Dissolution of Mechanism for Oral Immunization using Microsphere

微球口服免疫溶解机制

• 批准号: 11672285
• 负责人: UCHIDA Takahiro
• 金额: $ 2.18万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672285/
• 关键词: microsphere; vaccine; influenza; emulsion; protease; hepatitis B virus; emalsion; ワクチン; インフルエンザ; HBc抗原; マイクロカプセル; HA1; W; O

Purpose. To prepare poly (lactide-co-glycolide)(PLGA) microspheres containing model influenza antigen or 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 vaccine system in mice.Methods. For model 35 base antigen (FISEG FTWTG VTQNG GSNAC KRGPD SGFFS RLNWL) corresponding to HANA (hemagglutinin-neuraminidase) were synthesized and used as a model antigen for influenza vaccine. 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.Whereas in the case of HbcAg, shape confirmation of the HBcAg antigen was performed by a sucrose gradient velocity centrifugal technique. For in vivo study, prepared microspheres were administered subcutaneously to Balb/C mice, and the serum IgG level was determined by ELISA.Results. In the case of HANA antigen, molecular weight was proofed to be 3839 by measerement using mass spectrometry. But the stability was not so good. Therefore, addition of tioglycohlic acid was added to protect degradation of synthesized peptide. glutathioin was also effective for preventing from denaturation of peptide. On the other hand, 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. The prepared microspheres (4.27 mm + 1.23 mm) containing 0.15% HBcAg displayed burst release (50-60% within 2 days).Conclusions. In subcutaneous inoculation, the adjuvant effect of PLGA microspheres containing HbcAg or model HANA antigen was effective for increasing serum IgG level. Finally, the possibility of this microparticle system as a potent long-acting vaccine system was demonstrated.

目的。采用w/o/w乳液/溶剂蒸发法制备含有模型流感抗原或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强效长效疫苗系统的可能性。对于模型，合成了对应于HANA（血凝素-神经氨酸酶）的35个基础抗原（FISEG FTWTG VTQNG GSNAC KRGPD SGFFS RLNWL）并用作流感疫苗的模型抗原。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 测量从制备的微球中提取的介质中的 HBcAg 抗原性。而对于 HbcAg，通过蔗糖梯度速度离心技术进行 HBcAg 抗原的形状确认。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。在HANA抗原的情况下，通过使用质谱法测量证明分子量为3839。但稳定性不太好。因此，添加硫代乙醇酸以防止合成肽的降解。谷胱甘肽也能有效防止肽变性。另一方面，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%。制备的含有 0.15% HBcAg 的微球（4.27 mm + 1.23 mm）显示出爆发释放（2 天内 50-60%）。结论。皮下接种时，含有HbcAg或模型HANA抗原的PLGA微球的佐剂作用可有效提高血清IgG水平。最后，证明了这种微粒系统作为有效的长效疫苗系统的可能性。