Studies on the co-encapsulation, release and integrity of two subunit antigens: rV and rF1 from Yersinia pestis

In the development of combination or multiple sub-unit vaccines, determinat ion of the encapsulation, release and integrity of two or more proteins co- encapsulated within microspheres is an important issue. A new extraction me thod, which exhibits excellent protein recovery, has been developed which e nables samples to be used for sodium dodecyl sulphate polyacrylamide gel el ectrophoresis (SDS-PAGE:) and subsequent measurement of individual antigens encapsulated within microspheres. Using the new method, the protein loadin g of poly-(L-lactide) microspheres co-encapsulating two plague sub-unit ant igens was found to be 1.22% (w/w) for recombinant V antigen (rV) and 1.24% (w/w) for recombinant Fl (rF1) by SDS-PAGE. The total protein loading was 2 .49% (w/w) by bicinchoninic acid assay. The individual release of the two s ubunit antigens from the coencapsulated microspheres was determined by SDS- PAGE analysis and rF1 was found to have a higher burst release than rV. The integrity and immunological activity of both rF1 and rV antigens was shown to be unaffected by the microencapsulation process. This study shows that encapsulation of more than one antigen within poly-(L -lactide) microspheres is a viable method for the delivery of intact protei ns.