Development of an inhalational Bacillus anthracis exposure therapeutic model in cynomolgus macaques

Abstract

Appropriate animal models are required to test medical countermeasures to bioterrorist threats. To that end, we characterized a nonhuman primate (NHP) inhalational anthrax therapeutic model for use in testing anthrax therapeutic medical countermeasures according to the U.S. Food and Drug Administration Animal Rule. A clinical profile was recorded for each NHP exposed to a lethal dose of Bacillus anthracis Ames spores. Specific diagnostic parameters were detected relatively early in disease progression, i.e., by blood culture (∼37 h postchallenge) and the presence of circulating protective antigen (PA) detected by electrochemiluminescence (ECL) ∼38 h postchallenge, whereas nonspecific clinical signs of disease, i.e., changes in body temperature, hematologic parameters (ca. 52 to 66 h), and clinical observations, were delayed. To determine whether the presentation of antigenemia (PA in the blood) was an appropriate trigger for therapeutic intervention, a monoclonal antibody specific for PA was administered to 12 additional animals after the circulating levels of PA were detected by ECL. Seventy-five percent of the monoclonal antibody-treated animals survived compared to 17% of the untreated controls, suggesting that intervention at the onset of antigenemia is an appropriate treatment trigger for this model. Moreover, the onset of antigenemia correlated with bacteremia, and NHPs were treated in a therapeutic manner. Interestingly, brain lesions were observed by histopathology in the treated nonsurviving animals, whereas this observation was absent from 90% of the nonsurviving untreated animals. Our results support the use of the cynomolgus macaque as an appropriate therapeutic animal model for assessing the efficacy of medical countermeasures developed against anthrax when administered after a confirmation of infection.

Fig 1

Kaplan-Meier curves representing time to death from challenge and mortality data for each group. The time to death from challenge and mortality data for both challenged groups are shown. Solid line, group challenged with B. anthracis and then treated with antibody when ECL positive; dotted line, group challenged with B. anthracis and untreated.

Fig 2

Circulating levels of PA. The circulating PA levels as assessed by ELISA are presented for the untreated animals (open circles, average of nonsurvivors; closed circles, average of survivors) (A) and treated animals (open circles, average of nonsurvivors; closed circles, average of survivors) (B).

Fig 3

Kinetics of body temperature for challenged and unchallenged animals. The mean body temperatures (± the standard errors of the mean) for B. anthracis-challenged and treated (black), B. anthracis-challenged and untreated (red), and unchallenged (blue) groups are shown. The dotted line represents the average time to treatment for treated animals.

Fig 4

Kinetics of clinical hematology and CRP for challenged and unchallenged animals. Average WBC count (A), neutrophil count (B), lymphocyte count (C), and CRP (D) trends are depicted for treated, untreated, and unchallenged animals. Closed circles, animals challenged with B. anthracis and treated with antibody; open circles, animals challenged with B. anthracis and untreated; closed triangles, unchallenged animals. Group averages with standard errors of the mean are shown. ^, P < 0.05 compared to the unchallenged group; *, P < 0.05 compared to the treated group; #, P < 0.05 compared to the untreated group.

Fig 5

Correlation for the onset of clinical parameters. The correlations for the onset for a positive/abnormal ECL with bacteremia (A), PA-ELISA (B), SIBT (C), WBC (D), neutrophils (E), and CRP (F) are shown in scatter plots. The correlation coefficient for each comparison is included. *, P < 0.05.

Fig 6

Anthrax-related lesions in the livers, lymph nodes, and brains of treated and untreated NHPs. (A) Image at ×40 magnification of liver section from a monoclonal antibody-treated NHP that died 3 days after challenge. Mild sinusoidal leukocytosis and a few circulating B. anthracis organisms (arrow) are evident. (B) Image at ×40 magnification of a liver section from an untreated NHP that died 7 days after challenge. A focus of hepatocellular necrosis, fibrin exudation, suppurative inflammation, and numerous intralesional B. anthracis organisms can be seen in the lower left portion of the image. (C) Image at ×40 magnification of mediastinal lymph node from monoclonal antibody-treated NHP that died 3 days after challenge. Mild lymphoid depletion, subcapsular edema, minimal hemorrhage, and a few B. anthracis organisms (arrow) are apparent. (D) Image at ×40 magnification of mediastinal lymph node from an untreated NHP that died 5 days after challenge. Marked lymphoid necrosis, fibrin exudation, hemorrhage, and intralesional B. anthracis organisms (arrow) can be seen. (E) Image at ×20 magnification of the cerebral cortex from a monoclonal antibody-treated NHP that died 4 days after challenge. Suppurative meningitis, vasculitis, hemorrhage, and necrosis (n) are visible. (F) Image at ×20 magnification of the meninges and cerebral cortex from an untreated NHP that died 3 days after challenge. Note the typical lack of notable microscopic lesions. (G) Image at ×40 magnification of the cerebral meninges from a monoclonal antibody-treated NHP that died 4 days after challenge. Suppurative meningitis and hemorrhage with intralesional B. anthracis organisms (arrow) are evident. (H) Image at ×40 magnification of the cerebral meninges from an untreated NHP that died 3 days after challenge. Note the presence of intravascular B. anthracis organisms (arrow) but no other microscopic lesions. hematoxylin and eosin stain was used for all tissues.

Fig 7

Clinical profiles for treated (A), untreated (B), and unchallenged (C) animals. For panel A, the animal was challenged with B. anthracis and treated with antibody. For panel B, the animal was challenged with B. anthracis but not treated. Panel C shows the results for an unchallenged animal. Lines and symbols: solid lines with open circles, white blood cell count; dashed lines with open triangles, neutrophil/lymphocyte (N/L) ratio; dotted lines with squares, CRP levels; solid lines, body temperature; solid lines with open triangles, PA level; squares, positive blood culture.