**4. Safety evaluation of influenza vaccines on the basis of biomarker gene expression**

**Symbol Official full name Accession** *Cxcl11* Chemokine (C-X-C motif) ligand 11 NM\_019494 *Cxcl9* Chemokine (C-X-C motif) ligand 9 NM\_008599 *Zbp1* Z-DNA binding protein 1 NM\_021394 *Mx2* MX dynamin-like GTPase 2 NM\_013606 *Irf7* Interferon regulatory factor 7 NM\_016850 *Lgals9* Lectin, galactoside-binding, soluble, 9 NM\_010708 *Ifi47* Interferon gamma inducible protein 47 NM\_008330 *Tapbp* TAP binding protein (tapasin) NM\_001025313 *Csf1* Colony stimulating factor (macrophage) NM\_007778 *Timp1* Tissue inhibitor of metalloproteinase 1 NM\_001044384 *Trafd1* TRAF type zinc finger domain containing 1 NM\_001163470 *Lgals3bp* Lectin, galactoside-binding, soluble, 3 binding protein NM\_011150 *Psmb9* Proteasome (prosome and macropain) subunit, beta type, 9 NM\_013585 *C2* Complement component 2 NM\_013484 *Tap2* Transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) XM\_006525355 *Ifrd1* Interferon-related developmental regulator 1 NM\_013562 *Psme1* Proteasome (prosome and macropain) activator subunit 1 NM\_011189 *Ngfr* Nerve growth factor receptor NM\_033217

antibody production is considered insufficient to prevent the progression of influenza virus infection [30]. Historically, however, vaccines have been more effective than the existing split vaccines. The WPV is considered effective against influenza virus infections. This type of vaccine contains the whole influenza virus particle, including lipid and single-stranded RNA, and therefore, drives various immune responses. On the other hand, various WPVinduced immune responses also cause adverse reactions in humans [29]. Therefore, although WPV is a highly effective vaccine, it has lately not been employed as a seasonal influenza vaccine and is only partially manufactured as a pandemic influenza vaccine. We have carried out the searches for safety assessment marker genes of the HAV using two types of vaccines: the WPV and split influenza vaccine (SV). The WPV has high reactogenicity (effectiveness and toxicity) and therefore serves as a toxicity reference. The SV has low reactogenicity and frequency of adverse reactions and is therefore employed as a safety control. As a result, the clearest clustering of gene expression patterns in the lungs of animals by different types of vaccines was obtained [25]. In particular, the gene expression patterns in the lungs differed between the SV-treated and WPV-treated animals. Furthermore, the gene expression levels, which showed large differences between the SV- and WPV-treated animals, were estimated. As a result, 18 genes expressed in the lungs were identified as biomarker genes (**Table 1**) [25].

116 Influenza - Therapeutics and Challenges

**Table 1.** Marker genes for safety evaluation of influenza vaccines.

The utility of the identified biomarker genes has been verified. For seasonal influenza vaccines, the ATT has been regarded as a test for safety and quality control. Therefore, a safety assessment of SVs manufactured at four manufacturing plants was conducted by means of biomarker gene expression and body weight as parameters (ATT) and by the LTT, with a WPV as a control [33]. With respect to phenotypic changes, body weight loss rates of all the SVs were found to be equivalent, and leukocyte number reduction was hardly observed for the HAVs from all the manufacturers. Nevertheless, in case of one manufacturer's HAV, analyses of the expression of 18 biomarker genes in lungs showed a significant difference in gene expression levels from other manufacturers' HAV [33]. This result suggests that the biomarker genes identified by the microarray analysis can capture biological changes that cannot be detected by body weight changes and leukocyte number reductions. This finding indicates that the analysis of expression of biomarker genes is a more sensitive assay than the conventional safety and quality control tests (ATT and LTT). This evaluation method can be applied not only to predict the toxicity but also to evaluate the homogeneity among vaccines produced in different batches.

Subsequently, the safety assessment of trivalent virosome-type influenza vaccine (Inflexal Berna V) currently licensed in several European countries such as Switzerland and Italy was 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.

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

Genomic Approaches Enable Evaluation of the Safety and Quality of Influenza Vaccines…

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and R848, an oil/water emulsion adjuvant, and various other TLR-related adjuvants.

**6. Development of an alternative assay for the leukopenic toxicity** 

The biomarker genes for the safety assessment of an influenza vaccine are characterized by the biological activity that can be detected by the ATT and LTT. Specifically, biomarker gene expression levels and the WBC count with body weight changes show a negative correlation [31]. Momose *et al*. (2015) reported that a virosomal influenza vaccine caused only a body weight loss and did not cause leukopenia; however, some of the marker genes showed increased expression levels at that time point [31]. In other words, it seems that all the marker genes cannot respond uniformly via the same mechanism of action. Therefore, we considered whether the leukocytopenic activity could be evaluated with the expression of the marker genes responsible for the leukopenic activity, and we searched for biomarker genes associated with leukocytopenic activity. Furthermore, we devised a method for WBC count-predicting systems involving only the biomarker gene expression levels. If this method is established, it will be possible to set up the WBC number prediction using the biomarker gene expression and body weight loss evaluation by the ATT in one test system. This strategy will reduce the number of animals required and shorten the testing duration. We tried to identify the genes useful for the prediction of the WBC count from the biomarker gene set by multiple linear regression analysis and a stepwise method [32]. In the multiple regression analysis method,

**test based on biomarker gene expression**
