titers of influenza-specific antibodies measured by ELISA, using a cross-reactive biotinylated anti-mink IgG Figure 3: Percentage of peripheral blood lymphocytes positive for intracellular gamma-interferon staining after reactivation by 4 hours of culture in PMA. The group immunized with the combination vaccine + adjuvant exhibited lower levels of virus in the nasal washes at peak replication than the control groups or the groups immunized with the commercial vaccine only (fig.1). Groups vaccinated with or without the adjuvant both developed influenza-specific antibodies early during the immunization period (fig.2), but the group “vaccine + adjuvant” exhibited much higher titers than the group “vaccine only”, the latter displaying antibody levels similar to those obtained in the control groups after challenge. Finally, the group “vaccine + adjuvant” showed lower levels of gamma-IFN during the infection period (fig. 3), confirming the higher protective effect of the new combination. Conclusion: In this experiment, DDA/TDB was used as an adjuvant for the commercial influenza vaccine and gave a better protection than the vaccine alone, as shown by the reduction of the viral load and gamma-interferon levels during infection, and by the production of very high levels of influenza-specific antibodies early during immunization. Further investigations will be conducted to elucidate whether DDA/TBD induces CD8 response and confers cross-protection against other genetically drifted strains of influenza A virus.