| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
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.
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