Development of a high sentitive oral hepatitis B vaccine using microspheres

使用微球开发高灵敏度口服乙型肝炎疫苗

• 批准号: 09672342
• 负责人: UCHIDA Takahiro
• 金额: $ 1.47万
• 依托单位: Mukogawa Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672342/
• 关键词: hepatitis B Core antigen; microspheres; poly (lactide-co-glycolide); ELISA; vaccine; 肝炎; マイクロスフェア-

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.

目的。采用w/o/w乳液/溶剂蒸发法制备含有重组乙型肝炎核心抗原（HBcAg；Mw = 3,600,000）的聚丙交酯乙交酯（PLGA）微球，并评价该系统作为小鼠乙型肝炎核心抗原有效长效载体的可能性。在微囊化HBcAg的过程中，内部水相中掺入了各种添加剂，通过ELISA测定了从制备的微球中提取的介质中的HBcAg抗原性。通过蔗糖梯度速度离心技术对HBcAg抗原进行了形状确认。对于体内研究，将制备的微球皮下给予Balb/C小鼠，并通过ELISA测定血清IgG水平。结果。通过在制备过程中向内部水相中添加明胶 (4-8% (w/v))，可显着降低二氯甲烷对 HBcAg 的灭活作用。通过冷却，装载效率进一步提高至近 61% (4*)。制备的含有0.15％HBcAg的微球(4.27mum*1.23mum)表现出爆发释放(2天内50-60％)。皮下接种时，PLGA微球的佐剂作用与弗氏完全佐剂几乎相同。而使用微​​球口服接种效果不佳。结论。从各种稳定性测试和 CD 谱研究来看，添加明胶的 pH 值似乎是 HBcAg 稳定的关键。最后，证明了使用该系统作为有效的长效乙型肝炎疫苗的可能性。