Preservation of the immunogenicity of dry-powder influenza H5N1 whole inactivated virus vaccine at elevated storage temperatures

Abstract

Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20 degrees C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40 degrees C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40 degrees C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40 degrees C may also point to an extended shelf-life when stored at 4 degrees C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.

Fig. 1

Antibody responses induced by liquid and freeze-dried WIV vaccines before and after storage at ambient temperature. Serum H5N1-specific IgG titers were determined by ELISA and are given as geometric mean titers + standard error of the means (SEM), before storage, and after 3 months and 1 year storage (a). The serum capacity to inhibit agglutination of guinea pig erythrocytes by H5N1 vaccine virus is given in geometric mean HI titers + SEM, before storage, and after 3 months and 1 year of storage (b). Immunization groups are plotted on the x axis with the vaccines’ actual storage temperature in degrees Celsius: liquid liquid WIV, FD freeze-dried WIV without sugar, FDT freeze-dried WIV with trehalose, FDI freeze-dried WIV with inulin. HI titers below the detection limit were assigned half the value of the lowest detectable serum dilution, which was 8. *p < 0.05, **p < 0.01. nd not determined

Fig. 2

Effect of storage duration on the vaccines capacity to induce antibody responses. Pooled sera of each group of mice immunized at different time points with liquid or freeze-dried WIV vaccine were tested simultaneously on H5N1-specific IgG or hemagglutination-inhibiting capacity. Bars represent the average values of duplicate determinations (deviations were less than 0.2). Immunization groups are plotted and labeled as in Fig. 1. Storage duration: 0 day (white bars), 3 months (gray bars), 1 year (black bars). nd not determined

Fig. 3

Effect of high storage temperature on the immunogenic potency of the vaccines. Vaccines were stored for 3 months at 40°C and subsequently injected in mice. Serum H5N1-specific IgG titers (a) and HI titers (b) were determined, and are shown as geometric mean titers + SEM. Immunization groups are labeled as in Fig. 1. The group immunized with liquid WIV, which had not been subjected to the storage experiment, serves as a standard. *p < 0.05 when compared with the standard; ^# p < 0.05 ^## p < 0.01 when compared with liquid WIV stored at 40°C

Fig. 4

Effect of storage temperature on the vaccines’ capacity to induce antibody responses. Pooled sera of each group of mice immunized with liquid or freeze-dried WIV vaccine, which had been stored for 3 months at different temperatures, were tested simultaneously on H5N1-specific IgG or HI capacity. Bars represent the average values of duplicate determinations (deviations were less than 0.2). Immunization groups as in Fig. 1. Storage temperatures: 4°C (white bars), 20°C (gray bars), 40°C (black bars)