(A) serum haemagglutination inhibition (HI) titres, (B) single radial haemolysis (SRH) zone areas (mm2) and (C) virus neutralisation titres were measured at 3?weeks after the second vaccination against the homologous strain. humoral and cellular immune responses of two promising mucosal adjuvants (Chitosan and c\di\GMP) for intranasal influenza H5N1 vaccine in a murine model. Furthermore, we evaluated the concept of co\adjuvanting an experimental adjuvant (c\di\GMP) with chitosan. Methods? BALB/c mice were intranasally immunised with two doses of subunit NIBRG\14 (H5N1) vaccine (75, 15 or 03?g haemagglutinin (HA) adjuvanted with chitosan (CSN), c\di\GMP or both adjuvants. Results? All adjuvant formulations improved the serum and local antibody responses, with the highest responses observed in the 75?g HA 2′-O-beta-L-Galactopyranosylorientin CSN and c\di\GMP\adjuvanted groups. The c\di\GMP provided dose sparing with protective single radial haemolysis (SRH), and haemagglutination inhibition (HI) antibody responses found in the 03?g HA group. CSN elicited a Th2 response, whereas c\di\GMP induced higher frequencies of virus\specific CD4+ T cells producing one or more Th1 cytokines (IFN\+, IL\2+, TNF\+). A combination of the two adjuvants demonstrated effectiveness at 75?g HA and triggered a more balanced Th cytokine profile. Conclusion? These data show that combining adjuvants can modulate the Th response and in combination with ongoing studies of adjuvanted intranasal vaccines will dictate the way forward for optimal mucosal influenza vaccines. studies have also shown that chitosan may promote paracellular transport through a transient opening of intercellular tight junctions. 22 CSN is a safe mucosal adjuvant, 23 which augmented the immune response to intranasally administered influenza vaccine. 24 The bacterial second messenger (3, 5)\cyclic dimeric guanylic acid (c\di\GMP) has been identified in bacteria but not in higher eukaryotes (reviewed in Ref. 25), and several studies have emphasised its adjuvant potential. 26 , 27 , 28 , 29 The transmembrane protein stimulator of interferon genes (STING) was recently shown to function as a direct sensor for c\di\GMP and other cyclic dinucleotides. 30 , 31 A proposed mechanism for c\di\GMPs adjuvant properties is that STING ligation increases the production of type I interferons, 32 which 2′-O-beta-L-Galactopyranosylorientin in turn drives the adaptive immune response. In this study, we have evaluated CSN, c\di\GMP and a combination of the two adjuvants in a dose response study of an intranasal subunit (SU) influenza H5N1 vaccine. The humoral and cellular immune responses were evaluated and compared between the different vaccine formulations. Both adjuvants augmented the immune response, but the Th profile differed with CSN eliciting a Th2\biased response, c\di\GMP a Th1\biased response and the adjuvant combination a more balanced Th profile. The c\di\GMP adjuvant was most IgG2b Isotype Control antibody (PE) effective at boosting local and systemic humoral immune responses and allowed significant dose sparing. Materials and methods Materials Inactivated influenza subunit vaccine (NIBRG\14) and chitosan adjuvant (CSN, ChiSys?) were supplied by Archimedes Development Ltd., Reading, UK. The chitosan utilised in the study was chitosan glutamate 213 (manufactured by FMC BioPolymer AS, Drammen, Norway) which was 75C90% deacetylated and had a glutamate content of 35C50%. The bis\(3,5)\cyclic dimeric guanosine monophosphate (c\di\GMP) adjuvant was produced at the Helmholtz Centre for Infection Research as previously described. 28 The antigen was mixed with adjuvant immediately prior to vaccination. Animals and vaccination A dose\sparing study was conducted by intranasally immunising mice (twelve groups with five mice in each group) with two doses (21?days apart) of NIBRG\14 SU with or without CSN or c\di\GMP or a combination of the two adjuvants. The study was approved and conducted according to the Norwegian Animal Welfare Act. Six\ to eight\week\old female BALB/c mice (Taconic M&B, Denmark) were housed at the Vivarium, University of Bergen 2′-O-beta-L-Galactopyranosylorientin at a temperature of 21C with 12?hour light/dark cycles and food and water value? ?005 was considered to be statistically significant. T\cell distributions were compared using the Wilcoxon Signed Rank test integrated in SPICE. 38 Results This study aimed to investigate the quality and magnitude of the B\ and T\cell responses in mice after intranasal vaccination with an H5N1 subunit vaccine (NIBRG\14 SU). The effect of two different adjuvants (CSN and c\di\GMP) and a combination of the two adjuvants were evaluated. To assess the dose\sparing capabilities of the adjuvants, groups of mice were immunised with different doses (75, 15 or 03?g HA) of NIBRG\14 SU alone or with one or both of the adjuvants. Adjuvant augments the HI, SRH and VN antibody response. The serum influenza\specific humoral immune responses are commonly measured by the HI, SRH and VN assays. An HI titre 40 or SRH zone area of 25?mm2 continues to be connected with a 50% possibility of getting clinically protected against seasonal influenza, 39 and these trim\off beliefs are used being a surrogate correlate of security when evaluating applicant pandemic influenza vaccines. No correlate of security continues to be set up for VN, although titres of 20C80 have already been recommended for H5N1 infections. The post\vaccination HI, VN and SRH titres were measured in cardiac bloodstream collected 3?weeks following the second immunisation (Amount?1A,B,C). Open up in another window Amount 1 ?The serological antibody.