Consequently, there is a continuing need to design and develop a new generation of broadly protective and safe vaccines, especially for this age category. The anionic adjuvant Endocine™ was developed specifically to formulate intranasal vaccines. Endocine™ is

composed Cyclopamine nmr of endogenous lipids found ubiquitously in the human body and has been tested successfully in clinical trials with diphtheria, influenza and HIV [19], [20] and [21] (and unpublished data). The results of these trials showed that Endocine™ is safe and tolerable in humans, and in the influenza trial the Endocine™ adjuvanted whole virus vaccine fulfilled the EMA/CHMP HAI criteria for a seasonal influenza vaccine. Moreover, influenza-specific IgA was measured in nasal swabs and it was shown that the Endocine™ adjuvanted vaccine induced a significantly higher fold-increase in nasal IgA compared to the mock vaccine with Endocine™ alone [19]. In line with these observations, no adverse effects of the administration of Endocine™ were noted in pre-clinical toxicology or efficacy studies (unpublished

data). The two components of Endocine™, monoolein (monoglyceride) and oleic acid (fatty acid), are metabolites generated in mammalians when lipids (triglycerides) are mobilized and energy needed. Monoolein is composed of glycerol and oleic acid and is a nontoxic, biodegradable and biocompatible material which is included in the FDA Inactive Ingredients Guide and in nonparenteral

signaling pathway medicines licensed in the United Kingdom [53]. Oleic acid has been described as being the most abundant fatty acid in human adipose tissue and it is abundantly present in mammalian tissues including tissues from rat, chicken, pig and cow [54] and [55]. Both oleic acid and monoolein and are classified as GRAS (generally recognized as safe) by the FDA, US. A study in mice showed that Endocine™ mixed with a commercially available trivalent split influenza vaccine (Vaxigrip) significantly (p < 0.003–0.05) improved the humoral (HI, VN) and Phosphoprotein phosphatase cellular (IFNγ and IL-2 secreting cells) immunity upon nasal administration [21]. Furthermore, intranasal immunization with the Endocine™ formulated vaccine significantly increased the H1N1-specific IgA levels both in serum and nasal washings [21]. In the present study, we have shown that Endocine™ formulated inactivated pH1N1/09 influenza vaccines administered as nasal drops induced a protective systemic immune response in influenza naïve ferrets. Serum HI antibody titers of ≥40 (GMT) were already measured after one immunization, even at the lowest antigen dose of 5 μg HA split antigen. All animals in this study received three nasal immunizations, but optimal serological responses were already measured after two immunizations and the third immunization proved to be redundant for antibody induction.