Dissolution of Mechanism for Oral Immunization using Microsphere

目的。采用w/o/w乳状液/溶剂挥发法制备含模型流感抗原或重组乙肝核心抗原(HBcAg)的聚丙交酯-乙交酯(PLGA)微球，并评价该微球作为长效疫苗系统在小鼠体内应用的可能性。针对模型35，合成了与血凝素神经氨酸酶(HANA)对应的碱基抗原(FISEG、FTWTG、VTQNG、GSNAC、KRGPD、SGFFS、RLNWL)，并将其作为流感疫苗的模式抗原。在微囊化HBcAg的过程中，在内水相中加入不同的添加剂，用ELISA检测从微球中提取的介质中HBcAg的抗原性，而对于HbcAg，用蔗糖梯度速度离心法进行HBcAg抗原的形状确认。在体内实验中，将制备的微球皮下注射Balb/C小鼠，用ELISA法测定血清中的免疫球蛋白水平。以HANA抗原为例，用质谱仪测定其相对分子质量为3839。但稳定性并不是很好。因此，为了保护合成肽的降解，添加了聚乙二醇酸。谷胱甘肽还能有效地防止多肽变性。另一方面，在制备过程中向内水相中添加明胶(4-8%(w/v))，可显著减少二氯甲烷对HBcAg的灭活。由于冷却，加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天内有50%~60%的突释性释放。在皮下接种中，含HbcAg的PLGA微球和模型HANA抗原的PLGA微球均能有效提高血清中的免疫球蛋白水平。最后，论证了该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.