Development of a single-dose stabilized poly(d,l-lactic-co-glycolic acid) microspheres-based vaccine against hepatitis B

Abstract

The purpose of this study was to develop a stable single-dose vaccine based on recombinant hepatitis B surface antigen (HBsAg) in poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, in which HBsAg was stabilized by a protein stabilizer (trehalose) and an antacid (Mg(OH)2). The microspheres were prepared by the double emulsion method and characterized by scanning electron microscopy. To neutralize the acids liberated by the biodegradable lactic/glycolic acid based polymer, we coincorporated into the polymer an antacid, Mg(OH)2, which neutralized the acidity during degradation of the polymer and also prevented HBsAg structural losses and aggregation. The antigen integrity after encapsulation was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining, isoelectric focusing and Western blotting techniques, which confirmed that antigen remained intact after encapsulation. In-vitro release experiments were performed in phosphate-buffered saline (pH 7.4) and the release of antigen was found to be improved by the protein stabilizer (trehalose). In stability studies, performed at 37 degrees C, the microspheres were found to be stable for 16 days. The immunogenicity of stable microsphere formulations bearing HBsAg was compared with the conventional alum-absorbed HBsAg vaccine in a guinea-pig model. The antibody titre indicated that a single injection of stabilized HBsAg-PLGA microspheres produced a better immune response than two injections of alum-formulated HBsAg vaccine. The findings suggest that recombinant HBsAg can be stabilized by use of a protein stabilizer and antacid during entrapment, and this stabilized preparation can be useful for antigen delivery.

Peptide/protein vaccine delivery system based on PLGA particles

“…Many studies in different disease models, indicated that long lasting immunity can be achieved by administration of PLGA particles, which can avoid the requirement of multiple injections of the conventional vaccines. 36,58,80 For example, protective humoral and cellular immunities that were achieved by a single-shot HBsAg adsorbed PLGA MPs were similar to the response induced by booster injection of alum adsorbed HBsAg vaccine. 58 Furthermore, a single subcutaneous injection of HBsAgloaded PLGA MSs in mice led to comparable antibody responses to those of three injections of HBsAg aluminum vaccine.…”

“…Therefore, this delivery system is able to be administrated as a single-shot carrier adjuvant in development of vaccines and can prevent the need for multiple doses in next generation vaccines. 80 In addition, a remarkably high and long-lasting immune response associated with strong protective capacity was achieved with a single dose administration of SPf66 encapsulated PLGA MSs in Aotusmonkeys. 81 These results provide additional evidence that PLGA particles can be applied as a delivery system to induce protective immunity after a single dose administration of a vaccine.…”

“…Addition of a protein stabilizer and an antacid resulted in an enhancement in antigen stability during entrapment and release from the MSs. 80 It was found that a single injection of PLGA MSs gave rise to an almost equivalent anti-HBsAg antibody levels comparing to two injections of alum-adsorbed HBsAg vaccine in guinea-pigs. Therefore, this delivery system is able to be administrated as a single-shot carrier adjuvant in development of vaccines and can prevent the need for multiple doses in next generation vaccines.…”

Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response

“…This indicates that the preparation conditions did not cause any irreversible aggregation or cleavage of the antigen. In addition, in vitro immunogenicity was also measured by EIA/protein ratio (relative potency) determination as described in our previous studies (Jaganathan et al, 2004;Pawar et al, 2013). The CS and GC NPs showed an EIA/protein ratio of 1.0 ± 0.2 and 1.0 ± 0.1, respectively.…”

“…Proteins were visualized by coomassie blue staining. The same sample was also used to measure the in vitro antigenicity using an enzyme immunoassay (EIA) (Jaganathan et al, 2004;Pawar et al, 2013). Each sample (HBsAg extracted from the NPs) was diluted with 0.2% (w/v) BSA in PBS (pH 7.4) to get three different concentrations and examined against a linear fitting to the response of control standard sample.…”