Abstract
Cochleates are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. Proteoliposomes from bacteria may also be used as a source of negatively charged components, to induce calcium-cochleate formation. In this study, proteoliposomes from V. cholerae O1 (PLc) (sized 160.7±1.6 nm) were transformed into larger (16.3±4.6 µm) cochleate-like structures (named Adjuvant Finlay Cochleate 2, AFCo2) and evaluated by electron microscopy (EM). Measurements from transmission EM (TEM) showed the structures had a similar size to that previously reported using light microscopy, while observations from scanning electron microscopy (SEM) indicated that the structures were multilayered and of cochleate-like formation. The edges of the AFCo2 structures appeared to have spaces that allowed penetration of negative stain or Ovalbumin labeled with Texas Red (OVA-TR) observed by epi-fluorescence microscopy. In addition, freeze fracture electron microscopy confirmed that the AFCo2 structures consisted of multiple overlapping layers, which corresponds to previous descriptions of cochleates. TEM also showed that small vesicles co-existed with the larger cochleate structures, and in vitro treatment with a calcium chelator caused the AFCo2 to unfold and reassemble into small proteoliposome-like structures. Using OVA as a model antigen, we demonstrated the potential loading capacity of a heterologous antigen and in vivo studies showed that with simple admixing and administration via intragastric and intranasal routes AFCo2 provided enhanced adjuvant properties compared with PLc.
Highlights
Cochleates (Co) are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations such as calcium
We have expanded the Co formation strategy to other microorganisms such as Vibrio cholerae to develop a novel mucosal formulation against this and related enteric pathogens. This was achieved by preparing proteoliposomes from V. cholerae O1 El Tor Ogawa, C7258 strain and transforming them into new tubular structures designated AFCo2 [6]
The aim of this paper was to evaluate the molecular organization of AFCo2, using electron microscopy to gain a greater understanding of the formation of the membranes in AFCo2, to evaluate the interaction of AFCo2 with a heterologous antigen (OVA) and to assess the immune response induced when the antigen was admixed and administered via intragastric and nasal routes
Summary
Cochleates (Co) are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations such as calcium. They have a defined multilayered structure consisting of a solid, lipid bilayer sheet rolled up in a spiral. We have expanded the Co formation strategy to other microorganisms such as Vibrio cholerae to develop a novel mucosal formulation against this and related enteric pathogens This was achieved by preparing proteoliposomes from V. cholerae O1 El Tor Ogawa, C7258 strain (called PLc) and transforming them into new tubular structures designated AFCo2 [6]
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