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乳化/溶剂挥发法制备含重组B肝炎核心抗原（HBcAg ; Mw = 3，600，000）的聚（丙交酯-乙交酯）共聚物（PLGA）微球，并评价其作为B肝炎核心抗原（HBcAg）长效载体的可行性。在微囊化过程中，内水相中加入不同的添加剂，用ELISA法检测微球提取液中HBcAg抗原性，用蔗糖梯度离心法对HBcAg抗原进行形态确认。体内研究中，将制备的微球皮下注射给Balb/C小鼠，用ELISA法测定血清IgG水平。在制备过程中，通过在内水相中加入明胶（4-8%（w/v）），可显著降低二氯甲烷对HBcAg的灭活作用。通过冷却，装载效率进一步提高到近61%（4*）。所制备的含0.15%HBcAg的微球（4.27 μ m * 1.23 μ m）显示突释（2天内50-60%）。在皮下接种中，PLGA微球的佐剂效应与完全弗氏佐剂几乎相同。而口服微球接种效果不佳。从各种稳定性试验和CD光谱研究来看，加入明胶的pH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B型肝炎疫苗的可能性。