It is still unknown what factors induce these biomarkers in the HAv from manufacturer B. Further studies are needed to determine whether our biomarkers could predict the toxicity of influenza Oleamide vaccine by using different formulations of HAv. Using biomarkers from any grade characterized in this study, we could also predict the safety of influenza vaccines within 2 days whereas the conventional animal use safety test, ATT requires 7 days for evaluating batchto- batch consistency and vaccine safety. Further studies are needed to determine how these biomarkers can be used to evaluate the safety of HAv. To set the percent limit of up-regulation of each biomarker, it might be helpful to compare another conventional test such as LTT ] as well as a comparison of failed batches of HAv. LTT evaluates the peripheral leukocyte number reduction rate compared with WPv. In general, WPv induces a strong loss of peripheral leukocyte numbers 16 hours after WPv administration in mice. The test criteria of LTT is that the loss of leukocyte numbers in test samples must be no greater than 20% compared with a reference toxic vaccine such as WPv or less than 50% of SAtreated mice. These criteria may be applicable to set our biomarker expression limit. Further validation is required to set the limit the gene expression level. Influenza is a socially important infectious disease that causes seasonal flu outbreaks worldwide and has a pandemic status. Correspondingly, many types of influenza vaccine, have been developed to ensure efficacy and reduce toxicity. While some NVP 231 adjuvants have been developed and used to amplify vaccine efficacy, the safety of adjuvants is still of concern. Recently, several adjuvants developed and licensed for use only in pandemic influenza vaccines were under investigation for the occurrence of narcolepsy in vaccinated children in European countries. Conventional safety tests could be used to evaluate the safety of these vaccines, but it is still difficult to predict the safety and toxicity of influenza vaccines, adjuvants and additives. We demonstrated that usage of system biological approaches to evaluate safety might revolutionize vaccine testing methods. Most of the previously identified biomarkers were up-regulated and correlated with influenza infection, interferon responses, antigen presentation and antibody production. In addition, we found that several biomarkers, Cxcl9, Trafd1, and C2 were candidates for evaluating differences between alum-adjuvanted influenza vaccines and nonadjuvanted vaccines. Further studies, using several adjuvants, are needed to confirm the feasibility of these biomarkers in evaluating adjuvant safety. In addition to whole transcriptome analysis of vaccinated animals, recent advances in genome research enabled the acquisition of whole transcriptional data from vaccinated individuals and identification of gene expression after immunization with vaccines to yellow fever, measles, tularemia and tuberculosis. With a focus on the influenza vaccine, Bucasas et al. reported a 494 gene set, including biomarkers identified in our previous study that strongly correlated with antibody responses in humans. Wei et al. reported gene expression differences between HAv and live attenuated influenza vaccine.