Influenza vaccines might benefit from a dose sparing effect by bioneedles. Furthermore, while trehalose is present as lyoprotectant in the lyophilized vaccine formulations, it has shown no adjuvant activity in influenza vaccines before. The rate of vaccine dissolution and release may have had an effect on the immunogenicity. Bioneedles might induce a short-term depot effect, or alter the kinetics of antigen recognition and processing, resulting in an enhanced immunogenicity of the vaccine. Future studies, including biodistribution studies, are required to test these hypotheses. The vaccine antigens in the produced influenza bioneedles were expected to possess increased vaccine stability. The stability of the different types of influenza vaccines after lyophilization was assessed in vials. In order to retain the vaccine antigenicity after lyophilization and subsequent storage, trehalose was chosen as stabilizer. Trehalose is known for its excellent lyoprotective capacities in influenza vaccine formulations. Lyophilized vaccines had low residual moisture contents, positively affecting the glass transition temperature of the vaccine product. Glass transition temperatures calculated were above 95uC, indicating that the glassy matrix provided by trehalose was physically stable, and Nilotinib retained its glassy structure with low molecular mobility at the storage conditions evaluated in this study. Lyophilization of the influenza vaccines resulted in vaccine formulations that were heat stable at 60uC for at least one month. In sharp contrast, conventional liquid influenza vaccines showed a decrease in stability after one-month storage at 37uC. The liquid vaccines lost all HA1 content and antigenicity after storage at 60uC for one month, indicating that conventional liquid vaccines have limited heat stability. It should be noted that antigenic recoveries of freeze-dried vaccines dropped slightly after one-month storage at 37uC and 60uC, which could have an impact on immunogenicity of the product. However, a previous study by Geeraedts et al. concluded that storage of liquid WIV vaccine at 40uC for three months resulted in a significant loss of immunogenic potency of the vaccine, whereas lyophilized WIV vaccine remained stable under the same conditions. These results show a similar trend in vaccine degradation compared to this study, and demonstrates that freeze-dried influenza vaccines are still immunogenic after storage at elevated temperatures. In case of bioneedles, a previous study with hepatitis B antigen demonstrated that heat-stressed bioneedles induced similar immune responses compared to unstressed bioneedles, which underlines the stability of freeze-dried bioneedles. For the current study, it should be noted that the lyophilized vaccine product in the bioneedles might not behave exactly similar in terms of stability as the formulations lyophilized in vials. A previous study by Hirschberg et al. compared the heat stability of liquid tetanus toxoid with tetanus toxoid that was lyophilized in glass vials or bioneedles.