Liposomes as vaccine carriers. Incorporation of soluble and particulate antigens in giant vesicles.

Giant liposomes (mean diameter 5.5 microns) composed of egg phosphatidylcholine or distearoyl phosphatidylcholine, phosphatidyl glycerol, cholesterol and triolein were prepared by a double emulsion technique. They were then mixed with model particulate (killed Bacillus subtilis, and killed Bacille Calmette-Guérin) and soluble (tetanus toxoid) vaccines and freeze-dried. Rehydration of the powder resulted in the generation of vesicles of similar mean diameter and diameter range, containing up to 27% (mean value) of the materials used for entrapment. Separation of entrapped from non-entrapped material was carried out by sucrose gradient centrifugation (B. subtilis and BCG) or centrifugation at 600 x g (toxoid). Light microscopy of liposomes containing B. subtilis labelled with fluorescein isothiocyanate revealed the presence of bacteria in individual vesicles which, in separate studies, were also found to entrap latex particles (0.5 and 1.0 micron diameter). Bacteria-containing liposomes could be freeze-dried in the presence of trehalose with most (83-87%) of the entrapped material recovered within the vesicles on reconstitution with saline. Liposomes were also shown to retain quantitatively their content of B. subtilis and, to a lesser extent, toxoid in the presence of mouse plasma at 37 degrees C and in situ after intramuscular injection into mice, for up to 24 h. Since liposomes are known (Gregoriadis, G. (1990) Immunol. Today 11, 89) to act as immunological adjuvants and vaccine carriers, giant vesicles containing microbes (live or attenuated if needed since the conditions of entrapment are mild) and, when appropriate, soluble antigens, could be used as multiple vaccines to ensure simultaneous presentation of antigens to immunocompetent cells.