Characterization of Flagellin-Functionalized Liposomes as a Vaccine Carrier and Adjuvant.

Abstract

Since the recognition that the adjuvant capacity of flagellin is better harnessed when both flagellin and the antigen are delivered to the same cell, there has been a need to exploit flagellin in ways that fulfill this constraint. We propose a liposomal delivery system functionalized with Salmonella typhimurium flagellin (fliC) as a way to meet this need. Our goal is to characterize the fliC-functionalized liposome as a vaccine adjuvant and evaluate its ability to target cells expressing Toll Like receptor 5 to enhance the vaccine potential of a liposome-encapsulated antigen. Proinflammatory cytokine secretion and preferential cell association were evaluated in murine alveolar macrophage cell line and bone marrow-derived macrophages in vitro. Caspase-1 activation and IL-1β secretion were used to determine inflammasome activation in studies employing LLO to gain cytosolic access. After a prime-boost immunization regimen, humoral and CD8+ T cell adjuvant effect of functionalized liposomes in vivo were determined by quantifying antigen-specific IgG1 and IgG2c and tetramer staining of antigen-specific CD8+ T cells. We report that fliC-functionalized liposomes are able to elicit the proinflammatory cytokine, IL-6, with comparable efficacy to soluble protein in a TLR5-mediated manner from an alveolar macrophage cell line but not from bone marrow-derived macrophages. FliC-functionalized liposomes also demonstrate the capacity to preferentially associate with flagellin-responsive cells, enhance MHC class I –restricted peptide presentation in vitro, and elicit IgG1 and CD8+ T cell response specific to liposome-encapsulated antigen. Using LLO-encapsulating flagellin-bearing liposomes, we demonstrate that fliC delivery to the cytosol enhances inflammasome activation and fliC-functionalized LLO liposomes are able to stimulate antigen-specific IgG1 in immunized mice. The physicochemical stability of the flagellin-functionalized liposome and the immune profile it elicits recommend fliC-functionalized liposomes as feasible for vaccine carrier and adjuvant function.