Preparation and evaluation of a freeze-dried oral killed cholera vaccine formulation

Different oral liquid cholera vaccines have proved to be safe and effective, but their formulations present problems for use in low-income countries, since large package volumes have to be transported and cold chain maintenance is required. A solid state formulation would here be more advantageous, and consequently, the possibility to develop a dry cholera vaccine formulation by freeze-drying was investigated. The ability of sucrose, trehalose and mannitol to provide process stabilization during freeze-drying was tested on a formalin-killed whole-cell Vibrio cholerae model vaccine. A matrix of sucrose or trehalose prevented bacterial aggregation, preserved cell morphology and maintained practically completely the protective lipopolysaccharide (LPS) antigen on the cell surface and its reactivity with specific antibody in vitro. After reconstitution, this formulation also retained the capacity to elicit a strong serum and gut mucosal anti-LPS antibody response in orally immunized mice, as compared to the corresponding liquid vaccine formulation. The full preservation of the in vivo immunogenicity was also maintained when the internationally widely licensed oral cholera vaccine Dukoral™, which comprises a cocktail of inactivated V. cholerae together with cholera toxin B-subunit (CTB), was freeze-dried using sucrose for stabilization. Thus, we present a process generating a dry oral inactivated whole-cell cholera vaccine formulation with attractive features for public health use in cholera-afflicted settings.

Graphical abstractFreeze-drying of inactivated whole-cell Vibrio cholerae strain JS 1569 as model vaccine was tested in the absence and presence of different excipients. The stabilizing effect was clearly dependent on the type and concentration of the excipient. It was found that a matrix of sucrose prevented aggregation and fragmentation of the bacteria during freeze-drying, preserved the morphology of the bacteria and practically completely the protective lipopolysaccharide (LPS) antigen on the cell surface and its reactivity with specific antibody in vitro. After being reconstituted this dry vaccine formulation also retained the capacity to elicit a strong serum as well as gut mucosal anti-LPS response in orally immunized mice, as compared to the corresponding liquid vaccine formulation.Figure optionsDownload full-size imageDownload as PowerPoint slide