RECOMBINANT ROTAVIRUS PROTEINS FOR SUBUNIT VACCINE

THE OBJECTIVE OF PHASE I IS TO PRODUCE A ROTAVIRUS ASSEMBLED-SUBUNIT VACCINE USING RECOMBINANT DNA TECHNOLOGY. SPECIFICALLY, BACULOVIRUS-EXPRESSED ROTAVIRUS PROTEINS VP6 AND VP7 WILL BE PURIFIED AND ASSEMBLED, IN VITRO, INTO DOUBLE-SHELLED, VIRUS-LIKE PARTICLES, WITH THE INNER CAPSID COMPOSED OF VP6 AND THE OUTER CAPSID COMPOSED OF THE SEROTYPE-SPECIFIC PROTEIN, VP7. THE ASSEMBLY OF VP6 INTO SPHERICAL PARTICLES IS A PH-DEPENDENT PROCESS, WHILE THE ADDITION OF VP7 ONTO THESE PARTICLES IS A CALCIUM-DEPENDENT PROCESS. BOTH PROCEDURES HAVE BEEN USED TO SUCCESSFULLY REASSEMBLE THE NATIVE FORMS OF VP6 AND VP7 INTO STABLE, VIRUS-LIKE SPHERICAL PARTICLES. CHARACTERIZATION OF THE RECOMBINANT PARTICLES WILL BE BY IMMUNOPRECIPITATION, IMMUNOBLOT ELISA, AND ELECTRON MICROSCOPY USING SPECIFIC MONOCLONAL ANTIBODIES. THE ABILITY OF THESE PARTICLES TO ATTACH TO TARGET CELLS WILL BEINVESTIGATED BY CARRYING OUT BINDING STUDIES USING RADIOLABELED PARTICLES. THE IMMUNOGENICITY OF THE ASSEMBLED PARTICLES WILL ALSO BE ASSESSED AND COMPARED TO THAT OF THE NATIVE, DOUBLE-SHELLED VIRUS. THE POTENTIAL OF THIS APPROACH IS THAT IT MAY BE USED TO COASSEMBLE MORE THAN ONE SEROTYPE OF VP7 ONTO EACH VP6 SPHERICAL PARTICLE, THEREBY PRODUCING A HETEROTYPIC VACCINE. PHASE II STUDIES ARE DESIGNED TO EXAMINE THE POTENTIAL OF THIS VACCINE CANDIDATE TO PROTECT AGAINST ROTAVIRUS INFECTIONS.