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Diagnosis of bacillus anthracis infection based on detection of bacterial secreted biomarkers

The gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a rare fatal disease which is initiated, in its most severe form, by inhalation of infected spores.  Due to the severity of the disease, the ease of respiratory infection and the everlasting resistance of the spores to unfavorable environmental conditions, B. anthracis is considered a potential biological warfare agent, and in recent years the need for novel reliable diagnostic approaches, improved vaccination strategies, novel therapeutic targets and better understanding of the pathogenesis, have been widely acknowledged. Global surveys combined with serological study of B. anthracis were carried out at IIBR, for identification of vaccine and diagnostic marker candidates among extracellular (secreted or membranal) proteins.  These studies indeed revealed a list of proteins that can serve as potential biomarkers, based on their immunogenicity (which probes their in vivo expression), abundance under various culture conditions, and functional relatedness to infection. Furthermore, these studies identified novel virulence factors whose genetic disruption enabled development of novel vaccine strains (see attached IIBR references).

Identification of novel diagnostic markers for Bacillus anthracis infection in clinical samples

The presence of bacterial secreted proteins in the circulation of B. anthracis infected rabbits, was addressed using specific antibodies generated by DNA vaccination against the previously selected immunogenic proteins.  The search enabled the successful detection of three secreted proteins uniquely expressed by B. anthracis, HtrA (BA3660), endopeptidase BA1952 and a protein of unknown function (BA0796). All of these proteins are potential virulence-related factors. The three biomarkers appear to be present only in the secretome of B. anthracis and not in those of the related pathogens B. thuringiensis, B. cereus.  The presence of B. anthracis secreted proteins other than the bacterial toxin in the circulation of infected animals enabled establishment of a novel approach for early detection of B. anthracis infection in clinical samples.

Detection of HtrA, the NlpC/P60-domain endopeptidase and the product of gene BA0796 in blood culture using specific antibodies 

Detection of HtrA, the NlpC/P60-domain endopeptidase and the product of gene BA0796 in blood culture using specific antibodies.  ​


​Next-Generation Bacillus anthracis Live Attenuated Spore Vaccine Based on the htrA- (High Temperature Requirement A) Sterne Strain

Live attenuated anthrax vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. We demonstrated that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in a virulent Bacillus anthracis strain resulted in significant virulence attenuation in guinea pigs, rabbits and mice. Accordingly, htrA disruption was implemented for the development of a novel Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis strain secretes functional anthrax toxins but is 10-104-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits).  In spite of this attenuation, double or even single immunization elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

Guinea pigs vaccinated with a novel live attenuated anthrax vaccine are protected against respiratory infection with a fully vir 

Guinea pigs vaccinated with a novel live attenuated anthrax vaccine are protected against respiratory infection with a fully virulent strain of Bacillus anthracis.

References

Chitlaru T, Altboum Z, Reuveny S, Shafferman A. Progress and novel strategies in vaccine development and treatment of anthrax. Immunol Rev 239, 221-236 (2011).

Chitlaru T, Shafferman A. Proteomic studies of Bacillus anthracis. Future Microbiol 4, 983-998 (2009).

Chitlaru T, Ariel N, Zvi A, Lion M, Velan B, Shafferman A, Elhanany E. Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic analysis: prevalence of proteins containing S-layer homology domains. Proteomics 4, 677-691 (2004).

Chitlaru T, Gat O, Gozlan Y, Ariel N, Shafferman A. Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities. J Bacteriol 188, 3551-3571 (2006).

Chitlaru T, Gat O, Grosfeld H, Inbar I, Gozlan Y, Shafferman A. Identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the Bacillus anthracis secretome. Infect Immun 75, 2841-2852 (2007).

Gat O, et al. Search for Bacillus anthracis potential vaccine candidates by a functional genomic-serologic screen. Infect Immun 74, 3987-4001 (2006).

Sela-Abramovich S, Chitlaru T, Gat O, Grosfeld H, Cohen O, Shafferman A. Novel and unique diagnostic biomarkers for Bacillus anthracis infection. Appl Environ Microbiol 75, 6157-6167 (2009).

Chitlaru T, Zaide G, Ehrlich S, Inbar I, Cohen O, Shafferman A. HtrA is a major virulence determinant of Bacillus anthracis. Mol Microbiol 81, 1542-1559 (2011).

Chitlaru T, Israeli M, Bar-Haim E, Elia U, Rotem S, Ehrlich S, et al. Next-Generation Bacillus anthracis Live Attenuated Spore Vaccine Based on the htrA(-) (High Temperature Requirement A) Sterne Strain. Sci Rep. 2016;6:18908.

Chitlaru T, Zaide G, Ehrlich S, Inbar I, Cohen O, Shafferman A. HtrA is a major virulence determinant of Bacillus anthracis. Mol Microbiol. 2011;81:1542-59.

Chitlaru T, Israeli M, Rotem S, Elia U, Bar-Haim E, Ehrlich S, Cohen O, Shafferman. A. Novel Anthrax Live Attenuated Spore-Vaccine Based on an Acapsular Bacillus anthracis Sterne Strain Mutated in the htrA, lef and cya genes. 2017;35:6030-6040.