Development of a high sentitive oral hepatitis B vaccine using microspheres

目的。采用w/o/w乳剂/溶剂蒸发法制备重组乙型肝炎核心抗原（HBcAg, Mw = 3,600,000）聚乳酸-羟基乙酸酯（PLGA）微球，并评价该体系作为乙型肝炎核心抗原长效载体的可行性。在HBcAg微囊化过程中，内水相中加入各种添加剂，用ELISA法测定制备的微球提取的培养基中HBcAg的抗原性。采用蔗糖梯度速度离心技术对HBcAg抗原进行形状鉴定。在体内实验中，制备的微球皮下注射Balb/C小鼠，elisa法检测血清lgG水平。制备过程中，在内水相中加入明胶（4-8% (w/v)）可显著降低二氯甲烷对HBcAg的失活作用。通过冷却，负载效率进一步提高到近61%（4*）。制备的含0.15% HBcAg的微球（4.27 μ m*1.23 μ m）在2天内爆发释放（50-60%）。在皮下接种时，PLGA微球的佐剂效果与完全的弗氏佐剂几乎相同。而口服接种微球效果不佳。各种稳定性试验和CD谱研究表明，明胶的pH值是HBcAg稳定的关键。最后，验证了该系统作为长效乙肝疫苗的可行性。

Purpose. To prepare poly(lactide-co-glycolide) (PLGA) microspheres containing recombinant hepatitis B core antigen (HBcAg ; Mw = 3,600,000) by a w/o/w emulsion/solvent evaporation method and evaluate the possibility of this system as a potent long-acting carrier for hepatitis B core antigen in mice.Methods. Various additives had been incorporated in the internal aqueous phase during the process of microencapsulating HBcAg, HBcAg antigenicity in the medium extracted from the prepared microspheres were measured by ELISA.Shape confirmation of the HBcAg antigen was performed by asucrose gradient velocity centrifugal technique. For in vivo study, prepared microspheres were administered subcutaneously to Balb/C mice, and the serum lgG level was determined by ELISA.Results. The inactivation of HBcAg by methylene chloride was dramatically reduced by the addition of gelatin (4-8% (w/v)) to the internal aqueous phase during the preparation. Further improvement of the loading efficiency to almost 61% resulted with cooling (4*). The prepared microspheres (4.27 mum*1.23mum) containing 0.15% HBcAg displayed burst release (50-60% within 2 days). In subcutaneous inoculation, the adjuvant effect of PLGA microspheres was almost the same as that of the complete Freund's adjuvant. Whereas oral inoculation using the microspheres was not effective.Conclusions. The pH of the added gelatin seemed to be the key to the stabilization of HBcAg from various stability tests and CD spectrum study. Finally, the possibility of using this system as a potent long-acting hepatitis B vaccine was demonostrated.