Method of manufacturing CAF®09 liposomes affects immune responses induced by adjuvanted subunit proteins

The ability to induce antigen-specific CD4⁺ and CD8⁺T-cell responses is one of the fundamental requirements when developing new efficacious vaccines against challenging infectious diseases and cancer. However, no adjuvants are currently approved for human subunit vaccines that induce T-cell immunity. Here, we incorporated a Toll-like receptor 4 agonist, i.e., the ionizable lipidoid L₅N₁₂, in the liposomal cationic adjuvant formulation 09 (CAF®09), and found that modified CAF®09 liposomes possess preserved adjuvant function as compared to unmodified CAF®09. CAF®09 consists of the cationic lipid dimethyldioctadecylammonium (DDA), monomycoloyl glycerol analogue 1 (MMG-1), and polyinosinic:polycytidylic acid [poly(I:C)]. By using the microfluidic mixing technology for liposome preparation, we gradually replaced DDA with L₅N₁₂, while keeping the molar ratios of MMG-1 and poly(I:C) constant. We found that this type of modification resulted in colloidally stable liposomes, which were significantly smaller and displayed reduced surface charge as compared to unmodified CAF®09, prepared by using the conventional thin film method. We showed that incorporation of L₅N₁₂ decreases the membrane rigidity of CAF®09 liposomes. Furthermore, vaccination with antigen adjuvanted with L₅N₁₂-modified CAF®09 or antigen adjuvanted with unmodified CAF®09, respectively, induced comparable antigen-specific serum antibody titers. We found that antigen adjuvanted with L₅N₁₂-modified CAF®09 induced antigen-specific effector and memory CD4⁺ and CD8⁺T-cell responses in the spleen comparable to those induced when unmodified CAF®09 was used as adjuvant. However, incorporating L₅N₁₂ did not have a synergistic immunopotentiating effect on the antibody and T-cell responses induced by CAF®09. Moreover, vaccination with antigen adjuvanted with unmodified CAF®09, which was manufactured by using microfluidic mixing, induced significantly lower antigen-specific CD4⁺ and CD8⁺T-cell responses than vaccination with antigen adjuvanted with unmodified CAF®09, which was prepared by using the thin film method. These results show that the method of manufacturing affects CAF®09 liposome adjuvanted antigen-specific immune responses, which should be taken into consideration when evaluating immunogenicity of subunit protein vaccines.