**7. Possibility of application of genomic approaches to** *in vitro* **safety evaluation methods**

Currently, safety evaluation and parts of quality control of vaccines are carried out in animal experiments. The ATT and LTT are representative tests in this regard; they have been practiced for more than 50 years [48]. Due to the nature of the test, the ATT excludes the unintended toxicity of the vaccine; the test of lots having reactogenicity different from that of other reference lots is a final confirmatory test designed to prevent serious toxicity in humans [21]. Actually, the ATT has been incorporated into the lot release of vaccines, especially for inactivated influenza vaccines in Japan [19]. On the other hand, tests on animals are being replaced with *in vitro* evaluation systems involving cultured cells, from the viewpoint of animal welfare and cost [48]. In the safety evaluation of chemically synthesized medicines, there has been a notable development: human cultured hepatocytes and cardiomyocytes prepared from induced pluripotent stem cells [48–52]. The benefit of the *in vitro* evaluation system is not only the reduction in the number of animals used, but also the possibility of using human tissue or fluid samples, so that extrapolation to humans can be expected. For biologicals, a part of the rabbit pyrogen test was replaced with an endotoxin quantitative test. Nevertheless, this replacement has not yet been achieved for all biologicals. The endotoxin test could not be performed on some preparations because of the presence of interfering substances [53, 54]. Therefore, a rabbit pyrogen test has been carried out for these biologicals. In this test, the causative agent of the fever has been recognized as the endotoxin in some biologicals. Thus, switching to a method of directly quantitating endotoxins was introduced as an alternative for the rabbit pyrogen test. Nonetheless, because the ATT evaluates the weight loss of animals, it is difficult to determine from body weight changes what types of molecular signaling pathways or physiological reactions have been affected. Therefore, it has not been easy to develop an *in vitro* assay as an alternative for the ATT.

We have tried to create a safety evaluation system for an influenza vaccine using the genomics technology in animal models [22–25]. The marker genes identified in animal experiments are believed to be involved in the body weight loss of animals after WPV injection [25]. When considering an alternative assay, phenotypic changes in animals, such as body weight loss, cannot be assessed in cultured cells. On the contrary, marker genes linked to these bioactivities can be identified at the cultured-cell level. Biomarker genes can make it possible to link cellular data with biological reactions observed in animal phenotypes. We are currently working on demonstrating the usefulness of marker genes and their expression mechanisms. Most of the marker genes are involved in an immune response and are related to type 1 IFN signaling and innate immune responses [39]. According to these findings, it is possible that the usefulness of biomarker genes evaluated in animals can be extrapolated to cultured cells, if such cell lines as peripheral blood mononuclear cells, immune cells, and alveolar epithelial cell lines are employed in the assays. If an alternative (*in vitro* method) for the ATT and LTT is developed, it will be possible to secure the safety and quality of the current ATT and LTT by animal-free testing. This approach is expected to reduce the number of animals tested and to shorten the testing period.

increase. This increase in the WBC count could not be predicted from the biomarker gene expression levels. The possible reason is that multiple linear regression analysis was performed on the animals inoculated with vaccines with leukopenic activity. The physiological association of the extracted biomarker gene with leukopenia could be another reason for this phenomenon. We have identified biomarker genes highly correlating with leukocyte counts in mathematical terms, ignoring the functions of the genes. There is a report that apoptosis of leukocytes caused by type 1 interferons (IFNs) could be a mechanism underlying the WBC count reduction by WPVs [47]. The marker gene set contains many genes related to type 1 IFN. By contrast, in model 4 (**Table 2**), more than a half of the genes related to type 1 IFN were omitted because they lacked predictability. As a result, it is conceivable that a correlation cannot be obtained from only a simple expression level because of the time lag between gene expression and actual leukocyte depletion; furthermore, a gene itself forms a

**7. Possibility of application of genomic approaches to** *in vitro* **safety** 

it has not been easy to develop an *in vitro* assay as an alternative for the ATT.

We have tried to create a safety evaluation system for an influenza vaccine using the genomics technology in animal models [22–25]. The marker genes identified in animal experiments are believed to be involved in the body weight loss of animals after WPV injection [25]. When considering an

Currently, safety evaluation and parts of quality control of vaccines are carried out in animal experiments. The ATT and LTT are representative tests in this regard; they have been practiced for more than 50 years [48]. Due to the nature of the test, the ATT excludes the unintended toxicity of the vaccine; the test of lots having reactogenicity different from that of other reference lots is a final confirmatory test designed to prevent serious toxicity in humans [21]. Actually, the ATT has been incorporated into the lot release of vaccines, especially for inactivated influenza vaccines in Japan [19]. On the other hand, tests on animals are being replaced with *in vitro* evaluation systems involving cultured cells, from the viewpoint of animal welfare and cost [48]. In the safety evaluation of chemically synthesized medicines, there has been a notable development: human cultured hepatocytes and cardiomyocytes prepared from induced pluripotent stem cells [48–52]. The benefit of the *in vitro* evaluation system is not only the reduction in the number of animals used, but also the possibility of using human tissue or fluid samples, so that extrapolation to humans can be expected. For biologicals, a part of the rabbit pyrogen test was replaced with an endotoxin quantitative test. Nevertheless, this replacement has not yet been achieved for all biologicals. The endotoxin test could not be performed on some preparations because of the presence of interfering substances [53, 54]. Therefore, a rabbit pyrogen test has been carried out for these biologicals. In this test, the causative agent of the fever has been recognized as the endotoxin in some biologicals. Thus, switching to a method of directly quantitating endotoxins was introduced as an alternative for the rabbit pyrogen test. Nonetheless, because the ATT evaluates the weight loss of animals, it is difficult to determine from body weight changes what types of molecular signaling pathways or physiological reactions have been affected. Therefore,

complicated network.

122 Influenza - Therapeutics and Challenges

**evaluation methods**
