Genome-based bioinformatic selection of chromosomal Bacillus anthracis putative vaccine candidates coupled with proteomic identification of surface-associated antigens

Abstract

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.

FIG. 1.

Strategy for reductive selection of vaccine candidates from the B. anthracis chromosome draft sequence (version of February 2001). Flowchart of the computational analysis and the filtering steps, of the B. anthracis chromosome candidate selection (left panel). Filtering steps are noted by letters (a through d, as detailed in the right panel), and the resulting number of ORF products at each step is noted in a shaded box.

FIG. 2.

Serological proteome analysis of B. anthracis membranal proteins. B. anthracis ATCC Δ14185 (pXO1⁻, pXO2⁻) membranal proteins were separated by 2-DE (IEF on a pH 3 to 10 IPG strip). The gel was stained with Coomassie blue, for total protein spot detection. Twin gels were transferred to nitrocellulose membranes and probed with guinea-pig anti-B. anthracis immune sera (whole-cell lysate titer of 1:12,800). The Coomassie blue stain is shown on the left and the respective Western blot, on the right. The complete 2-DE gel (A) and enlarged sections (B through E) are shown. Western blots in panels A and regions B through D were probed with 1:1,000 diluted antiserum. For better resolution, the region depicted in E is taken from a 2-DE gel run on a pH 4 to 7 IPG strip (first dimension) and its respective Western blot developed with 1:300-diluted antiserum. The seropositive protein spots, are identified by running numbers. See Table 2 for the complete list of identified seropositive proteins detected in these experiments.