Objective To analyze the immunostimulatory effects of cyclic dinucleotides ( CDN) on immune responses to a nasal spray influenza split virus vaccine and to evaluate its potential as a mucosal ad-juvant. Methods A H1N1 influenza split virus vaccine combined with different CDN was used for mouse immunization. Each mouse was intranasally immunized twice with 4. 5μg of hemagglutinin (HA) and 10μg of CDN with an interval of 21 d. Titers of hemagglutination inhibition ( HI) antibodies in serum, secretory IgA ( sIgA) in bronchoalveolar lavage fluid and IgG in serum were detected 21 d after the last immunization. Immunostimulatory activities of different CDN were compared. Effects of cyclic di-GMP ( c-di-GMP) and ch-itosan (CSN) on the immunogenicity of H1N1 and H7N9 influenza split vaccines were analyzed and com-pared. H1N1 influenza split vaccine combined with c-di-GMP or CSN was used to immunize mice. Three weeks after the last immunization, these mice were challenged with 10 times the median lethal dose ( LD50 ) of A/Puerto Rico/8/34 (H1N1) influenza virus. Survival rates of the mice were observed for 14 d. Results All three CDN induced high levels of HI antibodies and IgG in serum and sIgA in BALF. HI antibody sero-conversion rates were also higher than those of the control groups. c-di-GMP was superior to CSN in enhan-cing the immunogenicity of H1N1 and H7N9 antigens as higher titers of HI antibodies in serum and sIgA in BALF were induced. Conclusions CDN could enhance the immunogenicity of influenza antigens with better efficacy than CSN adjuvant.

Objective: To evaluate the immunogenicity of split influenza H1N1 vaccine formulated with an oil-in-water nano-emulsion adjuvant in aged mice and young mice. Methods: A nano-emulsion adjuvant formulated split influenza H1N1 vaccine was used to immunize aged and young mice through intramuscular injection. Each mouse was immunized with 0.012 μg of hemagglutinin (HA) twice with an interval of 28 d. Hemagglutination inhibition (HI) titers in serum were measured 27 d after first immunization. Serum HI, IgG1 and IgG2a titers were detected 14 d after the last immunization. No adjuvant-formulated vaccine and normal saline (NS) were used to set up control groups. Virus challenge test was carried out using 10 times the median lethal dose (LD₅₀) of A/Puerto Rico/8/34 (H1N1) strain two weeks after the last immunization and the protective effects were assessed through measuring the dynamic changes in body weight and survival rate. Results: Higher levels of serum HI, IgG1 and IgG2a antibodies and higher HI antibody conversion rates were induced in the adjuvant groups, especially in the aged mice group, than in the control groups. Nano-emulsion adjuvant improved the immunogenicity of HA and mouse immunity to A/Puerto Rico/8/34 (H1N1). Conclusions Nano-emulsion adjuvant could enhance the immunogenicity of influenza antigens, especially in aged mice.