In contrast, the produced split and subunit vaccines showed heterodisperse particle sizes, indicating that the vaccines contained viral components in a less organized fashion. WIV and virosome vaccine retained their particle size after freeze-drying, while split and subunit vaccines displayed still a heterodisperse particle size. These differences however did not influence the survival of the trehalose/HBS stabilized vaccines after freeze-drying in vials; a complete recovery of HA1 protein and vaccine antigenicity was observed after freeze-drying. This indicates that influenza vaccine containing bioneedles can be produced without the loss of vaccine structure and antigenicity. Immunization of C57BL/6 mice with all influenza vaccine-filled bioneedles induced strong systemic humoral responses. Serum HI titers induced by vaccines delivered by bioneedles were higher than the HI titers induced by intramuscular administered subunit vaccine and comparable to HI titers induced by intramuscular administered WIV vaccine. Overall, bioneedle delivered influenza vaccines induced similar HI titers as their liquid counterparts, indicating that this alternative method of administration could be used for influenza vaccines. The HI titers induced after i.m. immunization with WIV or subunit vaccine were comparable to those found in the study by Hagenaars et al. This might indicate that in terms of HI titers, subcutaneous and intramuscular administered liquid influenza vaccines elicit comparable responses. The current correlate of protection for human influenza vaccines is indicated by an HI titer higher than 40 in humans after vaccination. However, previous studies have shown that cellular immune SCH772984 ERK inhibitor responses are important as well, especially for pandemic vaccine candidates. Additionally, it is believed that cellular responses may play an important role in the elderly, in which vaccines fail to elicit adequate antibody responses. Analysis of the cellular immune responses and IgG subtype profiling revealed that subunit, split and virosomal vaccines induced comparable mixed Th1/Th2 responses. In contrast, immunization with WIV vaccines resulted in an IgG subtype profile that was significantly skewed towards IgG2c, indicating a cellular response skewed towards Th1. This observation can be explained by the presence of viral ssRNA in the WIV vaccine, which is not present in subunit, split and virosomal vaccines. Administration by bioneedles did not alter the cellular or IgG subtype profile significantly, which indicates that the route and method of bioneedle administration have no impact on the quality or type of immune response induced by the influenza vaccines. From the data it can be concluded that influenza vaccine-filled bioneedles can induce immune responses that are similar to responses induced by subcutaneous and intramuscular influenza vaccines. Considering the virosome vaccine, there is an indication that bioneedles might actually improve the immunogenicity of influenza vaccines.