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Abstract
Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2−) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R.
Highlights
Bacillus anthracis (B. anthracis), the etiological agent causing anthrax, is a Gram-positive, spore-forming bacterium
In the culture supernatants of the A16R and Sterne strains, active protective antigen (PA) and lethal factor (LF) showed an increase at the beginning and a decrease
Anthrax caused by B. anthracis is an important endemic disease of public health concern
Summary
Bacillus anthracis (B. anthracis), the etiological agent causing anthrax, is a Gram-positive, spore-forming bacterium. It can be used in biowarfare or bioterror attacks and has been a top bioterrorism concern since the 2001 anthrax attacks in the USA [1,2]. Virulent forms of B. anthracis carry two large plasmids: pXO1 and pXO2. The pXO1 plasmid encodes anthrax toxins, and pXO2 encodes proteins that form the poly-D-glutamic acid capsule. Anthrax toxin (AT), including lethal toxin (LT) and edema toxin (ET), are Atype exotoxins each composed of two proteins. The A component is either the lethal factor (LF, kDa) or edema factor (EF, kDa), and the B component is the protective antigen (PA, 83 kDa) [3]. EF is a calmodulin-dependent adenylyl cyclase that elevates intracellular cAMP
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