Generation ofYersinia pestisAttenuated Strains by Signature-Tagged Mutagenesis in Search of Novel Vaccine Candidates

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Abstract
In a search for novel attenuated vaccine candidates for use againstYersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulentY. pestisKimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10−5to 10−7to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g.,purH,purK,dnaE, andgreA) or for hypothetical polypeptides, as well as for the virulence regulator genelcrF. One of the avirulent mutant strains (LD50, >107CFU) was found to be disrupted in thepcmlocus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcmmutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.