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

Abstract

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated 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. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA 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 with SterneΔhtrA spores 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.

Distinct Contribution of the HtrA Protease and PDZ Domains to Its Function in Stress Resilience and Virulence of Bacillus anthracis

“…Involvement of HtrA BA in the resilience of the bacteria to stress was demonstrated previously by determining the growth of Δ htrA strains under various stress conditions and is considered to represent the main reason for the virulence attenuation associated with htrA gene disruption (Chitlaru et al, 2011b, 2016). To determine the relative contribution of the proteolytic or the PDZ domains to the role of HtrA BA in the resilience of the bacteria to stress, Δ HtrA trans -complemented with the FL, ΔPDZ or S255A forms of HtrA BA were subjected to a growth dilution drop-assay addressing sensitivity to high temperature, oxidative stress and salt, as depicted in Figure 4 (see also Supplementary Figure S3 for temperature sensitivity complementation using liquid cultures).…”

“…Anthrax is acknowledged as a toxinogenic disease, owing to the lethality of pure toxin preparations and pivotal role of the toxins in B. anthracis virulence, yet, during infection, B. anthracis secretes a large number of proteins, many of which bear biological functions indicative of a role in the onset and progression of the disease (Ariel et al, 2002, 2003; Chitlaru et al, 2006, 2007). As of today, a number of proteins, other than the classic toxins, have been suggested to play an essential role during B. anthracis infection, based on the attenuated virulence of null mutants entailing targeted disruption of specific genes (Chitlaru et al, 2011a; see Chitlaru et al, 2016 for a list and discussion of reported B. anthracis attenuating mutations).…”

“…Disruption of the htrA gene in toxinogenic B. anthracis Vollum (pXO1 + ; pXO2 + ) or Sterne (pXO1 + ; pXO2 - ) strains resulted in a dramatic attenuation in the guinea pig, murine and rabbit models of anthrax (Chitlaru et al, 2011b, 2016). Previous studies of these htrA -disrupted B. anthracis strains (to be referred from hereon as Δ htrA , Chitlaru et al, 2010, 2011b, 2016, 2017) have demonstrated that while abrogation of HtrA BA synthesis does not affect the in vitro growth of the bacteria in culture, it results in a complex phenotype exhibiting the following characteristics: (i) in vitro increased sensitivity to stress (heat, oxidative, salt, and ethanol), (ii) failure to up-regulate the NprA secreted protease under nutritional deprivation, (iii) inability to assemble the typical B. anthracis S-layer (in strains cured of the pXO1 and pXO2 native plasmids), (iv) propagation delay in a macrophage-infection assay (suggested to reflect the increased sensitivity to oxidative stress). The significant attenuation of virulence of B. anthracis Δ htrA strains suggested that HtrA BA is essential for manifestation of B. anthracis pathogenicity and accordingly, represents a novel B. anthracis virulence determinant.…”

“…The significant attenuation of virulence of B. anthracis Δ htrA strains suggested that HtrA BA is essential for manifestation of B. anthracis pathogenicity and accordingly, represents a novel B. anthracis virulence determinant. Consequently, htrA -gene disruption in the non-capsular Sterne-strain served for the development of an efficacious and safe next-generation live attenuated anthrax spore-vaccine (Chitlaru et al, 2016, 2017). The role of HtrA BA in promoting tolerance to various stress stimuli, similar to that observed in a variety of Gram-positive and negative bacteria (Skórko-Glonek et al, 2013; Backert et al, 2018) may represent the basis for the dramatic virulence-attenuation of the htrA disrupted bacteria, reflecting the inability of the mutated cells to cope with stress conditions encountered in the host.…”

Transcriptome Sequencing Data of Bacillus anthracis Vollum Δ htrA and Its Parental Strain, Isolated under Oxidative Stress

“…Proteomic/serologic surveys of B. anthracis ( 1 – 3 ) showed that the secreted protease/chaperone high-temperature requirement (HtrA) (involved in protein synthesis quality control and necessary for tolerance to heat, oxidative, and other stress regimens) belongs to a class of immunogenic vaccine and disease biomarker candidates ( 4 ). Virulence-attenuating htrA gene disruption was implemented for the development of a live spore vaccine ( 5 – 7 ). Recently, we suggested that HtrA acts as both a protease/chaperone and a pleiotropic factor of gene expression ( 8 ).…”