Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. Rotavirus VLPs composed of simian SA11 strain VP2 and VP6 proteins (homologous 2/6-VLPs) were produced by cloning the rotavirus simian SA11 genes 2 and 6 into a single baculovirus transfer vector (pAcAB4). The overall yield of homologous 2/6-VLPs produced with the dual recombinant baculovirus was at least 30-fold higher than that of VLPs composed of bovine RF strain VP2 and simian SA11 strain VP6 (heterologous 2/6-VLPs), produced with single recombinant baculoviruses. Adult mice were immunized intramuscularly twice with various doses of homologous or heterologous 2/6-VLPs in QS-21, orally with or without cholera toxin (CT), or intranasally with mutant Escherichia coli heat-labile enterotoxin (LT-R192G). Both homologous and heterologous 2/6-VLPs were immunogenic and induced protection from challenge, with those administered parenterally or intranasally affording the highest mean protection from challenge. The 2/6-VLPs did not induce serum neutralizing antibody (N-Ab) responses, but these VLPs primed for a broad heterotypic N-Ab response, which was elicited after rotavirus challenge. Heterotypic N-Ab responses were not observed in 2/6-VLP vaccinated mice that were > or =94% protected from challenge. After challenge, control mice immunized with adjuvant alone developed only homotypic serum N-Ab responses. Similar results were obtained after challenge of rabbits immunized parenterally or intranasally with heterologous 2/6-VLPs. These results suggest that 2/6-VLPs prime the immune system to enhance the production of heterotypic N-Ab responses, but the induction of heterotypic N-Abs requires that virus replication occurs after challenge. The use of 2/6-VLPs expressed from a single recombinant baculovirus simplifies production and would reduce the cost of a VLP-based vaccine.