Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R

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.

Efficacy assessment of a triple anthrax chimeric antigen as a vaccine candidate in guinea pigs: challenge test with Bacillus anthracis 17 JB strain spores

“…PA comprises a basis for all anthrax vaccines [1]. Furthermore, LF and EF also induce the production of toxin neutralizing antibodies [11][12][13]. Live attenuated vaccines, such as vaccines based on the Sterne strain (34F2), have been approved for veterinary anthrax vaccines since the 1930s while they were only used for human vaccination in the past.…”

“…Meanwhile, China uses toxin-producing live-attenuated B. anthracis spores (A16R; pXO1þ, pXO2À) as a human anthrax vaccine. No evidence or reports have been released regarding the effectiveness, possible side effects, and usage limitations of these vaccines [11].…”

Analysis of Single Bacterium Dynamics in a Stochastic Model of Toxin-Producing Bacteria

“…We illustrate our results by focusing on the bacterium Bacillus anthracis and its anthrax toxins. For the A16R B. anthracis strain we quantify for the first time the rate of protective antigen (PA) production making use of published data from an in vitro experimental assay [28]. The resulting parametrized mathematical model serves to illustrate our techniques and allows us to consider antibiotic treatment.…”

Development of Enzyme Linked Immunosorbent Assay for humoral immuneresponse and infection monitoring of anthrax

“…Anthrax toxin is composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). All of which play a significant role in boosting the immune system's response to such vaccines; however, the PA (among the three component PA, LF and EF) is the most effective factor in increasing the immunogenicity in B. anthracis toxin (Zai et al, 2016). Although the existing vaccines such as AVA are safe and effective, there has been a surge in the need for anthrax vaccines due to the increased terroristic attacks in the recent decades.…”