Co-delivery of polyinosinic:polycytidylic acid and flagellin by poly(lactic-co-glycolic acid) MPs synergistically enhances immune response elicited by intranasally delivered hepatitis B surface antigen

The aim of the present work was to investigate the synergistic effect between toll-like receptor (TLR) 3 ligand polyinosinic:polycytidylic acid (pI:C) and TLR5 ligand flagellin (FLN) on immune responses induced by nasally delivered hepatitis B virus surface antigen (HBsAg). Mannan and chitosan oligosaccharide-modified, pH-responsive poly(lactic-co-glycolic acid) (MC-PLGA) microparticles (MPs) containing HBsAg, FLN, pI:C or both ligands were prepared with a double-emulsion method. In vitro uptake experiments show that cellular uptake of MC-PLGA MPs by macrophages was through energy-dependent, receptor-mediated endocytosis mechanism. After uptake of MPs by macrophages, MC-PLGA MPs existed both in the endo-some and in the cytoplasm. FLN and pI:C in solution or MP formulation could synergize to activate macrophages and induce higher pro-inflammatory cytokines interleukin (IL)-6, IL-12, interferon-γ and anti-inflammatory cytokines IL-10 compared to single TLR ligand (P<0.05). In vivo immunogenicity studies indicated that co-delivery of FLN and pI:C within MC-PLGA MPs synergistically induced higher serum anti-HBsAg IgG levels and Th1 cytokine levels compared with MC-PLGA MPs encapsulated single TLR ligand plus MPs encapsulated HBsAg (P<0.05). These results suggest that synergic TLR3 and TLR5 stimulation might be a promising novel tool for nasally delivered HBsAg.