The immunogenicity of WIV vaccine depends on the presence and integrity of the HA proteins and an intact viral particle structure (42
). The HA antigens provide the B-cell epitopes and define the specificity of the antibody response, while the viral particle acts as a vehicle for the vaccines primary immunopotentiating agent, the viral single-stranded RNA (ssRNA). Its immunopotentiating activity is exerted through binding to Toll-like receptor 7 (TLR7) in the endosomes of recipient host cells, and triggering of the innate immune system (41
). To a large extent, this mechanism accounts for the superior immunogenicity of WIV compared to split-virus formulations or subunit vaccines, which do not contain intact viral RNA (41
). TLR7 signaling also determines the response type, being characteristically of a Th1 type for WIV, with a high amount of IgG2a/c antibodies in mice.
In liquid WIV vaccine, the antigenic properties of HA and the immunopotentiating activity provided by the ssRNA appear to be remarkably stable. Our results show that the immunogenicity of WIV vaccine stored in buffer solution at 20°C remained well preserved with minimal loss of activity over a period of at least 12 months. In contrast, seasonal subunit vaccine (H3N2) stored in buffer solution at ambient (25°C) temperature was reported to lose potency already over a period of 12 to 20 weeks (28
). Compared to subunit vaccine, WIV, therefore, appears to be more stable. Yet, it should be noted that the comparability of the two studies is limited since they differ in the virus strains used for vaccine production and the method by which vaccine stability was assessed (in vivo
)). At high storage temperature (40°C), the immunogenic potency of liquid WIV rapidly deteriorated. Progressive degradation of HA antigens and/or loss of intact viral particles, as reflected in the strongly reduced hemagglutination and hemolytic activity of the vaccine, may likely be the cause. Yet, the antibody response remained Th1 skewed, which may indicate that a small amount of viral particles escaped degradation, as it was shown previously that even a very low dose of viral particles is sufficient for Th1 skewing of the response to WIV vaccine (44
At high storage temperature, dry-powder formulations were superior to liquid WIV, as reported by others (29
). With the use of sugar stabilization no substantial loss of immunogenicity was observed after storage of freeze-dried WIV for 3 months at 40°C. Furthermore, sugar stabilization played a critical role in preserving the Th1-skewing capacity of the vaccine during freeze-drying and subsequent storage. Freeze-drying in absence of sugar led to a mixed Th1/Th2 antibody response, which further shifted during storage at elevated temperature to an overt Th2 type response, while the characteristic Th1 phenotype of the WIV response was retained when sugars were used during freeze-drying. Sugar molecules obviously play a role in preserving effective TLR7 signaling by the ssRNA, probably by stabilizing the viral particle structure and protecting the viral ssRNA from degradation. Sugars are known to stabilize enveloped viruses during freeze-drying (49
), and trehalose and inulin have the capacity to stabilize lipid bilayers (35
). In freeze-drying experiments with ‘empty’ viral envelopes (virosomes), use of inulin was found to preserve the vesicular structure, while absence of sugar stabilization resulted in complete disintegration after rehydration (38
). The mode of action of trehalose and inulin is presumably by replacement of the water molecules situated in between the hydrophilic heads of the lipids. Hereby, a detrimental phase transition of the viral membrane upon rehydration is prevented and the vaccine particles are preserved (35
By assessing immunogenicity in vivo
we discovered an important role for sugar compounds in preserving not only the quantity but also the quality of the immune response to WIV after freeze-drying and storage. Previous storage studies that showed improved stability of freeze-dried WIV used the hemagglutination assay to determine vaccine stability, which provides a quantitative measure only (29
). The shift from a Th1 response to WIV before freeze-drying to a Th2 response to WIV after freeze-drying in absence of sugar-stabilization could not have been predicted from hemagglutination results, nor by other in vitro
tests that assess the integrity of the HA component, like the SRID. In vitro
HA stability tests alone are therefore insufficient to obtain a complete representation of WIV vaccine stability.
The efficacy of influenza vaccines is determined by the induction of an effective antibody response. The serum HI titer, which is a measure for the magnitude of the antibody response, is the principle correlate of protection used to evaluate the efficacy of current human seasonal and pre-pandemic influenza vaccines. For seasonal vaccines an HI titer of >40 is considered to be protective (52
). For the mouse model, no protective titer has been defined. Due to the intrinsic low immunogenicity of H5N1 virus and the single-dose immunization scheme the maximal titer reached in our experiments was 32. It has to be noted that immunogenicity in terms of HI and IgG titers does not necessarily correlate with protective efficacy. In the mouse model, the relative amounts of IgG1 and IgG2a (or IgG2c), expressed in the IgG2a/IgG1 ratio, appear to be decisive for protection (40
). IgG2a-dominated responses are clearly more effective than IgG1-dominated responses. Yet, in the absence of significant differences in HI and IgG titers and similar IgG subtype dominance, as observed, for example, for liquid WIV, FDI, and FDT during storage at ambient temperatures, protective efficacy is likely to be comparable.
The preservability of WIV vaccine is strongly increased by sugar stabilization and freeze-drying. Similarly, other formulations like subunit, split-virus, and virosome (31
) vaccines have been successfully freeze-dried and stabilized with sugars (summarized in (31
)). Yet, a combination of increased stability together with specific features of WIV which promote vaccine availability, like strong immunogenicity, dose sparing quality and manufacturing simplicity (46
), make dry-powder WIV an apt candidate formulation for pre-pandemic stockpiling. Based on the results of storage at 40°C it may also be predicted that dry-powder WIV will have a shelf-life exceeding that of liquid WIV, when kept under optimal refrigerated conditions (52
). An extended vaccine shelf-life could delay costly replacement of stocks when vaccines reach their expiry date. In case of a pandemic emergency, deployment of dry-powder vaccine stocks may be exerted without refrigeration unless the temperatures are extremely high. This could speed up pandemic intervention and reduce losses due to cold-chain failures.