Brucella is the etiologic agent of brucellosis, one of the most common and 
widely distributed zoonotic diseases. Its highly infectious nature, the 
insidious, systemic, chronic, debilitating aspects of the disease and the lack 
of an approved vaccine for human use in the United States are features that make 
Brucella a viable threat to public health. One of the main impediments to 
vaccine development is identification of suitable antigens. In order to identify 
antigens that could potentially be used in a vaccine formulation, we describe a 
multi-step antigen selection approach. We initially used an algorithm (Vaxign) 
to predict ORF encoding outer membrane proteins with antigenic determinants. 
Differential gene expression during acute infection and published evidence for a 
role in virulence were used as criteria for down-selection of the candidate 
antigens that resulted from in silico prediction. This approach resulted in the 
identification of nine Brucella melitensis outer membrane proteins, 5 of which 
were recombinantly expressed and used for validation. Omp22 and Hia had the 
highest in silico scores for adhesin probability and also conferred invasive 
capacity to E. coli overexpressing recombinant proteins. With the exception of 
FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, 
and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in 
splenocytes from immunized mice while BtuB and Hia elicited NO release from 
splenocytes of S19 immunized mice. The results support the applicability of the 
current approach to the identification of antigens with immunogenic and invasive 
properties. Studies to assess immunogenicity and protective efficacy of 
individual proteins in the mouse are currently underway.