Complementation ofBrucella abortusRB51 with a FunctionalwboAGene Results in O-Antigen Synthesis and Enhanced Vaccine Efficacy but No Change in Rough Phenotype and Attenuation

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Abstract
Brucella abortusRB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that thewboAgene in RB51 is disrupted by an IS711element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760–764, 1999). Disruption of thewboAgene in smooth, virulentB. abortus,Brucella melitensis, andBrucella suisresults in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether thewboAgene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functionalwboAgene, 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 cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics 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 supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of thewboAgene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible either directly or indirectly for the observed enhancement in the T-cell response.