Efficacy of ETI-204 Monoclonal Antibody as an Adjunct Therapy in a New Zealand White Rabbit Partial Survival Model for Inhalational Anthrax

Abstract

Inhalational anthrax is characterized by extensive bacteremia and toxemia as well as nonspecific to mild flu-like symptoms, until the onset of hypotension, shock, and mortality. Without treatment, the mortality rate approaches 100%. Antibiotic treatment is not always effective, and alternative treatments are needed, such as monotherapy for antibiotic-resistant inhalational anthrax or as an adjunct therapy in combination with antibiotics. The Bacillus anthracis antitoxin monoclonal antibody (MAb) ETI-204 is a high-affinity chimeric deimmunized antibody which targets the anthrax toxin protective antigen (PA). In this study, a partial protection New Zealand White (NZW) rabbit model was used to evaluate the protective efficacy of the adjunct therapy with the MAb. Following detection of PA in the blood, NZW rabbits were administered either an antibiotic (doxycycline) alone or the antibiotic in conjunction with ETI-204. Survival was evaluated to compare the efficacy of the combination adjunct therapy with that of an antibiotic alone in treating inhalational anthrax. Overall, the results from this study indicate that a subtherapeutic regimen consisting of an antibiotic in combination with an anti-PA MAb results in increased survival compared to the antibiotic alone and would provide an effective therapeutic strategy against symptomatic anthrax in nonvaccinated individuals.

INTRODUCTION

Bacillus anthracis is a Gram-positive, endospore-forming bacterial rod (1 to 10 μm) that causes the disease anthrax (1, 2). The most lethal route of exposure is via inhalation, and the disease is characterized by extensive bacteremia and toxemia which, without aggressive prophylaxis or intervention, results in a high mortality rate (3–5). Symptoms of inhalational anthrax present as nonspecific to mild flu-like symptoms until the onset of hypotension, shock, and sudden death (6–8).

Aerosolized B. anthracis is considered a serious biological threat, with potential use as a military or terrorist weapon (9, 10). Several countries are believed to have offensive biological weapons programs, and some independent terrorist groups have suggested their intent to use biological weapons (11, 12). In a bioterrorism attack in the United States in September 2001, B. anthracis spores were sent in letters to several locations via the U.S. Postal Service and resulted in 22 confirmed or suspect cases of cutaneous or inhalational anthrax infection and 5 deaths (13).

The capsule and two exotoxins, lethal toxin (LT) and edema toxin (ET), produced by B. anthracis are thought to be primarily responsible for the symptoms and pathogenesis of infection (14–16). The two enzymatically active toxin components, lethal factor and edema factor, are synthesized from different genes, but both associate with protective antigen (PA), which is the cell-binding component for each (17, 18). The key role of PA in anthrax infection and pathogenesis makes it a good target for therapeutic treatments (19, 20), as well as its use as a diagnostic biomarker for infection (7, 8, 14, 21, 22).

Prophylactic anthrax vaccination in the United States has been available to special populations for over 40 years, but the vaccine is not available to the general public except for postexposure prophylaxis scenarios (23). Also, vaccine administered after exposure will not be effective for the first 4 to 6 weeks postadministration (21, 22). Therefore, alternative therapeutic strategies in nonvaccinated individuals, especially for those already suffering from the onset of anthrax infection, are critically needed (3, 6, 24). Once clinical symptoms are apparent, mortality is nearly 100% in untreated cases (5, 9, 25, 26). Early diagnosis and aggressive treatment improve the prognosis. In the bioterrorism attack via the U.S. Postal Service in 2001, of the first 10 cases of inhalational anthrax, 4 patients were symptomatic at the time of hospital admission, were given multiple therapeutics, including antibiotics, but still succumbed to disease (13, 27). This strongly suggests that antibiotics are unable to prevent a fatal outcome in humans once the disease has reached a phase involving toxin production (11). Treatment to inactivate the toxins has been shown to be beneficial (19, 21, 28–30). Therefore, as recommended by the Centers for Disease and Control and Prevention (CDC) and shown with raxibacumab, the use of an antitoxin antibody in conjunction with antibiotic treatment could be an effective alternative therapy (24, 31).

Elusys Therapeutics, Inc., has developed ETI-204, a monoclonal antibody (MAb) against PA. ETI-204 contains human constant region sequences and deimmunized murine variable region sequences generated from 1H (32), an affinity-enhanced recombinant scFv (single-chain variable fragment) derived from the murine MAb 14B7 (33, 34). 1H is known to bind to domain 4 of PA, which is the domain responsible for the binding of PA to cell surface receptors (35). Based on the mechanism of action, we hypothesized that treatment with ETI-204 in combination with antibiotic would increase the survival above that with antibiotic alone as a therapeutic strategy against inhalational anthrax.

Well-characterized anthrax animal models are necessary to evaluate combination treatments of antibiotics with adjunct therapies. While nonhuman primates are often deemed the most desirable animal model of inhalation anthrax, the high cost and small number of laboratories capable of performing such trials are very limiting (20). Furthermore, the Animal Rule requires the utilization of more than one well-characterized animal model when the evaluation of a medical countermeasure's efficacy is not feasible in humans for the approval and licensing of medical countermeasures against certain biological agents (24, 36, 37). This study utilized NZW rabbits, an animal model which has been extensively used in inhalational anthrax research and is considered acceptable for evaluating the efficacy of therapeutics (19, 20, 37–39). NZW rabbits are susceptible to B. anthracis spores, regardless of route of exposure (2, 36, 39–42). Although the disease progresses more rapidly in rabbits than in humans and nonhuman primates, rabbits display most of the pathological changes that are reported in naturally occurring disease in humans (37, 40).

Doxycycline is a tetracycline antibiotic that offers a broad spectrum of activity which is primarily bacteriostatic, and it is active against Gram-positive as well as Gram-negative bacteria as it inhibits the elongation phase of protein synthesis (43, 44). Doxycycline is readily absorbed (45), with a bioavailability of more than 80%. Doxycycline is generally well tolerated with few adverse effects (44, 46). It is one of the drugs recommended by the CDC for anthrax and has been FDA approved for inhalational anthrax as postexposure prophylaxis in adults over 18 years of age (24, 47, 48). In this model, to evaluate whether or not ETI-204 can increase survival above that possible with administration of an antibiotic, a lower dose of antibiotic had to be given to achieve an overall survival rate of less than 100% in order to evaluate whether ETI-204 led to increased survival. Our previous studies indicated that a dosing regimen of a 0.5-mg/ml solution infused intravenously (i.v.) at 2.0 mg of doxycycline/kg of body weight at a rate of 4 mg/min will produce a survival rate of 40 to 60% in rabbits exposed to aerosolized B. anthracis (data not shown), and in this study we used the same dosing regimen for doxycycline treatment groups.

In conclusion, the results from this study support our hypothesis that the combination therapy of doxycycline and ETI-204 would result in increased survival compared to that provided by antibiotic alone and could provide an effective therapeutic strategy against symptomatic anthrax.

MATERIALS AND METHODS

Animals.

Twenty-four specific-pathogen-free (SPF) male (n = 13) and female (n = 11) New Zealand White rabbits (3.0 to 5.0 kg) were acquired from approved vendors. Animals were housed in individual cages and were acclimated to a 12/12-h light/dark cycle in a temperature- and humidity-controlled environment. Food and water were provided ad libitum. All rabbits were acclimated to the facility for at least 7 days prior to surgical implantation of a venous access port (VAP). VAPs were used to facilitate blood collections and intravenous administration of therapeutic agents. Rabbits were given at least 1 week to recover from surgery and were determined to be clinically healthy. Forty-eight hours before aerosol exposure, a complete blood count (CBC) and blood culture were completed to ensure there was no postoperation infection or high white blood count.

Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals (49). The facility where this research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

Spore preparation.

B. anthracis (Ames strain) spores were produced in flask cultures of Leighton and Doi medium. Spores were harvested by centrifugation, washed in sterile water for injection, and purified on gradients of 60% Hypaque-76. Spores were stored until use at 4°C in 1% phenol. Before aerosolization, phenol was removed, and spores were resuspended in sterile water for injection and heat shocked at 60°C for 50 min.

Aerosol exposure.

The study was performed in 2 iterations because of the large number of animals. On the day of aerosol challenge, NZW rabbits were placed in individual cat restraint bags in preparation for plethysmography. Whole-body head-out plethysmography (Buxco Research Systems, Wilmington, NC) was performed on conscious NZW rabbits immediately prior to exposure, in order to calculate the respiratory minute volume. Animals were exposed to target concentrations of B. anthracis Ames spores in a muzzle-only aerosol chamber within a class III biological safety cabinet. Aerosols were created by a 3-jet collision nebulizer (BGI, Waltham, MA) and controlled by using an automated bioaerosol exposure system (50). The temperature of the chamber during the spray was ambient, generally 22 to 28°C, and was measured by a temperature and humidity sensor (HX94C; Omega Engineering, Stamford, CT). The humidity of the chamber during the steady-state portion of the spray was between 40 and 70%. The concentration of aerosolized B. anthracis in the chamber was determined through constant sampling by using an all-glass impinger (AGI; Ace Glass, Vineland, NJ) containing sterile water. The viable spore concentrations in AGI samples were analyzed the same day as the aerosol exposure by performing a bacterial plating assay on sheep blood agar (SBA) plates to determine the CFU/ml. Actual presented doses were calculated by multiplying the total volume of experimental atmosphere inhaled times the aerosol concentration. The target aerosol challenge dose was 150 to 250 times the 50% lethal dose (LD₅₀) of B. anthracis (Ames strain) spores.

Clinical observations.

NZW rabbits were observed and scored at least twice a day after aerosol exposure. Rabbits were monitored for clinical signs and changes in appearance (coat condition, respiration, and body posture), natural behavior (mobility, alertness, food intake, and vocalization), and provoked behavior.

The scoring parameters were as follows: appearance, 0 to 2; natural behavior, 0 to 3; provoked behavior, 0 to 4. NZW rabbits with total scores between 4 and 6 were monitored with an increased frequency (three times/day), with the endpoint criterion for humane euthanasia, indicative of very poor health status, being a score of ≥7.

Blood collection.

Aseptic techniques and preparation methods were used throughout these procedures. Blood specimens were collected from all rabbits via a VAP. If the VAP was not functioning, rabbits were anesthetized and blood was collected via ear vein. Blood was collected immediately prior to the start of treatment for culture to document the bacteremic status at time of treatment as well as the PA level based on enhanced chemiluminescence (ECL).

Bacteremia.

For blood plate cultures, whole blood was withdrawn via syringe from the VAP and injected directly into a pediatric yellow top bacterial culture tube (Wampole bacterial isolator) and then was cultured on sheep blood agar by using the spread plate method. Blood samples were diluted to serial 1:10 dilutions for quantitative results, and plating was carried out to the 10⁻³ dilution, except for terminal cultures, which were plated to a 10⁻⁶ dilution. Plates were incubated for 18 to 24 h at 37°C. Colonies were counted and were recorded as CFU/ml approximately 24 h after sampling.

PA ECL assay.

PA was determined qualitatively, from whole blood, by ECL immunoassay (Bioveris M-Series M1M system analyzer; Bioveris, Gaithersburg, MD). Three PA-specific monoclonal antibodies (Critical Reagents Program, Aberdeen Proving Grounds Edgewood Area, Chesterton, MD) were used as a capture-antibody mix, and a polyclonal rabbit serum made against PA was used as a detector antibody to measure PA. Purified antibodies were labeled with biotin using standard coupling methods. Biotinylated antibodies were prebound to streptavidin-coated paramagnetic beads (2.8-μm diameter; M-280; Dynal Corp., Lake Success, NY). Whole blood (50 μl) from the NZW rabbits, as well as two negative controls and two levels of positive controls (whole blood spiked with PA), was added to the appropriate assay tubes along with 50 μl of BioVeris (BV) diluent. All samples were tested in duplicate and read using the M1M analyzer. PA-negative rabbit whole blood served as the negative control and was used to establish assay cutoffs. Samples were considered positive if the ECL signal-to-noise (background) ratio was ≥1.2 times the average of the negative control or >3 standard deviations of the negative controls, whichever was greater. PA results were determined within 1 h of collection of each blood sample.

Doxycycline preparation.

Doxycycline (APP Pharmaceuticals, LCC, Schaumburg, IL) was withdrawn at 100 mg per 10 ml and diluted in sterile water to a working concentration of 0.5 mg/ml for administration. Dosing calculations were determined based on body weight, such that each animal would receive an i.v. treatment concentration of 2.0 mg/kg at an infusion rate of 3.0 ml/min over 5 min with 0.5 mg/ml of doxycycline. Rabbits were treated twice a day for 3 days with doxycycline alone. Doxycycline dose formulation analysis and pharmacokinetics were not assessed.

ETI-204 preparation.

ETI-204, an investigational drug manufactured by Baxter Healthcare Corporation for Elusys Therapeutics, Inc. (Pine Brook, NJ) was provided as a sterile liquid and was withdrawn and diluted to a nominal concentration (in mg/ml) in sterile saline that would deliver a single dose of 8 mg/kg per animal. Treatment was initiated immediately following the first antibiotic dose and was administered via slow i.v. push. Rabbits were treated with a single dose of ETI-204. ETI-204 dose formulation analysis and ETI-204 pharmacokinetics were not assessed.

Pathology.

A necropsy of each animal was performed, and the following tissues were collected at necropsy: ear with tattoo identification, thoracic pluck (lungs, heart, trachea, esophagus, with attached mediastinum), spleen, kidneys, liver, and brain.

Histology.

Tissues were routinely processed and stained with hematoxylin and eosin (HE). The tissue samples were trimmed and embedded in paraffin. Paraffin-embedded tissues were sectioned at 5 μm for histology. The histology slides were deparaffined, stained with HE, covered with coverslips, and labeled.

Statistical analysis.

Group sizes were determined at a two-tailed α level of 0.05 and provided a >80% power to detect a minimum difference in percent survival between two groups of 70% based on a Fisher exact test. SAS version 9.3 was used to perform statistical analyses throughout. All statistical tests were performed at an α level of 0.05 with two-sided alternatives. Survival rates between doxycycline alone, doxycycline plus ETI-204, and saline-treated groups were compared using exact permutation Cochran-Armitage trend tests stratified by experimental iteration, with P values corrected by permutation to account for multiple comparisons. Pairwise comparisons of treatment groups were made with log-rank comparisons for ECL and bacteremia results.

RESULTS

Study design and presented doses.

When the data from two iterations were combined, the average presented dose was 378 ± 225 LD₅₀s, ranging from 33 to 773 LD₅₀s for individual rabbits (Table 1). Previous studies have indicated that time to death (TTD) is not challenge dose dependent in this model (39). Additionally, any dose in excess of 30 LD₅₀s would be greater than the determined LD₉₉ for B. anthracis in NZW rabbits (51). Therefore, the lowest dose of 33 LD₅₀s was considered to be within acceptable limits for challenge doses in these animals. Rabbits were divided into three groups and were administered doxycycline alone (for which a dose of 2.0 mg/kg in a 10-mg/ml solution for 3 days twice a day was previously determined to be the necessary dosing regimen for 40 to 60% survival [data not shown]), an adjunct therapy of doxycycline and MAb ETI-204, or saline only (vehicle control) following a positive PA result or at 30 h postexposure.

TABLE 1.

Inhaled doses for NZW rabbits exposed to aerosolized B. anthracis

Animal ID no.	Treatment	CFU/animal	Dose (no. of LD50s)
37	Saline	1.25E+7	118.7
39	Saline	5.29E+6	50.4
48	Saline	6.03E+6	57.5
57	Saline	4.91E+7	467.4
41	Doxycycline	2.03E+7	193.3
43	Doxycycline	1.47E+7	140.0
45	Doxycycline	3.53E+6	33.6
46	Doxycycline	1.81E+7	172.5
50	Doxycycline	3.94E+7	375.6
56	Doxycycline	5.84E+7	556.3
59	Doxycycline	6.11E+7	581.6
60	Doxycycline	4.01E+7	381.9
61	Doxycycline	7.44E+7	708.3
62	Doxycycline	7.01E+7	667.2
40	Doxycycline + ETI-204	2.97E+7	283.1
42	Doxycycline + ETI-204	3.07E+7	292.4
44	Doxycycline + ETI-204	3.63E+7	345.9
47	Doxycycline + ETI-204	2.20E+7	209.7
49	Doxycycline + ETI-204	4.37E+7	416.0
51	Doxycycline + ETI-204	4.96E+7	472.6
52	Doxycycline + ETI-204	5.60E+7	532.9
53	Doxycycline + ETI-204	6.57E+7	625.3
54	Doxycycline + ETI-204	6.67E+7	635.2
55	Doxycycline + ETI-204	8.12E+7	773.3

Survival.

Of the 24 NZW rabbits exposed, 5 out of 10 rabbits in the antibiotics-alone group survived, yielding a 50% survival rate for the antibiotic-only group. Of the 10 rabbits that received doxycyline in combination with ETI-204, there were 9 survivors, resulting in a survival rate of 90% for this group. The four animals placed in the saline control group all succumbed to disease, resulting in a survival rate of 0% for this group. The results demonstrated a statistically significant difference in the survival rates between the vehicle control and the adjunct therapy group (P = 0.0051), while the survival rates between the antibiotic-alone and vehicle control groups (P = 0.8446) as well as that between the adjunct therapy and antibiotic alone-treated groups (P = 0.1031) were not significantly different (Fig. 1).

FIG 1.

Percent survival versus TTD in NZW rabbits exposed to aerosolized B. anthracis. Survival rates between animals treated with doxycycline plus ETI-204 versus saline-treated groups were significantly different (P = 0.0051).

Of the two NZW rabbits that never tested positive for the presence of PA in whole blood or became bacteremic, only one survived; it was in the adjunct therapy group (animal 47), while the other PA-negative animal, animal 45 in the antibiotic-alone group, succumbed to disease at 9 days postexposure (PE). Both animals were treated at 30 h PE. For the other remaining rabbits that succumbed to disease, TTD occurred at approximately 1 to 2 days PE for control animals, approximately 6 to 9 days PE for animals in the antibiotic-only group, and 6 days PE for the animal that was euthanized from the doxycycline plus ETI-204 group. All surviving animals were euthanized on day 29 (Table 1).

Clinical observations.

Clinical observations for B. anthracis-challenged rabbits were similar to those reported previously (51), with no obvious, clear pattern in the overall clinical observations between groups. Rabbits that succumbed to infection exhibited scores ranging from normal to elevated. Rabbits with elevated scores (scores of 4 to 9) displayed lethargy or weakness and evidence of respiratory distress and were euthanized when scores were ≥7. Scores for rabbits that were found expired in their cages were within normal limits at the last clinical observation. This outcome can be attributed to the rapid progression of disease in the NZW rabbit (39). Of the survivors, all animals maintained clinical scores that were within normal limits throughout the duration of the study with no significant differences between treatment groups (data not shown).

Development of toxemia.

PA detection by ECL assay was easy and reproducible (15), and results were acquired within 1 h of blood collection. All NZW rabbits, with the exception of one rabbit in the antibiotic alone group (animal 45) and one rabbit from the adjunct therapy group (animal 47), displayed a positive PA result at the time of treatment (Table 2). Detection of PA by the ECL assay correlated with bacteremia plate culture results in all three groups. All rabbits in both treatment groups that were PA positive remained positive throughout the 3 days of treatment, with the exception of one rabbit in the adjunct therapy group (animal 49) (Table 2). Rabbit 49 was PA negative on the third and the last day of the treatment, while all the other PA-positive rabbits were still PA positive on the last day of the treatment. By the end of the study (day 29 PE), the presence of PA dissipated in five out of the nine rabbits in the antibiotic-alone group, while PA dissipated in nine out of nine rabbits in the adjunct therapy group, including the one animal that died in this group (Table 2).

TABLE 2.

ECL results for the presence of protective antigen against B. anthracis

^a+, ECL present; −, no ECL. Shaded regions indicate days on which there was no blood draw. On day 1, the assay was done every 4 h, and therefore the data in the 6 “day 1” columns represent consecutive time points.

^bNA, not applicable.

Bacteremia.

In the control group, all four rabbits were bacteremic on day 1 PE, and increased bacteremia was observed on day 2 PE, when all control animals succumbed to disease (Table 3). Administration of antibiotics alone or in combination with ETI-204 was associated with lower bacteremia levels between days 1 and 2. Bacteremia was not detected in any animals following the administration of doxycycline plus ETI-204 combination therapy during the rest of the study; in the antibiotic-alone group, transient bacteremia development between days 6 and 14 was observed in 3 of 9 animals surviving to day 6 (Fig. 2). All three of these animals died by day 8 (Table 3). When we compare pre- and posttreatment bacteremia levels between doxycycline and doxycycline plus ETI-204groups, there was a clear difference between these two groups for clearing bacteremia (Fig. 3).

TABLE 3.

Bacteremia results for NZW rabbits exposed to aerosolized B. anthracis

^aShaded regions indicate days on which there was no blood draw. On day 1, the assay was done every 4 h, and therefore the data in the 6 “day 1” columns represent consecutive time points.

^bNA, not applicable.

FIG 2.

Average concentration of B. anthracis in whole blood for each treatment group. Onset of bacteremia occurred between days 1 and 2 PE, with levels dissipating by day 3. Recurrence of bacteremia was noted within the doxycycline-treated group on day 6.

FIG 3.

Comparison of pre- and posttreatment average bacteremia levels between treatment groups. Group 1, doxycycline-only treatment group; group 2, doxycycline plus ETI-204 treatment group. *, absence of bacteremia.

Pathological findings.

Of the 24 exposed NZW rabbits, 10 animals succumbed to anthrax disease, and 14 animals survived challenge. Among those that succumbed to disease, saline-treated animals died within 1 to 2 days PE and had pathological changes consistent with documented cases of aerosolized B. anthracis in NZW rabbits. Six animals had delayed time to death: five animals treated with doxycycline only (6 to 9 days PE) and one animals treated with doxycycline plus ETI-204 (6 days PE).

Gross changes in most animals that succumbed included noncollapsing lungs that were sharply mottled red; an enlarged friable spleen and liver; hemorrhage, congestion, and/or edema in multiple tissues; and pleural effusion. Additionally, three animals, numbers 43, 60, and 55, had lesions consistent with meningitis and atypical of inhalational anthrax in NZW rabbits (37, 51).

Histologic changes included necrotizing splenitis; pneumonia (due to infection with B. anthracis); adrenal gland necrosis and hemorrhage; degeneration and necrosis of renal tubules; mediastinitis and meningitis; and bacilli noted within blood vessels of multiple tissues and organs. These findings are consistent with reported anthrax lesions in NZW rabbits. Atypical histologic findings included meningitis noted in three animals: numbers 43, 60, and 55 (Fig. 4). In two of the animals with meningitis, numbers 43 and 55, bacilli were only identified within the meninges.

FIG 4.

Histological findings. (a) Rabbit 57 (saline control) brain: normal brain histology with thin leptomeninges (arrows). HE, stain. Magnification, 200×. (b) Rabbit 55 (doxycycline plus ETI-204) brain. The leptomeninges (arrow) are markedly expanded by inflammation that multifocally extended into the brain parenchyma (asterisk), causing meningoencephalitis. HE stain. Magnification, ×100. (c) Rabbit 43 (doxycycline) brain. The inflammation was restricted to the leptomeninges in this animal (meningitis). HE stain. Magnification, ×200. (d) Rabbit 43 (doxycycline) brain. The images, at its higher magnification, show the leptomeninges expanded by numerous heterophils (arrow) and myriad bacilli. HE stain. Magnification, ×600.

Within the control group, all animals succumbed to disease within 2 days PE with typical anthrax lesions. Within the doxycycline group, five animals had delayed time to death (6 to 9 days PE), and five animals survived challenge with minimal pathological changes (Tables 2 and 3). Mild splenitis was evident in survivors of the doxycycline-treated group.

DISCUSSION

Currently, the CDC recommendations following potential exposure to aerosolized B. anthracis spores entail 60 days of oral antibiotics combined with a 3-dose series of the PA-based anthrax vaccine (48). Antibiotics are effective in killing bacteria, although they are unable to clear released toxins from the bloodstream. Unless exposure is diagnosed early enough for antibiotic treatment to prevent significant bacterial replication, patients succumb to toxin-induced disease even after elimination of all bacteria (13, 21, 52). Therefore, new treatments directed at neutralizing anthrax toxins are needed (19, 53). A high-affinity monoclonal antibody that targets anthrax toxins would be an important therapeutic addition in the treatment of anthrax. Six other monoclonal antibodies against PA are under investigation and are aimed at targeting PA to neutralize toxin production in anthrax infection (21, 28, 54, 55).

These MAbs offer additional protection against inhalational anthrax via alternative mechanisms from those of either antibiotics or immunization, thus aiding in reducing morbidity and mortality. In this study, a partial protection New Zealand White rabbit model was used to demonstrate that a subtherapeutic regimen consisting of antibiotics in combination with a high-affinity MAb, ETI-204 (29), resulted in greater efficacy of protection and increased survival from inhalational anthrax than that provided by antibiotic alone.

ETI-204 is in late-stage development for treatment of inhalational anthrax by i.v. infusion. ETI-204 has been tested previously in vitro as well as in vivo as both preexposure prophylaxis as well as postexposure prophylaxis to aerosolized B. anthracis (29). ETI-204 has high affinity for PA (K_d, 0.33 nM), unlike raxibaumab, which has a low binding affinity for PA at 2.78 nM (31, 54) and protected 94% of rabbits when administered intravenously prior to aerosol challenge with B. anthracis Ames spores (29). Partial protection was observed in rabbits injected intravenously at 24, 36, and 48 h after aerosol challenge with B. anthracis Ames spores, with survival decreasing the later the administration.

In the present study, doxycycline was given alone at 2.0 mg/kg twice a day for 3 days or in conjunction with a single dose of ETI-204. A trend was observed for increased survival in the adjunct therapy group (9/10) compared to the antibiotic-alone group (5/10). While this increase in survival was not supported by statistical analysis, presumably due to the small sample size, the trend indicates improved survival rates with the combination therapy. The four remaining vehicle control animals all succumbed to disease, resulting in a survival rate of 0% for this group. In the rabbits that succumbed to disease, pathological and histological findings determined that inhalational anthrax was the cause of death. In two out of the six rabbits that succumbed to exposure (animals 43 and 55), histological changes and distribution of bacilli were atypical for experimental anthrax in the rabbit model. Unique features included bacilli restricted to meninges, acute meningitis, normal to hyperplastic lymphoid tissue in spleen and lymph nodes, and relative lack of hemorrhage. Speculations found in the literature may explain the presence of acute meningitis in these rabbits. While meningitis is uncommon in untreated rabbits, it is a common finding in humans and nonhuman primates (2, 51, 56). The cause of this variation in animal models is unknown. This distinction may, at least in part, be associated with a slower progression of disease in humans and nonhuman primates compared to that in untreated rabbits. Antibiotic therapy alone (animal 43) as well as antibiotic in combination with ETI-204 (animal 55) extended survival time in both rabbits, potentially giving sufficient time for such a lesion to develop.

Analysis of the ECL data demonstrated that toxin levels, as indicated by the presence of PA via ECL, did not dissipate until after treatment in the antibiotic-alone group as well as in the adjunct therapy group. Bacteremia levels in both groups diminished over the course of the study and remained absent in all survivors. Reemergence of bacteremia was not captured in all antibiotic-alone group animals that succumbed (2 of 5). Due to the rapid disease progression in rabbits (29, 39, 40), it is possible that the animals expired before a blood culture could be obtained. These results suggest that, while the antibiotic alone was able to eliminate bacterial growth, it was not sufficient to ensure rapid elimination of anthrax toxins (57), which led to decreased survival.

In previous studies animals challenged with aerosolized anthrax spores after administration of ETI-204 via intravenous injection developed toxin-neutralizing antibodies against PA (29). Rabbits in the adjunct therapy group were able to eliminate bacterial growth as well as eliminate the presence of PA in the bloodstream. This is significant, because it demonstrates that the adjunct therapy is not only able to eradicate bacterial growth but may also allow for the clearance of PA, thus increasing survival by preventing reemergence of bacterial growth and toxin development.

Overall, the results from this study indicate that in the rabbit model of inhalation anthrax, a subtherapeutic regimen consisting of antibiotics in combination with an adjunct therapy, ETI-204, results in a greater efficacy of protection against reemergence of bacteremia and PA production and overall increased survival than that provided by an antibiotic alone (doxycycline) against inhalational anthrax. Thus, the results from this study support our hypothesis that a therapy approach of doxycycline in combination with a high-affinity monoclonal antibody (ETI-204) against the PA component of anthrax toxin results in increased survival compared to that provided by antibiotic alone, and this could provide an effective therapeutic strategy against symptomatic anthrax in nonvaccinated individuals.