Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine

Abstract

A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick injuries, and (iii) ensure better protection against pertussis as compared to vaccines containing acellular pertussis antigens. An approach to obtain a hexavalent vaccine might be reconstituting lyophilized polio vaccine (IPV-LYO) with liquid pentavalent vaccine just before intramuscular delivery. The potential limitations of this approach were investigated including thermostability of IPV as measured by D-antigen ELISA and rat potency, the compatibility of fluid and lyophilized IPV in combination with thimerosal and thimerosal containing hexavalent vaccine. The rat potency of polio type 3 in IPV-LYO was 2 to 3-fold lower than standardized on the D-antigen content, suggesting an alteration of the polio type 3 D-antigen particle by lyophilization. Type 1 and 2 had unaffected antigenicity/immunogenicity ratios. Alteration of type 3 D-antigen could be detected by showing reduced thermostability at 45°C compared to type 3 in non-lyophilized liquid controls. Reconstituting IPV-LYO in the presence of thimerosal (TM) resulted in a fast temperature dependent loss of polio type 1-3 D-antigen. The presence of 0.005% TM reduced the D-antigen content by ∼20% (polio type 2/3) and ∼60% (polio type 1) in 6h at 25°C, which are WHO open vial policy conditions. At 37°C, D-antigen was diminished even faster, suggesting that very fast, i.e., immediately after preparation, intramuscular delivery of the conceived hexavalent vaccine would not be a feasible option. Use of the TM-scavenger, l-cysteine, to bind TM (or mercury containing TM degradation products), resulted in a hexavalent vaccine mixture in which polio D-antigen was more stable.

Keywords: Hexavalent vaccine; Inactivated polio vaccine; Lyophilization; Rat potency; Thimerosal.

Fig. 1

Mean virus-neutralizing (VN) titers of serum from rats (n = 10) immunized with 1/45 human dose (panel A) or 1/15 human dose (panel B) of liquid IPV (L IPV, in black) or IPV-LYO (in red) directly after preparation or after subsequent two weeks storage at 45 °C. Individual VN titers specific for serotype 1 (circles), 2 (triangles) and 3 (diamonds) were shown. Mean values were depicted as horizontal line and error bars showed 95% confidence interval (CI) values. Asterisks indicate significant differences between groups (^*p < 0.01, ^**p < 0.001, ^***p < 0.0001).

Fig. 2

Rat potency of liquid IPV (L IPV) and IPV-LYO after incubation for a short period of time; 2 weeks at 45 °C or one month at 25 °C or 37 °C (panel A), or long period of time (6 months at 4 °C, 25 °C or 37 °C) (panel B). The rat potency is calculated based on a theoretical composition of 40, 8, 32 DU for type 1, 2, and 3, respectively.

Fig. 3

Stability of reconstituted IPV-LYO. Liquid IPV (L IPV, black bars), formulated (liquid) IPV prior to lyophilization (L IPV:form, striped bars), and reconstituted IPV-LYO (red bars) were incubated for 24 h at 4 °C, 37 °C or 45 °C. Subsequently, D-antigen recoveries were determined by ELISA, specific for type 1 (panel A), type 2 (panel B) and type 3 (panel C), and normalized for D-antigen recoveries directly after lyophilization. Mean values (n = 3) and SD are shown.

Fig. 4

Effect of pentavalent vaccine on IPV-LYO. IPV-LYO was reconstituted with pentavalent vaccine containing 0.005% of thimerosal at temperatures of 2-8 °C (closed circles, grey), 25 °C (closed squares, blue) or 37 °C (closed triangles, red) for up to 24 h. Subsequently, D-antigen recoveries were determined by ELISA, specific for type 1 (panel A), type 2 (panel B) and type 3 (panel C). Mean values (n = 3) and SD are shown.

Fig. 5

Effect of cysteine on the stability of IPV-LYO mixed with pentavalent vaccine. IPV-LYO was mixed with pentavalent vaccine pre-incubated for one hour in the absence or presence of l-cysteine. Subsequently, D-antigen recoveries directly after mixing (panel A) and after 24 h incubation at 37 °C (panel B) were determined by ELISA, specific for type 1 (black bars), type 2 (striped bars) and type 3 (red bars), and normalized for D-antigen recoveries of IPV-LYO prior to mixing and incubation. Mean values (n = 3) and SD are shown.