Dissolution of Mechanism for Oral Immunization using Microsphere

目的。采用w/o/w乳剂/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）的聚乳酸-羟基乙酸酯（PLGA）微球，并评价该体系作为长效疫苗体系在小鼠体内的可行性。合成了与HANA（血凝素-神经氨酸酶）相对应的模型35碱基抗原（FISEG FTWTG VTQNG GSNAC KRGPD SGFFS RLNWL）作为流感疫苗模型抗原。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。而在HbcAg的情况下，HbcAg抗原的形状确认是通过蔗糖梯度速度离心技术进行的。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IgG水平。在HANA抗原的情况下，通过质谱测量证明分子量为3839。但是稳定性不是很好。因此，为了保护合成肽的降解，添加了巯基乙酸。谷胱甘肽对多肽的变性也有一定的抑制作用。另一方面，在制备过程中，在内水相中加入明胶（4-8% (w/v)）可以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%。含0.15% HBcAg的微球（4.27 mm + 1.23 mm）在2天内释放率达到50% ~ 60%。皮下接种时，含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一种有效的长效疫苗系统的可能性。

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.