Strategies to design delivery vehicles are critical in modern vaccine-adjuvant development. Nanoparticles (NPs) encapsulating antigen(s) and adjuvant(s) are promising vehicles to deliver antigen(s) and adjuvant(s) to antigen-presenting cells (APCs), allowing optimal immune responses against a specific pathogen. In this study, we developed a novel adjuvant delivery approach for induction of efficient in vivo immune responses. Polyethylenimine (PEI) was physically conjugated to poly(lactic-co-glycolic) acid (PLGA) to form PLGA/PEI NPs. This complex was encapsulated with resiquimod (R848) as toll-like receptor (TLR) 7/8 agonist, or monophosphoryl lipid A (MPLA) as TLR4 agonist and co-assembled with cytosine–phosphorothioate–guanine oligodeoxynucleotide (CpG ODN) as TLR9 agonist to form a tripartite formulation [two TLR agonists (inside and outside NPs) and PLGA/PEI NPs as delivery system]. The physicochemical characteristics, cytotoxicity and cellular uptake of these synthesized delivery vehicles were investigated. Cellular viability test revealed no pronounced cytotoxicity as well as increased cellular uptake compared to control groups in murine macrophage cells (J774 cell line). In the next step, PLGA (MPLA or R848)/PEI (CpG ODN) were co-delivered with ovalbumin (OVA) encapsulated into PLGA NPs to enhance the induction of immune responses. The immunogenicity properties of these co-delivery formulations were examined in vivo by evaluating the cytokine (IFN-γ, IL-4, and IL-1β) secretion and antibody (IgG1, IgG2a) production. Robust and efficient immune responses were achieved after in vivo administration of PLGA (MPLA or R848)/PEI (CpG ODN) co-delivered with OVA encapsulated in PLGA NPs in BALB/c mice. Our results demonstrate a rational design of using dual TLR agonists in a context-dependent manner for efficient nanoparticulate adjuvant-vaccine development.

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In the current study, soluble proteins prepared from 200 mature Echinococcus granulosus and protoscolices of sheep hydatid cysts were applied to immunize sheep and mice respectively. The samples were mechanically homogenized in a blender, sonicated and the final yield was maintained at -20 degrees C until analysis. Hydatid fluid was isolated from liver or lung of sheep under sterile conditions. In the first experiment, 15 mice were randomly allocated to three groups of five mice each. Each mouse in groups 1 and 2 was immunized with 100 microg of hydatid fluid and protoscolex proteins in 100 microl of phosphate-buffered saline (PBS) and emulsified with an equal volume of Freund's complete adjuvant (FCA) respectively. The mice of group 3 were immunized with adjuvant in PBS. The mice were boosted 4 weeks after the first vaccination with the same preparation except that FCA was replaced by Freund's incomplete adjuvant (FIA). In the second experiment, eight male or female lambs 4-6 months of age, were allocated to two groups of four lambs each. Each lamb in the test group was vaccinated subcutaneously in the neck with a 2-ml dose of vaccine (1 mg of whole body protein of E. granulosus dissolved in 1 ml of PBS plus 1 ml of FCA). Control lambs were vaccinated with adjuvant in PBS. Lambs were boosted the same way as in the first experiment. Three weeks after the second vaccination, each mouse and lamb received a challenge infection with 2000 protoscolices intraperitoneally and each lamb additionally received 10 gravid E. granulosus. All mice and sheep were killed after 7 months and examined for hydatid cysts. In these studies, protective immunity was induced in mice with protoscolex protein and with hydatid fluid, and in sheep with whole-body homogenate of E. granulosus and the levels of protection afforded were found to be 72.1, 82.6 and 90.9% respectively.

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