ABSTRACT
Cationic liposomes are potential adjuvants for influenza vaccines. In a previous study we reported that among a panel of cationic liposomes loaded with influenza hemagglutinin (HA), DC-Chol:DPPC (1:1 molar ratio) liposomes induced the strongest immune response. However, it is not clear whether the cholesterol (Chol) backbone or the tertiary amine head group of DC-Chol was responsible for this. Therefore, in the present work we studied the influence of Chol in the lipid bilayer of cationic liposomes. Moreover, we investigated the effect of the HA loading method (adsorption versus encapsulation) and the encapsulation of immune modulators in DC-Chol liposomes on the immunogenicity of HA. Liposomes consisting of a neutral lipid (DPPC or Chol) and a cationic compound (DC-Chol, DDA, or eDPPC) were produced by film hydration-extrusion with/without an encapsulated immune modulator (CpG or imiquimod). The liposomes generally showed comparable size distribution, zeta potential and HA loading. In vitro studies with monocyte-derived human dendritic cells and immunization studies in C57Bl/6 mice showed that: (1) liposome-adsorbed HA is more immunogenic than encapsulated HA; (2) the incorporation of Chol in the bilayer of cationic liposomes enhances their adjuvant effect; and (3) CpG loaded liposomes are more efficient at enhancing HA-specific humoral responses than plain liposomes or Alhydrogel.
ABSTRACT
Cationic liposomes are known as potent adjuvants for subunit vaccines. The purpose of this work was to study whether the content and the physicochemical properties of the positively charged compound affect the adjuvanticity of cationic liposomes. Cationic liposomes containing a cationic compound (DDA, DPTAP, DC-Chol, or eDPPC) and a neutral phospholipid (DPPC) were prepared by the film hydration-extrusion method and loaded with influenza hemagglutinin (HA) by adsorption. The liposomes were characterized (hydrodynamic diameter, zeta potential, membrane fluidity, HA loading) and their adjuvanticity was tested in mice. The formulations were administered twice subcutaneously and mouse sera were analyzed for HA-specific antibodies by ELISA and for HA-neutralizing antibodies by hemagglutination inhibition (HI) assay. First, the influence of cationic lipid concentration in the DC-Chol/DPPC liposomes (10 vs. 50 mol%) was investigated. The DC-Chol/DPPC (50:50) liposomes showed a higher zeta potential and HA loading, resulting in stronger immunogenicity of the HA/DC-Chol/DPPC (50:50) liposomes compared to the corresponding (10:90) liposomes. Next, we used liposomes composed of 50 mol% cationic lipids to investigate the influence of the nature of the cationic compound on the adjuvant effect. Liposomes made of the four cationic compounds showed similar hydrodynamic diameters (between 100 and 170 nm), zeta potentials (between +40 and +50 mV), HA loading (between 55% and 76%) and melting temperatures (between 40 and 55 °C), except for the DC-Chol liposomes, which did not show any phase transition. HA adjuvanted with the DC-Chol/DPPC (50:50) liposomes elicited significantly higher total IgG1 and IgG2a titers compared to the other liposomal HA formulations and non-adjuvanted HA. A similar trend was observed for the HI titers. These results show that the adjuvanticity of cationic liposomes depends on both the content and the physicochemical properties of the charged compound.
