Comprehensive characterization of toxins defines progression and stages in a non-human primate model of inhalation anthrax

Abstract

Inhalation anthrax has three clinical stages: early-prodromal, intermediate-progressive and late- fulminant. The toxins produced during infection exert pathologic effects leading to death, but they have not been comprehensively characterized throughout the course of infection. Mass spectrometry methods for anthrax toxins, total-protective antigen (PA), total-lethal factor (LF), total-edema factor (EF), and toxin complexes, lethal toxin and edema toxin were used to characterize the stages of inhalation anthrax in 23 cynomolgus macaques. The target aerosol dose was 200 LD50 B. anthracis Ames spores. 22 animals died during the study. Different patterns of toxemia and bacteremia were observed in 11 animals with the shortest survival times (fast progression), the 11 animals with longer survival (slow progression), and the one survivor. Toxemia and bacteremia were predominantly triphasic with an early rise (phase-1), a plateau/decline (phase-2), and a final rapid rise (phase-3). The patterns were consistent for all toxins. The end-of-phase-1 LF was higher in fast progression [median(lower quartile- upper quartile)] of [195(57.4-326)-ng/mL], than in slow progression [23.8(15.6-26.3)-ng/mL] (p=0.0001), or the surviving animal [11.1-ng/mL]. End-of-phase-1 EF was also higher in fast [22.2(2.7- 42.8)-ng/mL] than slow progression [0.17(0.064-0.066)-ng/mL] (p=0.0005), or the surviving animal [0.040-ng/mL]. Animals with slow progression and lower end-of-phase-1 toxemia, had an extended plateau/decline ([≥]24-hours), with low variability of PA, LF, and LTx across all animals. Its characterization revealed an upper threshold; a limit for exiting phase-2 and entering the critical phase-3, 342-ng/mL (PA), 35.8-ng/mL (LF), and 1.10-ng/mL (EF). The thresholds were exceeded early in animals with fast progression (38.3{+/-}7.4-hours) and later in slow progression (78.7{+/-}14.1-hours). Once the threshold was passed toxin levels rose rapidly, differences in toxemia were reduced, and the duration to terminal was rapid and similar; 21.0{+/-}7.3-hours for fast and 20.4{+/-}7.3-hours for slow. This first comprehensive evaluation of anthrax toxins defined all stages, providing insights into disease progression. Author SummaryThe comprehensive analysis of all major anthrax toxins and bacteremia in a non-human primate model of inhalation anthrax revealed a triphasic kinetics of toxemia that aligns with the three clinical stages, early-prodromal, intermediate-progressive and late-fulminant. End of phase-1 toxin levels may predict the subsequent speed of progression. Phase-2 toxemia helped define critical thresholds representing the entry to phase-3. Exceeding these thresholds was associated with a short remaining survival time (about 21 hours). This first comprehensive characterization of toxemia provides knowledge and guidance for better management of anthrax.