The stability during storage of outer membrane vesicles (OMVs) of Neisseria meningitidis group B was studied. Three types of OMVs were compared for their stability, containing either one (monovalent) or three different PorA subtypes (trivalent), the latter with and without class 4 outer membrane protein (OMO, RmpM). Aqueous formulations were stored freeze-dried (4 degrees C), frozen (-70 degrees C) and in liquid form at 4, 37 and 56 degrees C. Physico-chemical properties and immunogenicity of the OMVs as well as PorA conformation and antigenicity (P1.7-2,4, the subtype present in all formulations) were monitored during 1 year. At -70 or 4 degrees C, the structure and immunogenicity of OMVs was preserved. Storage of OMVs at high temperatures (37 or 56 degrees C) induced destruction of the OMV structure and denaturation of PorA, followed by chemical degradation. Immunogenicity decreased or was lost completely. Changes observed in the fluorescence spectra of degraded OMVs were also seen in tryptophan (Trp) and tyrosine (Tyr) derivatives incubated at 56 degrees C, indicating the occurrence of chemical degradation of tryptophan and tyrosine residues in PorA. Trivalent OMVs were slightly more stable at 37 degrees C than monovalent OMVs as assessed by in vitro methods, but these differences did not result in differences in the immunogenicity. The stability of trivalent OMVs was not affected by the presence of RmpM. Both trivalent and monovalent OMVs could be freeze-dried with preservation of their immunogenicity. In conclusion, OMVs are sensitive to elevated temperatures, but are stable in the frozen or freeze-dried state or when stored at 4 degrees C in the liquid state.