To date, the LTT, mouse body weight loss test, rabbit pyrogen test, and ATT have been used in Japan as safety tests for influenza vaccines1. However, conventional methods cannot assess the physiological effects of vaccines. In this study, we analyzed mouse urine to verify whether metabolomics analysis can be applied as a safety assessment tool for influenza vaccines. The reason for using urine is that it can be collected noninvasively; therefore, urine is also suitable for clinical applications13. Moreover, it is relatively easy to use urine for NMR analysis because it is less contaminated with proteins and lipids compared with serum and other biofluids13.

In this study, RE (comprised of WPV) and HAV were used as model vaccines. As reported, WPV induces leucopenia that peaks after 16 h and produces body weight loss after intraperitoneal injection into mice24. Additionally, in this study, RE vaccination produced marked leukocyte reduction and significant body weight loss compared with that observed in the SA group (Fig. 1b,c). Leukocyte reduction has been reported to occur in a manner dependent on IFNα production2. Thus, it has been thought that leukopenic toxicity caused by WPV occurs by an immunological mechanism2.

In the blood biochemical analyses, a slight increase in serum ALT levels, which is a marker of hepatocyte death, was observed (Fig. 2). Although it is difficult to determine whether fulminant liver injury is caused by RE administration on the basis of elevated levels of ALT (approximately average 60 U/l) (Fig. 2), it is presumed that mild damage or physiological stress was caused in the liver. In line with this result, autoimmune hepatitis caused by influenza vaccination in humans has been reported25. Furthermore, acute hepatitis and liver failure associated with influenza A infection in children has also been reported26. These reports suggest that influenza virus or its structural components (e.g., vaccine antigens) have the potential to affect liver functions or immune responses. However, the mechanism involved in hepatic dysfunction caused by influenza virus and its antigens has not been revealed. Further studies are needed to clarify the underlying mechanism. On the other hand, serum BUN levels were mainly used as a kidney injury biomarker. No significant increases in BUN were observed in mice administered 1.0 U of RE compared with that in mice in the SA group (Fig. 2). However, a decrease in BUN was observed in mice administered 1.0 U of RE (Fig. 2). Decreases in BUN are not notable changes in terms of kidney toxicity; however, a decrease in serum BUN levels is often observed in combination with low protein intake and decreased liver function27. Considering that ALT levels were increased by RE administration (Fig. 2), it is presumed that the decrease in BUN levels was caused by a decrease in liver function.

In addition to liver function, the serum glucose levels in the RE group showed a significantly decrease compared with those in the SA group (Fig. 2). However, the serum TG levels showed no marked change in the RE group (Fig. 2), suggesting that RE administration did not induce noticeable starvation. Because body weight loss in animals can be presumed to be caused directly by changes in food intake, it can be considered that the amount of activity in animals might be decreased because of an inflammatory reaction owing to leukopenic toxicity (Fig. 1b) and a slight increase in ALT levels (Fig. 2). However, in the HAV-administered group, there were no changes in blood biochemical markers suspected of being associated with toxicity (Fig. 2). Considering the above data, mild toxicity in the liver and changes in glucose metabolism caused by RE treatment were presumed to present according to the general toxicity markers.

The strain-specific toxicities of the influenza vaccines were examined. For the 3 different strains and 2 types of vaccine (HAV and WPV), strain-specific toxicities were not detected in terms of body weight changes, WBC changes and plasma ALT levels (Table 1 and Supplementary Fig. 1). Urine samples from vaccinated animals could not be analyzed in the present study because of the limited availability of samples for further analysis. However, there is a possibility that metabolomics analysis could detect toxicity that does not appear in the form of phenotypic changes (body weight changes, WBC changes and plasma ALT elevation); therefore, this should be investigated in future research.

Metabolites detected in urine have been known to be changed by damage to organs such as the liver and kidney and by cell metabolism13,14,15. Many studies have considered these changes as indicators of toxicity16,17,18,19. The liver is one of the major organs involved in endogenous metabolism (sugars, amino acids, and lipids) and the synthesis and excretion of endogenous metabolites. Metabolites excreted or released from hepatocytes are excreted in the urine through the portal vein17. The two metabolites that were increased in the RE administration group, 1-methylnicotinamide and trimethylamine N-oxide, are considered to be derived from the liver. Nicotinamide is known as niacin (vitamin B3) and is a component of nicotinamide adenine dinucleotide (NAD+) and its 2′-phosphate ester (NADP+), which is involved in the redox reaction in energy metabolism28. On the other hand, hepatocytes play a role in the metabolic excretion of nicotinamide. The methylation of nicotinamide is mainly performed in the liver29,30. Thus, 1-methylnicotinamide is produced in hepatocytes followed by its excretion in urine (Fig. 7a). Trimethylamine N-oxide is produced by the methylation of trimethylamine during the gut microbiome-mediated metabolism of amino acids31 (Fig. 7b). The metabolism of trimethylamine into trimethylamine N-oxide is mediated by flavin-containing monooxygenase (FMO) 3. Because FMO3 is strongly expressed in the liver32, trimethylamine N-oxide detected in urine would be mainly derived from the liver. The mechanism that causes these two metabolites to be increased in urine after RE vaccination is unclear. 1-Methylnicotinamide has been reported to be increased in urine from patients with liver cirrhosis compared with that in urine from healthy people33. It has been reported that the expression level of FMO3 in the liver is increased by liver injuries34,35. Because a slight increase in serum ALT levels was observed after RE administration (Fig. 2), it was also speculated that the increase in urinary trimethylamine N-oxide concentrations may have been due to the stressing of hepatocytes by RE administration. Collectively, it has been speculated that the elevation of 1-methylnicotinamide and trimethylamine N-oxide in urine may reflect slight liver damage caused by the administration of 1.0 U of RE. In terms of their usefulness as biomarkers for hepatic physiological changes, the sensitivity of these two metabolites is comparable to that of serum ALT levels (Figs 2 and 4). Therefore, urinary 1-methylnicotinamide and trimethylamine N-oxide levels cannot be used as highly sensitive biomarkers of hepatic physiological changes.

Figure 7 Representation of influenza vaccine-induced changes in urine metabolites related to nicotinamide (a) trimethylamine metabolism (b) and the tricarboxylic acid (TCA) cycle. (c) Each of the graphs shows a comparison of the urine metabolite levels from mice treated with sterilized physical saline (SA), 15 μg hemagglutinin (HA)-containing hemagglutinin split vaccine (HAV), or 1.0 U of the toxicity reference vaccine (RE). *p < 0.05 and **p < 0.01 (n = 5 in the SA or RE group and n = 3 in the HAV group). Full size image

Urinary metabolites include intermediates in the TCA cycle and glycolytic systems. Among them, some metabolites showed changes that depended on the type of influenza vaccine administered (Fig. 7c). The urinary pyruvate concentration showed a significant increase after the administration of 1.0 U of RE. On the other hand, there was no change in the urinary lactate concentration in any of the groups. This suggests that although glycolysis still took place after vaccination with 1.0 U of RE, anaerobic metabolism did not occur. Pyruvate is synthesized from acetyl-CoA, which is an intermediate in the citric acid cycle. Three metabolites were detected that serve as intermediates in the citric acid cycle (Fig. 7c). Although no significant changes were observed in these 3 metabolites because of vaccination, a slight tendency towards increases in the 2-oxoglutarate and succinate concentrations was observed in the group administered 1.0 U of RE (Fig. 7c). Thus, it is suggested that during RE vaccination, glycolysis proceeds, and as a result, citric acid cycle metabolism may be slightly enhanced.

On the other hand, two urinary metabolites showed a significant reduction specifically in the 15 μg HAV vaccine-administered group: N-carbamoyl β-alanine and creatine (Fig. 4). N-carbamoyl β-alanine is metabolized by the urea cycle into arginine36,37. In the same manner as N-carbamoyl β-alanine, creatine is also metabolized into arginine by the urea cycle38. Considering that the urea cycle generates fumarate for the TCA cycle35, the decrease of both metabolites result in enhancing the TCA cycle. However, metabolites related to the TCA cycle were not increased in the HAV-treated group (Fig. 7c). Because of this, it is inferred that decreases in urine N-carbamoyl β-alanine and creatine in the HAV group did not directly reflect the enhancement of the TCA cycle. The reasons for these metabolite reductions by HAV vaccine administration could not be clarified by the metabolomics analysis conducted in this study.

By subjecting the detected metabolite concentrations to hierarchical clustering analysis, it was possible to classify the administered vaccines by profiling the metabolites in urine (Fig. 5). Two clusters, which were divided into the SA and HAV group and the RE group, were generated (Fig. 5). On the other hand, some clusters that did not conform to the vaccine types were also found, suggesting that individual differences among animals in terms of urine metabolites were influential in the hierarchical cluster analysis (HCA). This indicates that it was difficult to utilize urinary metabolites as highly sensitive indicators of the safety of influenza vaccines as an alternative to mouse body weight loss tests and LTTs.

This study is the first to report that changes in the profiles of metabolites in urine are caused by influenza vaccination and that liver-derived metabolites are responsible for leukopenic toxicity and body weight loss after RE administration. One new finding was that RE caused liver metabolic changes, suggesting that the liver is the principal organ impacted by the toxicological and physiological effects of influenza vaccines. This knowledge will aid in the development of alternative safety test methods for vaccines, as typified by the LTT and mouse body weight loss test, by utilizing the metabolomics analysis of in vitro cultured hepatocytes.