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Influenza becomes epidemic worldwide every year, and many individuals receive vaccination annually. Quality control relating to safety and potency of influenza vaccines is important to maintain public confidence. The safety of influenza vaccines has been assessed by clinical trials, and animal safety tests are performed to monitor the consistent quality between vaccines used for clinical trials and marketing; the biological responses in vaccinated animals are evaluated, including changes in body weight and white blood cell count. Animal safety tests have been contributing to the quality relating to the safety of influenza vaccines for decades, but improvements are needed. Although precise mechanisms involving biological changes in animal safety tests have not been fully elucidated, the application of cDNA microarray technology make it possible to reliably identify genes related to biological responses in vaccinated animals. From analysis of the expression profile of >10,000 genes of lung in animals treated with an inactivated whole virion influenza vaccine, we identified 17 marker genes whose expression patterns correlated well to changes in body weight and leukocyte count in vaccinated animals. In influenza HA vaccine-treated animals exhibiting subtle changes in biological responses, a robust expression pattern of marker genes was found. Furthermore, these marker genes could also be used in the evaluation of adjuvanted influenza vaccines. The expression profile of marker genes is expected to be an alternative indicator for safety control of various influenza vaccines conferring high sensitivity and short turnaround time. Thus, gene expression profiling may be a powerful tool for safety control of vaccines in the future.

Vaccines are administered to healthy humans, including infants, so the safety and efficacy must be very high. Therefore, evaluating vaccine safety in preclinical and clinical studies, according to World Health Organization guidelines, is crucial for vaccine development and clinical use. A change in the route of administration is considered to alter a vaccine’s immunogenicity. Several adjuvants have also been developed and approved for use in vaccines. However, the addition of adjuvants to vaccines may cause unwanted immune responses, including facial nerve paralysis and narcolepsy. Therefore, a more accurate and comprehensive strategy must be used to develope next-generation vaccines for ensuring vaccine safety. Previously, we have developed a system with which to evaluate vaccine safety in rats using a systematic vaccinological approach and 20 marker genes. In this study, we developed a safety evaluation system for nasally administered influenza vaccines and adjuvanted influenza vaccines using these marker genes. Expression of these genes increased dose-dependent manner when mice were intranasally administered the toxicity reference vaccine. When the adjuvant CpG K3 or a CpG-K3-combined influenza vaccine was administered intranasally, marker gene expression increased in a CpG-K3-dose-dependent way. A histopathological analysis indicated that marker gene expression correlated with vaccine- or adjuvant-induced phenotypic changes in the lung and nasal mucosa. We believe that the marker genes expression analyses will be useful in preclinical testing, adjuvant development, and selecting the appropriate dose of adjuvant in nasal administration vaccines.

Although vaccines are routinely used to prevent infectious diseases, little is known about the comprehensive influences caused by vaccines. In this study, we showed, using comprehensive gene expression analysis, that pertussis vaccine affected many genes in multiple organs of vaccine-treated animals. In particular, lung was revealed to be the most suitable target to evaluate pertussis vaccine toxicity. The 13 genes identified from the analysis of vaccine-treated lung at day 1 showed a clear dendrogram corresponding to pertussis vaccine toxicity. Furthermore, quantitative analysis of these genes revealed a positive correlation between their respective expression levels and the degree of toxic effects observed in samples that had been treated with various doses of reference pertussis vaccines. The quantification of this 13 gene-set is an indicator of the vaccine toxicity-related reaction.

Conventional animal tests such as leukocytosis promoting tests have been used for decades to evaluate toxicity of pertussis vaccine. Here, we examined gene expression in relation to the vaccine toxicity using a DNA microarray. Comparison of conventional animal test data with the DNA microarray-based gene expression data revealed a gene expression pattern highly correlated with leukocytosis in animals. Of 10,490 rat genes analyzed, two genes, alpha1-acid-glycoprotein (Agp) and hemopexin (Hpx), were found up-regulated by the toxin administration in a dose-dependent manner (assayed by a quantitative PCR based on the microarray). Variation of the gene expression was very small amongst the test animals, and the results were highly reproducible. These findings suggest that gene expression analysis of vaccine-treated animals can be used as an accurate and simple method of pertussis vaccine safety assessment.