Evaluation of unmarked deletion mutants as improved Brucella vaccine strains in the mouse and goat models

Historical data suggests that prolonged survival of Brucella vaccine organisms in the target host enhances immune protection. Recent research has focused upon the development of rough vaccine strains to avoid interference with standard diagnostic tests. Rough organisms are rapidly cleared from the host, however. In an effort to develop improved vaccine strains, we have screened signature tagged mutagenesis banks to identify mutants with varying survival characteristics. We hypothesize that in order for a vaccine to be efficacious, it must survive in the host. In order to test this, we constructed marked and unmarked deletion mutants of B. abortus and B. melitensis in genes previously demonstrated by transposon mutagenesis to attenuate in vivo and in vitro virulence. Survival and efficacy of these novel deletion mutants were then evaluated in the mouse model. The asp24 mutants, which persist for extended periods in vivo, appear superior as a vaccine candidate compared to approved vaccine strains S19 and Rev1 in the mouse model against either homologous or heterologous challenges. Once enhanced protection against infection was demonstrated in the mouse, components of immune function that appeared to be most important were identified to correlate the immune response with the observed protection. We demonstrated that the most persistent mutant, delta-asp24, affords the greatest protection in mice against virulent challenge. In order to evaluate safety of the novel vaccine strains as well as protection against infection and abortion, we tested selected B. melitensis unmarked deletion mutants in a natural host, the goat. The delta-asp24 mutant was shown to be safe in pregnant goats while providing significant protection against infection and abortion.