Research Articles
Pharmaceutical and Immunological Evaluation of a Single-Shot Hepatitis B Vaccine Formulated With PLGA Microspheres

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Abstract

A single-shot Hepatitis B vaccine formulation using poly(d, l)-lactide-co-glycolide acid (PLGA) microspheres as a delivery system was examined using a variety of biophysical and biochemical techniques as well as immunological evaluation in C3H mice. PLGA microsphere encapsulation of the Hepatitis B surface antigen (HBsAg), a lipoprotein particle, resulted in good recoveries of protein mass, protein particle conformational integrity, and in vitro antigenicity. Some partial delipidation of the HBsAg, however, was observed. The loading and encapsulation efficiency of HBsAg into the PLGA microspheres were measured along with the morphology and size distribution of the vaccine-loaded PLGA microspheres. The in vitro release kinetics of HBsAg from the PLGA microspheres was evaluated and found to be affected by experimental conditions such as stirring rate. HBsAg showed enhanced storage stability at 37°C in the slightly acidic pH range reported to be found inside PLGA microspheres; thus, the antigen is relatively stable under conditions of temperature and pH that may mimic in vivo conditions. The immunogenicity of the microsphere formulations of HBsAg was compared with conventional aluminum adjuvant formulated HBsAg vaccine in C3H mice. Comparisons were made between aluminum formulations (one and two injections), PLGA microsphere formulations (single injection), and a mixture of aluminum and PLGA microsphere formulations (single injection). The nine-month serum antibody titers indicate that a single injection of a mixture of aluminum and PLGA-formulated HBsAg results in equal or better immune responses than two injections of aluminum-formulated HBsAg vaccine. Based on these in vitro and in vivo studies, it is concluded that HBsAg can be successfully encapsulated and recovered from the PLGA microspheres and a mixture of aluminum-adjuvanted and PLGA-formulated HBsAg can auto-boost an immune response in manner comparable to multiple injections of an aluminum-formulated vaccine.

Section snippets

INTRODUCTION

The evaluation of controlled-release formulations for protein-based vaccines has become an active area of vaccine pharmaceutical research.1, 2, 3, 4, 5, 6, 7, 8, 9 Currently, parenteral administration of purified protein-based vaccine antigens involves multiple injections to achieve an optimum immune response in humans. For example, recombinant Hepatitis B surface antigen (HBsAg) adsorbed to aluminum adjuvant is typically administered with multiple injections over many months to obtain a

Polymer and HBsAg

Poly(d,l)-lactide-co-glycolide acid (PLGA) with a lactide/glycolide (L/G) ratio of 65/35 (MW 75 000) and PLGA with a L/G ratio of 50/50 (MW 75 000) were purchased from Birmingham Polymers, Inc. In this paper, the corresponding polymers are referenced as PLGA65/35 and PLGA50/50, respectively. Recombinant Hepatitis B surface antigen (HBsAg) S protein was expressed in yeast and purified as outlined previously.41 The purified HBsAg used for PLGA encapsulation was dialyzed and concentrated to 1–2

Physical Characterization of PLGA Microspheres Containing HBsAg

PLGA microsphere preparation by the “double emulsion” method requires the PLGA polymer to be initially dissolved in an organic solvent, mixed and homogenized with an aqueous protein solution to form a w/o emulsion, subsequently mixed in a second solvent to form a w/o/w or double emulsion, followed by collection and drying of the protein-containing microspheres48, 49, 50, 51, 52 (see Materials and Method section). The PLGA microspheres prepared by the double emulsion method in this work were

CONCLUSIONS

The purpose of this study was to pharmaceutically characterize a HBsAg vaccine formulation with PLGA microspheres. HBsAg has been successfully encapsulated in PLGA microspheres and active antigen has been recovered upon release from the microspheres. The in vitro release studies indicate a controlled (delayed) release of HBsAg from PLGA microspheres, but that the in vitro release kinetics can be varied by experimental conditions, such as stirring rate. The biophysical and bioanalytical

Acknowledgements

The authors thank Hongkee Sah for helping to prepare some of the initial PLGA microsphere formulations containing HBsAg, Nirpal Dhanjal and Zheng Luo for carrying out some of the initial HBsAg stability studies and in vitro antigenicity measurements, Su Wang for assisting with the mouse serum analysis, Mei Tang and Mohinder Sadana for performing the amino acid analysis, and Alan Shaw and John Donnelly for helpful discussions.

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