**1. Introduction**

250,000–500,000 people die from influenza or bacterial infections every year following influenza infection (www.who.int/influenza/en/). Viral spread also results in considerable days of illness and the loss of millions of work days annually. Influenza A virus is an RNA virus with a segmented genome of eight genes. The two surface proteins hemagglutinin (HA) and neuraminidase (NA) are the main targets for the neutralizing antibodies. The combination of these two antigens (20 different serotypes of HA [HA1 to HA20] and eleven NA in [NA1 to NA11]) with the two most recent bat influenzas identified, greatly determines the variability between the influenza virus strains [1]. Human vaccination using these immunodominant antigens is a primary method of influenza prevention that is used to control both seasonal and pandemic influenza strains [2]. When unchecked, seasonal and pandemic influenza both strongly affect the elderly who are especially sensitive to complications following influenza infection. Furthermore, existing influenza vaccines are less effective in the elderly when compared to younger people. The development of mucosally administered live or killed inactivated adjuvanted vaccines would be one way to create vaccines that are more conveniently delivered efficiently to the elderly [3].

It would be highly desirable to develop influenza vaccines that provide broader influenza-specific immune responses than what can be obtained with the currently available commercial inactivated flu-vaccines. If stronger and more long-lasting, cell-mediated and humoral flu-specific immunity could be obtained, it would be more likely that the obtained immunity could better protect against disease in future epidemics. In preclinical models, it has been reported that the killed formalin-inactivated influenza vaccines nasally given induce immunity almost equally with or without adjuvants [4–6]. Nevertheless, it would be advantageous to broaden the often elicited homosubtypic immunity into a heterosubtypic immune response recognizing more divergent influenza virus strains. Several recent preclinical studies in mice suggest that this is possible [7–10] by using virus-like particle vaccines [11]. It seems clear that both arms of the humoral adaptive immune system will need to be employed to broaden vaccine immunity, with influenza A neutralizing serum IgG, and also preferably with mucosal immunity consisting of secretory IgA towards the outer envelope proteins HA and NA [12]. This should then be combined with a systemic cell-mediated immunity as the second line of defense, which consists of CD8+ T cells recognizing conserved internal influenza virus epitopes [13], as well as a broad repertoire of memory CD4+ Th cells, which are critical to the maintenance of long-lasting humoral and CD8+ T cell immunity [14–16].

A new pandemic would probably be more rapidly spread and extensive than the Spanish flu of 1918–1919 when considering growing human populations and ease of international travel [17]. Indeed, these factors appear to have facilitated the emergence of the recent A/H1N1/"Swine" influenza, which appears to be a mixture of influenza viruses previously not seen in man, such as three triple reassorted genes from north American swine and human, three genes from classical swine influenza, and two genes from Eurasian swine [18]. When another pandemic appears, there will be many challenges to overcome in order to rapidly develop an effective vaccine against influenza, especially if they evolve from complex reassorted gene mixtures, as seen with the 2009 Swine-origin 2009 A (H1N1) influenza viruses. The time it takes to produce influenza vaccines needs to be decreased as well as issues of immunogenicity, quality control, and safety. Development and testing of vaccines while using new technologies are to be lauded. However, they can introduce unquantified risk in the development chain slowing vaccine development. Finding ways to improve upon existing technologies may be a way of mitigating development risk, while at the same time improving immunogenicity, safety, and production speed. The aim of this study was to use a licensed, existing whole formalin-inactivated influenza A virus (WIV) as a source of antigen and improve upon its ability to elicit immune responses by the addition of adjuvants. WIV vaccines are well known to induce poor cellular immune responses, unless combined with adjuvants [19]. This study investigated how additon lipid and genetic adjuvant(s) could be formulated to contain several critical components that cooperate to provide both a strong humoral, systemic and mucosal, as well as systemic cell-mediated heterosubtypic immune response, in both

inbred mice (C57BL/6) and the outbred NMRI mice. Flagellin is an agonist of TLR5, but it is also directly recognized by the cytosolic Nod-like receptor family member Naip5, which signals through NLRC4 to form an inflammasome. Soluble flagellin has been shown to be a potent adjuvant in numerous studies and triggers numerous immune responses [20]. In previous studies, the presence and uptake of the bacterial flagellin proteins by CD103+ dendritic cells (DC) have resulted in their increased presence in mesenteric lymphnodes. Further, the flagellin-proteins have been shown to increase B-cells to switsch into IgA secreting cells, thereby enhancing the mucosal B and T cell responses against antigenic proteins [21]. However, there are few studies on the adjuvant effects of DNA-encoded flagellin [19]. The novelty of the present vaccination design of inactivated influenza A virus is the combination of previously never studied combined adjuvants. Thus, the adjuvants, the cationic lipid N3 alone, or N3 lipid mixed with DNA-plasmid expressing the TLR5-agonistic, de-glycosylated flagellin C-protein mixed with the WIV/Salomon Island/2006 A/H1N1-antigen prepared as an emulsion for nasal mucosal administration in two strains of aging mice.

The reasons for proposing mucosal administration would be to obtain mucosal immunity in the nasal and respiratory organs, where respiratory viral infections enter the body, to provide mucosal first-line barrier immunity. Furthermore, the immunization protocol aimed to study the long-term protective effect in an aging target-group of elderly individuals of mice reaching 22–23 months of age (representing human ages 60–70 yrs), where the influenza specific systemic and mucosal Th1/Th2-type immune pattern responses were followed and they could be correlated with different vaccination designs and levels of heterologous influenza A virus protection.

Here, we evaluated the potential benefits of combining the WIV vaccine with an experimental mucosal cationic oleic oil-based adjuvant alone and with a DNA-plasmid expressing a secreted form of flagellin from *Salmonella typhimurium*.
