**8. Establishment of a new evaluation method for vaccine or adjuvant bioactivity based on biomarker gene function**

Analysis by the genomics technology can be applied not only to the search for biomarkers but also to mechanistic analyses. Besides, it is possible to classify each biological reaction by hierarchical clustering analysis, according to microarray analysis results. Microarray analysis is the most information-rich assay; however, it is inefficient in terms of cost and labor. In the case of a clear-purpose test such as safety evaluation and quality control, it is expected that robust results will be obtained by using only selected highly important genes for evaluation. Therefore, if we consider the function of the genes identified as safety or quality evaluation markers for influenza vaccines, then the biological activity profile of the vaccine may be predicted. For example, genes such as *Irf7* are induced by type 1 IFN [55], genes such as *Psmb9* and *Tap2* are involved in antigen presentation [56], and *Csf1* participates in the activation of monocytes and macrophages [57]. Thus, expression levels of these genes could serve as indicators of the mode of action and help in the development of a biological activation assessment tool. These genes are thought to be involved in innate immunity, in which responses are observed at a relatively early time point after vaccination. Indeed, expression of these genes was assayed 16 h after vaccination. Therefore, long-term toxicity due to the vaccine (e.g., autoimmune and chronic inflammatory reactions) cannot be assessed. Safety evaluation by means of these biomarker genes is helpful for the development of adjuvant-containing vaccines. This is because most adjuvants are designed to activate the innate immune system. Adjuvants enhance innate immunity via cytokine production and activation of antigen-presenting cells; however, strong activation of innate immunity causes uncontrollable inflammatory reactions. This problem could lead to a fever, pain, and swelling, which appear as adverse reactions. Thus, adjuvants are required to have strong innate-immunity–activating effects, but at the same time, good safety. On the other hand, it is difficult to distinguish between the effectiveness and safety of vaccines. For example, interleukin (IL)-6 and type 1 IFN are important for the induction of adaptive immunity and are favorable for vaccine efficacy [58, 59]. Nevertheless, excess production of IL-6 or type 1 IFN causes a cytokine storm. Thus, safety can be evaluated with the same biological vector as that of effectiveness. In other words, if the factor of effectiveness becomes excessive, toxic effects may appear. We are currently working on establishing a safety assessment system based on the WPV as a toxicity indicator [60]. The WPV is an effective and excellent vaccine, but the frequency of adverse reactions is high, and currently, it is only rarely used, especially as a prepandemic vaccine. Therefore, we believe that the WPV can be a safety indicator. In other words, we think that there is a high probability that adjuvants and vaccines with innate-immunity– inducing activities that exceed the activity (toxicity) of WPVs will cause adverse reactions [60].

to their human counterparts, except for *Ifi47*. This observation suggests that some phenomena common to the tested animals and humans may be identified via the animal experiments by means of marker genes. To test this hypothesis and to develop an *in vitro* assay system, the use of human peripheral blood mononuclear cell-based or alveolar-epithelial-cell-based methods is necessary. Thus, extrapolation of the results of these evaluations to humans can be partially

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

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It has been established that information that could not be obtained by conventional phenotypic analyses can be obtained by genomic analyses. Research conducted on the safety of vaccines and adjuvants using toxicogenomics has been less likely to be reported, and such data about chemically synthesized drugs have mostly been limited. Since the late 2000s, we have been trying to apply the genomics technology to the safety assessment of vaccines, and to demonstrate its sensitivity, ability to yield abundant toxicological and mechanistic information, the possibility of extrapolating its results to humans, and its potential for application to *in vitro* evaluation systems. In addition, we have shown that the newly developed evaluation system may be employed in analyses involving a biomarker gene(s) as an indicator, instead of the conventional quality control or safety test. It can also be assumed that these technologies can be utilized for adjuvant development, and it is expected that a wide range of genomics technologies will be applied in the future to the development of quality

The research summarized in this review was supported by AMED, grant numbers

Department of Safety Research on Blood and Biological Products, National Institute of

achieved by bridging the species differences with the marker genes.

**11. Conclusions**

control and safety testing.

**Acknowledgements**

**Conflict of interest**

**Author details**

JP17ak0101071 and JP17fk0108124.

The authors declare that they have no conflicts of interest.

Eita Sasaki, Takuo Mizukami\* and Isao Hamaguchi

Infectious Diseases, Musashimurayama, Tokyo, Japan

\*Address all correspondence to: tmiz@nih.go.jp

## **9. Future perspectives of safety evaluation of vaccines and adjuvants**

According to the abovementioned concept, various evaluations of adjuvanted vaccines have been carried out. Furthermore, we have focused on the functions of biomarker genes. We have attempted to compile gene clusters based on the function of each gene. Such an assay is currently at the development stage, and further examination of the evaluation method and validation should be conducted in the future. Such an assay is considered applicable to the development of novel adjuvants.

For the development of low-molecular-weight synthetic drugs, a seed compound having a desired bioactivity is searched for by a screening system in compound libraries [61]. For promoting adjuvant development, to create as many prominent novel adjuvants as possible, finding seed compounds that are likely to become adjuvants is a crucial step in adjuvant development. Conventionally, to demonstrate whether a compound has adjuvant activities, animals are inoculated with one of the compounds, and the antibody titer and infection prevention rate are then assessed. This evaluation is not as efficient as the seed search and compound screening because the assessment process takes more than 1 month. In contrast, if we introduce an evaluation method involving a biomarker gene or genes, then prediction of safety and of the biological activity profile for compounds may be achieved in animals or cultured cells. Such an assessment may make it possible to search for effective adjuvant seeds that are safer than WPVs and more effective than the HAV.
