Category: Preclinical Development
Purpose: To increase vaccine immunogenicity, modern vaccines incorporate adjuvants, which serve to enhance immune cross-protection, improve humoral and cell-mediated immunity, and promote antigen dose sparing. Pattern recognition receptors (PRRs), including the Toll-like receptor (TLR) family are promising targets for development of agonist formulations for use as vaccine adjuvants. Combinations of co-delivered TLR4 and TLR7/8 ligands have been demonstrated to have synergistic effects on innate and adaptive immune response. Here, we create liposomes which stably co-encapsulate, CRX-601, a TLR4 agonist, and UM-3004, a lipidated TLR7/8 agonist, within the liposomal bilayer in order to achieve adjuvant co-delivery and induce in vitro and in vivo immune synergy.
Methods: To formulate optimal TLR4 and TLR7/8 agonist co-encapsulated liposomes, the total amount of lipid excipients was tuned to maximize CRX-601 and UM-3004 incorporation and recovery. The resulting CRX-601 and UM-3004 co-encapsulated liposomes were characterized by physical properties, including size, polydispersity, surface charge, and recovery of agonist after filtration. The ratio of CRX-601 to UM-3004 was tuned to maximize immune synergy, which was measured in vitro by innate TNFa, IL-1b, IFNa, and IL-12p70 release from human peripheral blood mononuclear cells (hPBMCs) and in vivo by antigen-specific total IgG, IgG1, and IgG2a antibody responses to the monovalent detergent-split influenza vaccine A/Victoria/210/2009-H3N2 (A/Vic) after primary and secondary immunization in BALB/c mice. To determine if co-encapsulation of TLR agonists was advantageous compared to admixtures, TLR co-encapsulated liposomes were compared to admixtures of single agonist liposomes all in vitro and in vivo studies.
Results: While increasing total amount of the lipid excipients 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol reduced particle size and polydispersity, 40:10 mg/mL DOPC:cholesterol lead to maximum recovery of both CRX-601 (85.4 ±3.3%) and UM-3004 (77.7 ±4.9%) after sterile filtration. Both co-encapsulation and admixed liposomes containing CRX-601 and UM-3004 demonstrate a synergistic increase in IL-12p70 cytokine output in vitro in hPBMCs which is absent in single agonist formulations. Target molar ratios of 1:1, 10:1, and 100:1 TLR7/8:TLR4 all demonstrate synergistic production of IL-12p70 in both co-encapsulated and admixed formulations. Strikingly, a liposome formulation of co-encapsulated CRX-601 and UM-3004 leads to a 14-fold increase in IgG2a titer when compared to liposomal vehicle + A/Vic (p < 0.01) and a 6.5-fold increase in titer when compared to an admix of CRX-601 and UM-3004 liposomes + A/Vic (p < 0.01) after a single immunization.
Conclusion: Liposomal co-encapsulation may be a useful and flexible tool for vaccine adjuvant formulation of TLR4 and TLR7/8 agonists. Co-encapsulation of CRX-601 and UM-3004 within the bilayer of a liposome can promote synergistic IL-12p70 production and antigen-specific IgG2a production which leads to improved early immune response in a vaccine setting. Importantly, innate and adaptive immune responses can be altered by adjuvant formulation strategy which can result in more potent and efficacious subunit vaccines without altering agonist components.
Kristopher Short– Missoula, Montana
Kristopher Short– Missoula, Montana
Shannon Miller– Missoula, Montana
Lois Walsh– Missoula, Montana
Van Cybulski– Missoula, Montana
Hélène Bazin– Missoula, Montana
Jay Evans– Research Professor, Center for Translational Medicine, Department of Biological Sciences, University of Montana, Missoula, Montana
David Burkhart– Research Professor, Center for Translational Medicine, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana