Enhancement of T-helper type I immune responses against hepatitis B surface antigen by LPS derivatives adjuvanted liposomes delivery system

Currently, there is a clinical need for more effective vaccine for hepatitis B that induces robust cell-mediated immune response capable of viral clearance in chronic hepatitis B infection. In the present study, hepatitis B vaccines formulations were designed by loading the hepatitis B surface antigen into liposomes adjuvanted with rough lipopolysaccharide (Re-LPS) and lpxL1 LPS using conventional rotatory evaporation method and were characterized for various parameters, such as vesicle shape and surface morphology, size and size distribution, entrapment efficiency, turbidity, and in vitro release pattern. The immunoreactivity in mice was evaluated by measuring anti-HBs IgG titer and compared with alum-adsorbed HBsAg solution, plain HBsAg, and liposomal HBsAg formulations. The formulations were also evaluated for cell-mediated immune response by HBsAg specific proliferation of spleenocytes after secondary immunization and re-stimulation in vitro with the same antigen. Simultaneous estimation of cytokines (IL-4, IFN-) was also carried out. Ex vivo cellular uptake study was performed by fluorescence microscopy. Results indicate that the serum IgG titer obtained after i.m administration of Re-LPS- and lpxL1 LPS-adjuvanted liposomal HBsAg formulation was equivalent to alum-adsorbed HBsAg formulation but was more responsive, sustained, and significantly higher than the corresponding liposomal HBsAg and plain HBsAg formulations. Incorporation of lpxL1 LPS into the liposomal HBsAg increased the stimulation index (SI) 6-10 times as compared with plain HBsAg. Re-LPS- and lpxL1 LPS-adjuvanted liposomal HBsAg formulations induced stronger cellular immune response with a predominant Interferon-gamma (IFN-) level than those induced by free HBsAg alone, alum-adsorbed HBsAg, and non-adjuvanted liposomal HBsAg. Probably, the possible mechanism for the enhancement of cellular immunity in addition to humoral immunity by LPS-adjuvanted liposomal HBsAg formulation is due to marked enhancement of immunological presentation and recruitment of antigen via macrophage and antigen-presenting cells (APCs).