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Infection and Immunity, February 2004, p. 908-915, Vol. 72, No. 2
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.2.908-915.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Generation of Yersinia pestis Attenuated Strains by Signature-Tagged Mutagenesis in Search of Novel Vaccine Candidates

Yehuda Flashner,{dagger}* Emanuelle Mamroud,{dagger} Avital Tidhar, Raphael Ber, Moshe Aftalion, David Gur, Shirley Lazar, Anat Zvi, Tamar Bino, Naomi Ariel, Baruch Velan, Avigdor Shafferman, and Sara Cohen*

Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100, Israel

Received 11 August 2003/ Returned for modification 22 September 2003/ Accepted 21 October 2003

In a search for novel attenuated vaccine candidates for use against Yersinia 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 virulent Y. pestis Kimberley53 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 10-5 to 10-7 to 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, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, >107 CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant 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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona, 74100, Israel. Phone for Yehuda Flashner: 972-8-9381526. Fax: 972-8-9401404. E-mail: yflash{at}netvision.net.il. Phone for Sara Cohen: 972-8-9381718. Fax: 972-8-9401404. E-mail: cohens{at}iibr.gov.il.

Editor: D. L. Burns

{dagger} Y.F and E.M. contributed equally to this work.

Infection and Immunity, February 2004, p. 908-915, Vol. 72, No. 2
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.2.908-915.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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