Oral immunization against hepatitis B using bile salt stabilized vesicles (bilosomes)

Abstract

PURPOSE: Presently available hepatitis B vaccine is administered through parenteral route and fails to induce mucosal antibody response. Oral immunization appears to be an effective and attractive alternative to the parenteral immunization. However, the problem of degradation of antigen in harsh and hostile environment of the gastrointestinal tract consequently requires larger dose and more frequent dosing of antigen. Furthermore, much larger dose can induce systemic tolerance. In order to overcome these problems bile salt stabilized vesicles (bilosomes) can be used, which could provide both protection to antigen as well as transmucosal uptake and subsequent resultant systemic immunity. Another purpose of this study was to determine the dose that could produce comparable serum antibody titre against hepatitis B through oral route as obtained after intramuscular immunization. METHODS: In the present study bilosomes containing recombinant hepatitis B surface antigen were prepared by cast film method. HBsAg loaded bilosomes were characterized in vitro for their shape, size, percent entrapment and stability. The fluorescence microscopy was carried out to confirm the uptake of bilosomes by gut associated lymphoid tissues (GALT). The in vivo study was comprised of estimation of anti-HBsAg IgG response in serum and anti-HBsAg sIgA in various body secretions using specific ELISA techniques following oral immunization with low dose loaded bilosomes (BSSV1, 10 µg), intermediate dose loaded bilosomes (BSSV2, 20 µg) and high dose loaded bilosomes (BSSV3, 50 µg) in Balb/c mice. RESULTS: The fluorescence microscopy suggests that an increase in fluorescence intensity following the uptake of bilosomes entrapped FITC-BSA in gut associated lymphoid tissues. The high dose HBsAg bilosomes (BSSV3, 50 µg) produced comparable anti-HBsAg IgG levels in serum to those observed in the case of intramuscular administration of alum adsorbed HBsAg (10 µg). However, alum adsorbed HBsAg was unable to produce sIgA in mucosal secretions, whilst, all bilosomal preparations could elicit measurable sIgA in mucosal secretions, where the highest responses were observed in high dose HBsAg bilosomes (BSSV3, 50µg). CONCLUSIONS: HBsAg loaded bilosomes produced both systemic as well as mucosal antibody responses upon oral administration. Thus bilosomes may emerge out to be potential carriers for non invasive mucosal immunization.