**5. Safety evaluation of the nasal influenza vaccine using biomarker gene expression**

Nasal vaccines have been attracting attention as promising strategies against influenza virus infection. This is because nasal vaccines can predominantly induce mucosal immunity, when compared with conventional subcutaneous vaccines or intramuscularly injectable vaccines [34]. In nasal vaccines, IgA antibody production and secretion in the bronchial and intranasal cavities are observed, and this approach seems to be effective for the prevention of influenza infection [35–37]. For this reason, several newly developed vaccines have been designed on the premise of nasal inoculation. It is important to develop a safety assessment method for nasal vaccines by assays that are different from the conventional intramuscular and subcutaneous injections. This is because there are case control studies on the use of the inactivated intranasal influenza vaccine, which is composed of influenza antigens in a virosomal formulation with an *E. coli*-derived LT adjuvant, and the risk of Bell's palsy in Switzerland [38]. Therefore, to determine the relation between the expression of the 18 biomarker genes and the safety evaluation of the nasal inoculation influenza vaccine, an assay was devised. Mice were nasally inoculated with an influenza vaccine, and biomarker gene expression levels in the lungs were analyzed [39]. As described earlier, this biomarker gene has been identified based on the gene expression profile obtained when the vaccine was inoculated intraperitoneally. After the administration of the nasal influenza vaccines, there was an increase in the WPVdependent expression of the biomarker gene; the evaluation of the HAV based on WPV was shown to be possible by nasal inoculation and by analysis of marker genes [39]. Furthermore, the biomarker expression level positively correlated with lymphoproliferation in nasal-associated lymphoid tissue [39], and it was inferred that this formulation induces the activity of mucosal immunity. Furthermore, in recent years, the development of an adjuvant-containing vaccine has been advanced for the purpose of enhancing the effectiveness of SVs [37]. The same trend in nasal vaccines has also been seen [37] because of the ability to induce IgA production by SVs alone is not enough to prevent infection with an influenza virus. Therefore, there has been active development of adjuvanted influenza vaccines. Although adjuvants increase the effectiveness of vaccines, strong adjuvant bioactivity is thought to lead to toxicity. The strong bioactivity of the adjuvant will ensure increased effectiveness of vaccines. In some cases, however, highly reactogenic adjuvants can cause toxicity in humans. For example, poly I:C is known to function as an excellent vaccine adjuvant. On the other hand, it is known to cause exothermic reactions and cytokine storms [40–42]. Additionally, in the past, poly I:C has been discontinued due to adverse reactions such as a fever and arthritis in clinical trials [40]. Even other adjuvants such as R848, a Toll-like receptor (TLR)7/8 agonist, are known to cause cold-like symptoms, including a fever [43–45]. Such compounds are **excellent in terms of enhancing the** effectiveness of the vaccine; however, the risk of developing toxicity remains high. Therefore, we hypothesized that the expression of 18 biomarker genes could be applied to the safety assessment of adjuvanted vaccines. The objective of this safety test is to identify an adjuvant that has high reactogenicity and toxicity such as poly I:C and R848. The risks of adverse reactions caused by adjuvanted vaccines as test products were compared with those of the WPV. In the case of a nasal vaccine, expression of some biomarker genes was higher when animals were inoculated with the TLR9 agonist CpG K3-adjuvanted HAV, than when the animals were inoculated with the HAV alone [39]. Nonetheless, the marker gene expression levels were markedly lower than those of the WPV. Thus, the CpG K3 adjuvant did not have high reactogenicity accompanied by toxicity. The CpG K3 adjuvant is under development for use with malaria vaccines [46]; no adverse reactions have been reported so far. The authors of these reports presumed that the risk of toxicity would not be high in humans. Currently, the authors are working on building a database for constructing an adjuvant evaluation system based on an influenza vaccine that includes various adjuvants including poly I:C and R848, an oil/water emulsion adjuvant, and various other TLR-related adjuvants.

performed by means of the biomarker genes. The virosome-type influenza vaccine is similar to the WPV but does not contain viral RNA. Leukopenic reactions were not noticeable when the animals were vaccinated with the virosome-type influenza vaccine; however, a body weight loss was observed, accompanied by an increase in the expression of some biomarker genes [31]. It is thought that some biological activities of this vaccine may be close to those of the WPV, because just like the WPV, Inflexal Berna V consists of a virosomal formulation. Genes whose increased expression levels were induced by the virosomal type influenza vaccine include *Tap2* and *Psmb9*, which are involved in antigen presentation and antigen digestion, suggesting that the antigen-presenting ability is higher for the virosomal-type influenza vaccine than for the HAV [31]. Consequently, it is likely that biomarker genes obtained by

genomic analysis can elucidate the mechanistic details of bioactivity and toxicity.

**gene expression**

118 Influenza - Therapeutics and Challenges

**5. Safety evaluation of the nasal influenza vaccine using biomarker** 

Nasal vaccines have been attracting attention as promising strategies against influenza virus infection. This is because nasal vaccines can predominantly induce mucosal immunity, when compared with conventional subcutaneous vaccines or intramuscularly injectable vaccines [34]. In nasal vaccines, IgA antibody production and secretion in the bronchial and intranasal cavities are observed, and this approach seems to be effective for the prevention of influenza infection [35–37]. For this reason, several newly developed vaccines have been designed on the premise of nasal inoculation. It is important to develop a safety assessment method for nasal vaccines by assays that are different from the conventional intramuscular and subcutaneous injections. This is because there are case control studies on the use of the inactivated intranasal influenza vaccine, which is composed of influenza antigens in a virosomal formulation with an *E. coli*-derived LT adjuvant, and the risk of Bell's palsy in Switzerland [38]. Therefore, to determine the relation between the expression of the 18 biomarker genes and the safety evaluation of the nasal inoculation influenza vaccine, an assay was devised. Mice were nasally inoculated with an influenza vaccine, and biomarker gene expression levels in the lungs were analyzed [39]. As described earlier, this biomarker gene has been identified based on the gene expression profile obtained when the vaccine was inoculated intraperitoneally. After the administration of the nasal influenza vaccines, there was an increase in the WPVdependent expression of the biomarker gene; the evaluation of the HAV based on WPV was shown to be possible by nasal inoculation and by analysis of marker genes [39]. Furthermore, the biomarker expression level positively correlated with lymphoproliferation in nasal-associated lymphoid tissue [39], and it was inferred that this formulation induces the activity of mucosal immunity. Furthermore, in recent years, the development of an adjuvant-containing vaccine has been advanced for the purpose of enhancing the effectiveness of SVs [37]. The same trend in nasal vaccines has also been seen [37] because of the ability to induce IgA production by SVs alone is not enough to prevent infection with an influenza virus. Therefore, there has been active development of adjuvanted influenza vaccines. Although adjuvants increase the effectiveness of vaccines, strong adjuvant bioactivity is thought to lead to toxicity.
