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Infection and Immunity, August 2003, p. 4563-4579, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4563-4579.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genome-Based Bioinformatic Selection of Chromosomal Bacillus anthracis Putative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens

N. Ariel,1* A. Zvi,1 K. S. Makarova,2 T. Chitlaru,1 E. Elhanany,1 B. Velan,1 S. Cohen,1 A. M. Friedlander,3 and A. Shafferman1*

Israel Institute for Biological Research, Ness Ziona 74100, Israel,1 National Center for Biotechnology Information, National Library of Medicine, National Institute of Health, Bethesda Maryland 20894,2 United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 217023

Received 14 January 2003/ Returned for modification 19 March 2003/ Accepted 1 May 2003

Bacillus anthracis (Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins, were selected as B. anthracis-specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version, 460 contigs, The Institute for Genomic Research, Rockville, Md.). The selection procedure combined preliminary annotation (sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen and additional filtering criteria (size, number of paralogs). The reductive strategy, combined with manual curation, resulted in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function. Proteomic analysis of two-dimensional gels of a B. anthracis membrane fraction, verified the expression of some gene products. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera from B. anthracis immunized animals. These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose expression and immunogenicity was not reported before. Five of these 8 immunogens were preselected by the bioinformatic analysis (EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates. This study demonstrates that a combination of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine.

* Corresponding authors. Mailing address for Naomi Ariel: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, P. O. Box 19, Ness Ziona 74100, Israel. Phone: 972-8-9381529. Fax: 972-8-9401404. E-mail ariel{at}iibr.gov.il. Mailing address for Avigdor Shafferman: Israel Institute for Biological Research, P. O. Box 19, Ness Ziona 74100, Israel. Phone: 972-8-9381595. Fax: 972-8-9401404. E-mail avigdor{at}iibr.gov.il.

Editor: D. L. Burns

Infection and Immunity, August 2003, p. 4563-4579, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4563-4579.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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