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, determination of the encapsulation, release and integrity of two or more proteins co-encapsulated within microspheres is an important issue. A new extraction method, which exhibits excellent protein recovery, has been developed which enables samples to be used for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent measurement of individual antigens encapsulated within microspheres. Using the new method, the protein loading of poly-(L-lactide) microspheres co-encapsulating two plague sub-unit antigens was found to be 1.22% (w/w) for recombinant V antigen (rV) and 1.24% (w/w) for recombinant F1 (rF1) by SDS-PAGE. The total protein loading was 2.49% (w/w) by bicinchoninic acid assay. The individual release of the two subunit antigens from the co-encapsulated 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 proteins.