Adjuvant Interactions with Lipid Membranes and Their Effect on Cellular Immune Responses

Abstract

Adjuvants are commonly included in vaccines and have been invaluable in making them safer and more robust. Despite their prolific use, adjuvant mechanisms of action remain poorly understood. Many receptor-mediated mechanisms have been proposed for adjuvants, and many likely contribute to their mechanisms of action, but several adjuvants also interact with the plasma membrane. Although few have considered how lipid-mediated interactions contribute to adjuvanticity, previous studies suggested aluminum-based adjuvants (ABAs) have high affinity for sphingomyelin and cholesterol, which allowed them to activate dendritic cells exclusively through lipid sorting. This dissertation sought to understand how lipid interactions contribute to the immunostimulatory properties of adjuvants. The membrane interaction of Alhydrogel (AH) and Adju-Phos (AP) was initially investigated in a simple lipid monolayer representative of the outer leaflet of the plasma membrane. AH and AP interacted with the model monolayer and promoted lipid clustering, although the physiochemical properties of each adjuvant caused them to interact differently. In a more complex lipid system containing sphingomyelin and cholesterol, the lipid interaction behavior was consistent and revealed AH and AP stabilized sphingomyelin- and cholesterol-rich lipid domains even in the presence of an antigen. Lipid raft clustering observed in dendritic cells exposed to ABAs in vitro was reminiscent of domain clustering observed in the monolayer and corresponded to conditions which enhanced cell activation, suggesting membrane interactions and lipid sorting could indeed contribute to ABA mechanisms of action. Lipid-interactions were also considered while designing an adjuvant-based antigen-specific immunotherapy (ASIT). An MF59-analog (MF59a) made in our lab was selected to co-deliver ovalbumin and dexamethasone based on its ability to solubilize dexamethasone, extend its release, and enhance its membrane permeability and internalization. The combination of MF59a, ovalbumin, and dexamethasone inhibited several pro-inflammatory cytokines in dendritic cells and ovalbumin-educated splenocytes, and proved emulsion adjuvants could provide an ideal vehicle to create targeted, tolerizing ASITs. Therefore, lipid interactions can provide valuable insight while selecting the physiochemical properties of an adjuvant for pro- and anti-inflammatory applications. Our results provide compelling evidence that lipid interactions participate in adjuvant mechanisms of action, and should be considered when developing novel vaccines and adjuvants.