Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation

Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain d erived from the virulent strain 2308. Recently, we demonstrated that the wb oA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J R, McQu iston, G. C. Schurig, N, Srirauganathan, S. M. Halling, and S. M. Boyle, Cl in, Diagn. Lab. Immunol, 6:760-764, 1999), Disruption of the wboA gene in s mooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen), In this study, we explored whether the wboA gene disruption is re sponsible for the rough phenotype of RB51, We complemented RB51 with a func tional wboA gene, and the resulting strain was designated RB51WboA, Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cy toplasm, Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic character istics similar to those of RB51, Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a ) and IgG3 isotypes, After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatan ts indicated that RB51WboA immunization induced higher levels of gamma inte rferon than immunization with RB51, Mice vaccinated with RB51WboA were bett er protected against a challenge infection with the virulent strain 2308 th an those vaccinated with RB51, These studies indicate that in addition to t he disruption of the wboA gene there Is at least one other mutation in RB51 responsible for its rough phenotype, These studies also suggest that the e xpressed O antigen in RB51WboA is responsible either directly or indirectly for the observed enhancement in the T-cell response.