The capsular polysaccharide of Neisseria meningitidis group B is an autoantigen, whereas noncapsular antigens are highly variable. These factors present formidable challenges for development of a broadly protective and safe group B vaccine. Mice and guinea pigs were sequentially immunized with three doses of micovesicles or outer membrane vesicles prepared from three meningococcal strains that were each antigenically heterologous with respect to the two major porin proteins, PorA and PorB, and the group capsular polysaccharide. The resulting antisera conferred passive protection against meningococcal group B bacteremia in infant rats and elicited complement-mediated bactericidal activity against genetically diverse group B strains that were either homologous or heterologous with respect to PorA of the strains used to prepare the vaccine. By using knockout strains, a portion of the bactericidal antibody was directed against the highly conserved protein, neisserial surface protein A (NspA). Further, an anti-NspA monoclonal antibody elicited by the sequential immunization was highly bactericidal against strains that were previously shown to be resistant to bacteriolysis by anti-NspA antibodies produced by immunization with recombinant NspA. Sequential immunization with heterologous vesicle preparations offers a novel approach to eliciting broadly protective immunity against N. meningitidis strains. An NspA-based vaccine prepared from protein expressed by Neisseria also may be more effective than the corresponding recombinant protein made in Escherichia coli.