Protective antigen antibody augments hemodynamic support in anthrax lethal toxin shock in canines

Abstract

Background: Anthrax-associated shock is closely linked to lethal toxin (LT) release and is highly lethal despite conventional hemodynamic support. We investigated whether protective antigen-directed monoclonal antibody (PA-mAb) treatment further augments titrated hemodynamic support.

Methods and results: Forty sedated, mechanically ventilated, instrumented canines challenged with anthrax LT were assigned to no treatment (controls), hemodynamic support alone (protocol-titrated fluids and norepinephrine), PA-mAb alone (administered at start of LT infusion [0 hours] or 9 or 12 hours later), or both, and observed for 96 hours. Although all 8 controls died, 2 of 8 animals receiving hemodynamic support alone survived (median survival times 65 vs 85 hours, respectively; P = .03). PA-mAb alone at 0 hour improved survival (5 of 5 animals survived), but efficacy decreased progressively with delayed treatment (9 hours, 2 of 3 survived; 12 hours, 0 of 4 survived) (P = .004 comparing survival across treatment times). However, combined treatment increased survival irrespective of PA-mAb administration time (0 hours, 4 of 5 animals; 9 hours, 3 of 3 animals; and 12 hours, 4 of 5 animals survived) (P = .95 comparing treatment times). Compared to hemodynamic support alone, when combined over PA-mAb treatment times (0, 9, and 12 hours), combination therapy produced higher survival (P = .008), central venous pressures, and left ventricular ejection fractions, and lower heart rates, norepinephrine requirements and fluid retention (P ≤ .03).

Conclusions: PA-mAb may augment conventional hemodynamic support during anthrax LT-associated shock.

Figure 1.

Timeline of experimental interventions, measurements, and treatments. As outlined in “Methods,” at the initiation of 24-h lethal toxin infusions, animals were randomized to receive hemodynamic support alone, protective antigen–directed monoclonal antibody (PA-mAb) at the time of or 9 or 12 h after starting toxin infusion, hemodynamic support combined with PA-mAb (administered at 0, 9, or 12 h), or no treatment (control). Hemodynamic support included a single bolus of 20 mL/kg of normal saline if the pulmonary artery occlusion pressure (PAOP, checked every 2 h for the first 8 h and every 4 h thereafter) was <10 mmHg. Also, if at any time the mean arterial blood pressure (MAP) decreased to <80 mmHg for >5 min, a norepinephrine (NE) infusion was initiated at 0.2 μg/kg/min and, if necessary, increased in a stepwise fashion every 5 min to 0.6, 1, or a maximum of 2 μg/kg/min. NE was titrated down in a stepwise fashion if MAP was >100 mmHg for >5 min. Abbreviations: ABG, arterial blood gas; CBC, complete blood count; CVP, central venous pressure; HR, heart rate; LVEF, left ventricular ejection fraction (measured with echocardiography).

Figure 2.

Proportion of animals surviving over time after a 24-h lethal toxin challenge (shaded area) and treated with conventional hemodynamic support alone (Hemo) or protective antigen–directed monoclonal antibody (PA-mAb) alone (mAb), or both (Hemo + mAb) or neither (placebo alone, control). The 3 panels in Figure 2A show experiments in which PA-mAb or placebo was administered at 0, 9, or 12 h, respectively. The efficacy of PA-mAb alone decreased significantly with delay in administration (P = .004 for the trend test) but not when it was combined with hemodynamic support (P = .95, trend test). Figure 2B displays proportional survival in animals in the 3 groups shown combined over all 10 experiments, irrespective of timing of PA-mAb. Because the effects of PA-mAb alone differed significantly with time of administration, survival data from this group could not be combined over experiments and are not presented in panel B. Two-group comparison P values labeled in Figure 2B were based on exact log-rank tests.

Figure 3.

Mean (± standard error [SE]) changes from baseline over the study period in mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), hemoglobin (Hb), and left ventricular ejection fraction (LVEF, as measured by echocardiography) in canines challenged with a 24-h infusion of lethal toxin (shaded area) and treated with either conventional hemodynamic support alone (Hemo) or in combination with protective antigen directed monoclonal antibody (Hemo + mAb) compared with placebo-treated controls (no therapy). Except for LVEF, these parameters were measured either continuously or multiple times every 24 h, and data shown at various time points are daily averages of values recorded in the preceding 24-h period. Number of animals from which data are gathered every day may be determined from the survival plots in Figure 2. Data shown are least-square means, and comparisons are made between groups every 24 h; corresponding P values (from linear mixed models) are shown below each graph, with P ≤ .05 considered significant.

Figure 4.

Mean (± standard error [SE]) fluid intake, urine output, and net fluid balance over each 24-h period of the study in lethal toxin (LT)–challenged canines randomized to no therapy (control), conventional hemodynamic support alone (Hemo), or in combination with protective antigen–directed monoclonal antibody (Hemo + mAb). The format for this figure is similar to the previous ones. Shaded areas represent time over which LT was infused, and number of animals contributing to data at each time point may be determined from survival plots in Figure 2. All P values were based on linear mixed models, and P ≤ .05 were considered significant.

Figure 5.

A, Mean (± standard error [SE]) amounts of norepinephrine (NE) usage averaged over each 24-h period required to maintain mean arterial blood pressure (MAP) ≥80 mmHg in the 2 groups of LT-challenged animals randomized to this treatment—Hemo and Hemo + mAb. Inset shows corresponding changes form baseline in MAP in the 2 groups, similar to Figure 3A. The Hemo group required a significantly higher amount of NE to maintain similar MAP levels compared with the Hemo + mAb group over the second half of the study (P values for comparisons below figure). B, Because MAP and NE usage are interdependent, we present a combined score, termed the shock index (SI), by calculating the difference between normalized values of MAP and NE usage. Values progressively greater than zero denote a less severely ill state, whereas values less than zero denote more severe illness. The Hemo + mAb group did significantly better than the Hemo group over the second half of the study period (P ≤ .008). All P values were based on linear mixed models, and P ≤ .05 were considered significant.

Figure 6.

Mean (± standard error [SE]) changes from baseline over study period in blood urea nitrogen (BUN), creatinine, lactate, pH, activated partial thromboplastin time (aPTT), and fibrinogen (F) measurements in canines challenged with a 24-h infusion of lethal toxin (shaded areas) and treated with placebo (control), or conventional hemodynamic support alone (Hemo) or in combination with protective antigen–directed monoclonal antibody (Hemo + mAb). Data shown are least square means; number of animals contributing data at each time point may be determined from the survival plots in Figure 2. Comparisons between groups are made using averaged data at 24-h intervals; corresponding P values are shown below each figure. Note that for lactate and aPTT, values plotted as change from baseline are log values. All P values were based on linear mixed models, and P ≤ .05 were considered significant.

Figure 7.

Mean (± standard error [SE]) changes from baseline over the study period in mean arterial blood pressure (MAP), heart rate (HR), central venous pressure (CVP), left ventricular ejection fraction (LVEF), and hemoglobin (Hb) in canines challenged with a 24-h infusion of lethal toxin (shaded area) and treated with either protective antigen–directed monoclonal antibody (PA-mAb; mAb in the figure) alone or a combination of hemodynamic support and PA-mAb (Hemo + mAb). Data for the combination therapy group were combined across all PA-mAb administration times [0 h (n = 5), 9 h (n = 3), or 12 h (n = 4)]. Animals treated with PA-mAb alone are divided into 2 groups on the basis of early administration of treatment [0 h (n = 5)] or later administration [9 h and 12 h (n = 7)]. The format for this figure is similar to Figure 3. All P values were based on linear mixed models, and P ≤ .05 was considered significant.